Note: Descriptions are shown in the official language in which they were submitted.
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IMMUNOGENIC COMPOSITIONS AND USES THEREOF
Sequence Listing
The instant application contains a Sequence Listing which has been submitted
electronically in
ASCII format and is hereby incorporated by reference in its entirety. Said
ASCII copy, created on
September 2, 2021, is named 51509-045W02 Sequence Listing 9 2 21 ST25 and is
52,787 bytes in
size.
Background
Vaccination has made an enormous contribution to both human and animal health.
Since the
invention of the first vaccine in 1796, vaccines have come to be considered
the most successful method
for preventing many infectious diseases by provoking an immune response in a
subject. According to the
World Health Organization, immunization currently prevents 2-3 million deaths
every year across all age
groups. Today, vaccines have been developed to prevent and control the spread
of more than 20
infectious diseases, including diphtheria, tetanus, pertussis, influenza, and
measles, and have led to the
complete eradication of smallpox. There remains a need to develop new and
improved immunogenic
compositions and uses thereof.
Summary
This disclosure provides compositions, pharmaceutical preparations, and uses
of
polyribonucleotides (e.g., circular or linear polyribonucleotides) encoding
one or more immunogens. In
particular, the disclosure provides circular polyribonucleotides encoding
multiple immunogens, circular
polyribonucleotides encoding at least one immunogen and further encoding at
least one adjuvant, and
compositions including multiple circular polyribonucleotides. This disclosure
further relates to methods of
using the circular polyribonucleotides encoding one or more polypeptide
immunogens. Compositions and
pharmaceutical preparations of circular polyribonucleotides described herein
may induce an immune
response in a subject upon administration. Compositions and pharmaceutical
preparations of circular
polyribonucleotides described herein may be used to treat or prevent a
disease, disorder, or condition in a
subject.
In one aspect, provided herein is a circular polyribonucleotide including a
plurality of sequences,
each sequence encoding a polypeptide immunogen, wherein at least two (e.g., at
least three, at least
four, at least five, at least six, at least seven, at least eight, and at
least nine) of the polypeptide
immunogens have at least 90% (e.g., at least 91%, at least 92%, at least 93%,
at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, and at least 99%) amino acid
sequence identity. In some
embodiments, the at least two polypeptide immunogens have at least 95% (e.g.,
96%, at least 97%, at
least 98%, and at least 99%) amino acid sequence identity. In some
embodiments, the at least two
polypeptide immunogens have less than 100% amino acid sequence identity. In
some embodiments,
each of the polypeptide immunogens have at least 90% (e.g., at least 91%, at
least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at
least 99%) amino acid sequence
identity. In some embodiments, each of the polypeptide immunogens have at
least 95% (e.g., 96%, at
least 97%, at least 98%, and at least 99%) amino acid sequence identity. In
some embodiments, each of
the polypeptide immunogens have less than 100% amino acid sequence identity.
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In some embodiments, each of the polypeptide immunogens includes one or more
epitopes that
identifies a target. In some embodiments, the target is a pathogen. In some
embodiments, the pathogen
is a virus, a bacterium, a fungus, or a parasite. In some embodiments, the
pathogen is a virus and each
polypeptide immunogen includes one or more epitopes (e.g., two, three, four,
five, six, seven, eight, nine,
ten or more) corresponding to a viral immunogen. In some embodiments, the
pathogen is a bacterium
and each polypeptide immunogen includes one or more (e.g., two, three, four,
five, six, seven, eight, nine,
ten or more) epitopes corresponding to a bacterial immunogen. In some
embodiments, the target is a
cancer cell. In some embodiments, polypeptide immunogen includes one or more
(e.g., two, three, four,
five, six, seven, eight, nine, and ten or more) epitopes corresponding to a
tumor antigen. In some
embodiments, the target is a toxin or an allergen. In some embodiments, the
target is associated with a
disease, disorder, or condition. In some embodiments, the disease, disorder,
or condition is a viral
infection. In some embodiments, the disease, disorder, or condition is a
bacterial infection. In some
embodiments, the disease, disorder, or condition is a cancer.
In another aspect, the disclosure provides a circular polyribonucleotide
including a plurality of
.. sequences, each sequence encoding a polypeptide immunogen, wherein at least
two (e.g., at least three,
at least four, at least five, at least six, at least seven, at least eight, or
at least nine) of the polypeptide
immunogens identify different proteins, wherein each of the different proteins
identifies the same target.
In some embodiments, each of the polypeptide immunogens identifies a different
protein. In
some embodiments, the target is a pathogen. In some embodiments, the pathogen
is a virus, a
bacterium, a fungus, or a parasite. In some embodiments, the pathogen is a
virus and each of the
different proteins is a viral protein associated with the virus. In some
embodiments, the pathogen is a
bacterium and each of the different proteins is a bacterial protein associated
with the bacteria. In some
embodiments the target is a cancer cell. In some embodiments, each of the
different proteins is a
different tumor antigen associate with the cancer cell. In some embodiments,
the target is an allergen or
.. a toxin.
In another aspect, the disclosure provides a circular polyribonucleotide
including a plurality of
sequences, each sequence encoding a polypeptide immunogen, wherein at least
two (e.g., at least three,
at least four, at least five, at least six, at least seven, at least eight, or
at least nine) of the polypeptide
immunogens identify different targets. In some embodiments, each of the
polypeptide immunogens
.. identifies a different target. In some embodiments, each target is a
different pathogen. In some
embodiments, each target is, independently, a cancer cell, a virus, a
bacterium, a fungus, or a parasite.
In some embodiments, each target is a different virus. In some embodiments,
each target is a different
bacterium. In some embodiments, the targets include a virus and a bacterium.
In some embodiments of any one of the circular polyribonucleotides described
herein, the circular
polyribonucleotide includes between 500 and 20,000 ribonucleotides (e.g.,
between 500 and 10,000, 500
and 9,000, 500 and 8,000, 500 and 5,000, 500 and 4,000, 500 and 3,000, 1000
and 10,000, 1,000 and
8,000, 1,000 and 5,000, 3,000 and 8,000, 4,000 and 9,000, or 10,000 and
20,000)). In some
embodiments, the circular polyribonucleotide includes between 500 and 5,000.
In some embodiments,
the circular polyribonucleotide includes between 1,000 and 5,000
ribonucleotides. In some embodiments,
the circular polyribonucleotide includes between 5,000 and 10,000
ribonucleotides In some
embodiments, the circular polyribonucleotide includes at least 500
ribonucleotides (e.g. at least 600, at
least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at
least 3500, at least 4000, at least
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4500, at least 5000, at least 5500, at least 6000, at least 6500, at least
7000, at least 7500, at least 8000,
at least 8500, at least 9000, or at least 9500 ribonucleotides).
In some embodiments, the circular polyribonucleotide includes at least three,
at least four, at
least five, at least six, at least seven, at least eight, or at least nine
sequences, each sequence encoding
a polypeptide immunogen. In some embodiments, the circular polyribonucleotide
includes two or three,
between two and five (e.g., two, three or four), or between five and ten
sequences (e.g., five, six, seven,
eight, nine, or sequences), each sequence encoding a polypeptide immunogen.
In some embodiments, at least one sequence encoding a polypeptide immunogen
further
encodes a signal sequence. In some embodiments, each sequence encoding a
polypeptide immunogen
further encodes a signal sequence. In some embodiments, each of the sequences
encoding each of the
polypeptide immunogens is operably linked to an internal ribosomal entry site
(IRES). In some
embodiments, the circular polyribonucleotide includes a single !RES. In some
embodiments, each of the
polypeptide immunogens is encoded by a single open-reading frame operably
linked to the single IRES,
wherein the expression of the open reading frame produces a polypeptide
including the amino acid
sequence of each the polypeptide immunogens.
In some embodiments, the polypeptide immunogens are each separated by a
polypeptide linker.
In some embodiments, the polypeptide immunogens are each separated by a
cleavage domain. In some
embodiments, each stagger element is a 2A self-cleaving peptide. In some
embodiments, the circular
polyribonucleotide includes a plurality of IRESs. In some embodiments, each
IRES is operably linked to
an open reading frame including a sequence encoding a polypeptide immunogen.
In some embodiments, the circular polyribonucleotide further includes at least
one sequence
(e.g., at least two, at least three, at least four, at least five, at least
six, at least seven, at least, eight, at
least nine, and at least ten sequences) encoding an adjuvant. In some
embodiments, the at least one
(e.g., at least two, at least three, at least four, at least five, at least
six, at least seven, at least, eight, at
least nine, and at least ten sequences) sequence encoding the adjuvant is
operably linked to an IRES
In another aspect the disclosure provides a circular polyribonucleotide
including a sequence
encoding a polypeptide immunogen and a sequence encoding an adjuvant or a
sequence that is an
innate immune system stimulator. In some embodiments, the sequence encoding
the polypeptide
immunogen is operably linked to an !RES. In some embodiments, the sequence
encoding the adjuvant is
operably linked to an !RES. In some embodiments, the polypeptide immunogen and
the adjuvant
polypeptide are encoded by a single open-reading frame operably linked to the
single IRES, wherein the
expression of the open reading frame produces a polypeptide including the
amino acid sequence of each
of the polypeptide immunogen and the adjuvant polypeptide. In some
embodiments, the polypeptide
immunogen and the adjuvant polypeptide are each separated by a polypeptide
linker. In some
embodiments, the polypeptide immunogen and the adjuvant polypeptide are each
separated by a
cleavage domain. In some embodiments, each stagger element is a 2A self-
cleaving peptide. In some
embodiments, the circular polyribonucleotide includes a plurality of IRESs. In
some embodiments, a first
IRES is operably linked to the sequence encoding the polypeptide immunogen and
a second IRES is
operably linked to the sequence encoding the adjuvant polypeptide.
In some embodiments, wherein the circular polyribonucleotide encodes at least
one polypeptide
immunogen and at least one adjuvant (e.g., a polypeptide adjuvant), the
expression of the sequence
encoding the polypeptide immunogen is at least 2-fold, 5-fold, 10-fold, 20-
fold, 50-fold, or 100-fold greater
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than the expression of the sequence encoding the adjuvant. In some
embodiments, the expression of the
sequence encoding the polypeptide immunogen is substantially the same as the
expression of the
sequence encoding the adjuvant.
In another aspect the disclosure provides a composition including a plurality
of circular
polyribonucleotides, wherein the composition includes at least a first
circular polyribonucleotide including
a sequence encoding a polypeptide immunogen and at least a second circular
polyribonucleotide
including a sequence encoding an adjuvant or a sequence that is an innate
immune system stimulator. In
some embodiments, the polypeptide immunogen and the adjuvant are each operably
linked to an !RES.
In some embodiments, the adjuvant is a polypeptide. In some embodiments, the
adjuvant is a
cytokine, a chemokine, a costimulatory molecule, an innate immune stimulator,
a signaling molecule, a
transcriptional activator, a cytokine receptor, a bacterial component, or a
component of the innate
immune system. In some embodiments, the cytokine is a pro-inflammatory
cytokine. In some
embodiments, the pro-inflammatory cytokine is selected from GM-CSF, IL-1
alpha, IL-1 beta, TGF-beta,
TNF-alpha, and TNF-beta. In some embodiments, cytokine is a Th-1 inducing
cytokine. In some
embodiments, the Th-1 inducing cytokine is selected from IFN-gamma, IL-2, IL-
12, IL-15, and IL-18. In
some embodiments, the cytokine is a Th-2 inducing cytokine. In some
embodiments, the Th-2 inducing
cytokine is selected from IFN-gamma, IL-4, IL-5, IL-6, IL-10, and IL-13. In
some embodiments, the
chemokine is selected from MCP-1, MIP-1alpha, MIP-1beta, Rantes, and TCA-3. In
some embodiments,
the costimulatory molecule is selected from CD80, 0D86, CD4O-L, CD70, and
0D27. In some
embodiments, the innate immune stimulator is selected from STING (e.g., a
constitutively active STING,
such as STINGv155m, STINGR284M, STING R284M/V147L/N154SN155M , or STING
V147L/N154S/V155M
) TLR3, TLR4,
TLR9, TLR7, TLR8, TLR7, RIG-I/DDX58, and MDA-5/IFIH1. In some embodiments, the
innate immune
stimulator is a constitutively active mutant. In some embodiments, the
signaling molecule is selected
from STING (e.g., a constitutively active STING, such as STING V155M ,
STINGR284m,
STING R284MN147UN 154S/V155M or , STINGv147uN154sivissm,) TRIF, TRAM,
MyD88, IPS1, ASC, MAVS, MAPKs,
IKK-alpha, IKK complex, TBK1, B-catenin, and caspase 1. In some embodiments,
the transcriptional
activator is selected from AP1, NF-kappa B, IRF3, IRF7, IRF1, and IRF5. In
some embodiments, the
cytokine receptor is selected from IL-2betaR, IFN-gammaR, and IL-6R. In some
embodiments, the
bacterial component is selected from flagellin and MBL.
In some embodiments of any one of the circular polyribonucleotides described
herein, at least
one sequence encoding the adjuvant further encodes a signal sequence. In some
embodiments, each
sequence encoding an immunogen further encodes a signal sequence. In some
embodiments, at least
one sequence encoding an immunogen does not encode a signal sequence. In some
embodiments,
none of the sequences encoding an immunogen further encodes a signal sequence.
In some embodiments, the circular polyribonucleotide further includes a
sequence that is an
innate immune system stimulator. In some embodiments, the sequence that is an
innate immune system
stimulator is a GU-rich motif, an AU-rich motif, a structured region including
dsRNA, or an aptamer.
In some embodiments of any one of the circular polyribonucleotides described
herein, wherein
the circular polyribonucleotide includes a first polyribonucleotide with a 5'
end and a 3' end, wherein 5'
end and 3' end are each hybridized to a second polynucleotide, there by
linking the 5' end and the 3' end
of the first polyribonucleotide to form a circular polyribonucleotide. In some
embodiments, the circular
polyribonucleotide is produced by splint ligation. In some embodiments, the
circular polyribonucleotide is
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produced by providing a linear polyribonucleotide having a 3' end and a 5'
end, wherein the 3' end and
the 5' end each include a portion of an intron, and wherein the intron potion
of the 3' end and the intron
portion of the 5' end catalyze a splicing reaction thereby covalently
conjugating the 5' end and the 3' end
to produce a circular polyribonucleotide. In some embodiments, the intron is a
Group I self-splicing
intron.
In another aspect the disclosure provides, a composition including a plurality
of circular
polyribonucleotides, each including a sequence encoding a polypeptide
immunogen. In some
embodiments, each of the plurality of circular polyribonucleotides is a
circular polyribonucleotide
described herein. In some embodiments, the composition includes at least a
first circular
polyribonucleotide including a sequence encoding a first polypeptide immunogen
and at least a second
circular polyribonucleotide including a sequence encoding a second polypeptide
immunogen, wherein the
first and second polypeptide immunogens have at least 90% (e.g., at least 91%,
at least 92%, at least
93%, at last 94%, at least 95%, at least 96%, at least 97%, at least 98%, and
at least 99%) amino acid
sequence identity. In some embodiments, the first and second polypeptide
immunogens have at least
95% (at least 96%, at least 97%, at least 98%, and at least 99%) amino acid
sequence identity. In some
embodiments, the first and second polypeptide immunogens have less than 100%
amino acid sequence
identity. In some embodiments, each of the polypeptide immunogens includes one
or more epitopes that
identifies a target. In some embodiments, the composition includes at least a
first circular
polyribonucleotide including a sequence encoding a first polypeptide immunogen
and at least a second
circular polyribonucleotide including a sequence encoding a second polypeptide
immunogen, wherein the
first and second polypeptide immunogens identify different proteins, wherein
each different protein
identifies the same target. In some embodiments, the composition includes at
least a first circular
polyribonucleotide including a sequence encoding a first polypeptide immunogen
and at least a second
circular polyribonucleotide including a sequence encoding a second polypeptide
immunogen, wherein the
first polypeptide immunogens identifies a first target and the second
polypeptide immunogen identifies a
second target. In some embodiments, each target is, independently, a cancer
cell, a virus, a bacterium, a
fungus, a parasite, a toxin, or an allergen. In some embodiments, the target
is a pathogen. In some
embodiments, the pathogen is a virus, a bacterium, a fungus, or a parasite. In
some embodiments, the
target is a cancer cell, an allergen, or a toxin. In some embodiments, each
polypeptide immunogen is
operably linked to an !RES.
In another aspect, the disclosure provides a pharmaceutical preparation of a
circular
polyribonucleotide, wherein the circular polyribonucleotide includes a
sequence encoding a polypeptide
immunogen, and wherein the circular polyribonucleotide constitutes at least
25% (w/w) (e.g., at least
30 /0(w/w), 40 /0(w/w), 50% (w/w), 60% (w/w), 70% (w/w), 75% (w/w), 80% (w/w),
85% (w/w), 90% (w/w),
91% (w/w), 92% (w/w), 93% (w/w), 94% (w/w), 95% (w/w), 96% (w/w), 97% (w/w),
98% (w/w) or 99%
(w/w)) of total ribonucleotide molecules in the pharmaceutical preparation. In
some embodiments, the
circular polyribonucleotide constitutes at least 70% (w/w), 75% (w/w), 80%
(w/w), 85% (w/w), 90% (w/w),
91% (w/w), 92% (w/w), 93% (w/w), 94% (w/w), 95% (w/w), 96% (w/w), 97% (w/w),
98% (w/w) or 99%
(w/w) of total ribonucleotide molecules in the pharmaceutical preparation. In
some embodiments, the
pharmaceutical preparation includes no more than 75% (w/w), 60% (w/w), 50%
(w/w), 40% (w/w), 30%
(w/w), 20% (w/w), 15% (w/w), 10% (w/w), 5% (w/w), or 1% (w/w) linear
polyribonucleotide molecules of
the total ribonucleotide molecules in the pharmaceutical preparation. In some
embodiments, the
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pharmaceutical preparation includes no more than 0.5% (w/w) (e.g., 0.4% (w/w),
0.3% (w/w), 0.2%
(w/w), 0.1 (w/w), and 0.05% (w/w) linear polyribonucleotide molecules of the
total ribonucleotide
molecules in the pharmaceutical preparation. In some embodiments, the
pharmaceutical preparation
includes at least 1% (w/w) linear polyribonucleotide. In some embodiments, the
pharmaceutical
preparation includes between 30%-40% (w/w) circular polyribonucleotide and
between 1%-10% (w/w),
10%-20% (w/w), 20%-30% (w/w), 30%-40% (w/w), 40%-50% (w/w), 50%-60% (w/w), or
60%-70% (w/w)
linear polyribonucleotide. In some embodiments, the pharmaceutical preparation
includes between
40%-60% (w/w) circular polyribonucleotide and between 1%-10% (w/w), 10%-20%
(w/w), 20%-30%
(w/w), 30%-40% (w/w), 40%-50% (w/w), 50%-60% (w/w) linear polyribonucleotide.
In some
embodiments, the pharmaceutical preparation includes between 60%-80% (w/w)
circular
polyribonucleotide and between 1%-10% (w/w), 10%-20% (w/w), 20%-30% (w/w), 30%-
40% (w/w) linear
polyribonucleotide.
In another aspect, the disclosure provides a pharmaceutical preparation of a
circular
polyribonucleotide, wherein the circular polyribonucleotide includes a
sequence encoding a polypeptide
immunogen, and wherein the pharmaceutical preparation includes no more than
75% (w/w), 60% (w/w),
50% (w/w), 40% (w/w), 30% (w/w), 20% (w/w), 15% (w/w), 10% (w/w), 5% (w/w), or
1% (w/w) linear
polyribonucleotide molecules of the total ribonucleotide molecules in the
pharmaceutical preparation. In
some embodiments, the pharmaceutical preparation includes no more than 20%
(w/w), 15% (w/w), 10%
(w/w), 5% (w/w), 1% (w/w), or 0.5% (w/w) linear polyribonucleotide molecules
of the total ribonucleotide
molecules in the pharmaceutical preparation.
In some embodiments, the preparation includes no more than 15% (w/w) (e.g.,
15% (w/w), 10%
(w/w), 9% (w/w), 8% (w/w), 7% (w/w), 6% (w/w), 5% (w/w), 4% (w/w), 3% (w/w),
2% (w/w), 1% (w/w), and
0.5% (w/w)) nicked polyribonucleotide molecules of the total ribonucleotide
molecules in the
pharmaceutical preparation. In some embodiments, the preparation includes no
more than 8% (w/w), 7%
(w/w), 6% (w/w), 5% (w/w), 4% (w/w), 3% (w/w), 2% (w/w), 1% (w/w), or 0.5%
(w/w) nicked
polyribonucleotide molecules of the total ribonucleotide molecules in the
pharmaceutical preparation.
In another aspect, the disclosure provides a pharmaceutical preparation of a
circular
polyribonucleotide, wherein the circular polyribonucleotide includes a
sequence encoding a polypeptide
immunogen, and wherein the pharmaceutical preparation includes no more than
15% (w/w) (e.g., 15%
(w/w), 10% (w/w), 9% (w/w), 8% (w/w), 7% (w/w), 6% (w/w), 5% (w/w), 4% (w/w),
3% (w/w), 2% (w/w),
1% (w/w), and 0.5% (w/w)) nicked polyribonucleotide molecules of the total
ribonucleotide molecules in
the pharmaceutical preparation. In some embodiments, the preparation includes
no more than 8%
(w/w), 7% (w/w), 6% (w/w), 5% (w/w), 4% (w/w), 3% (w/w), 2% (w/w), 1% (w/w),
or 0.5% (w/w) nicked
polyribonucleotide molecules of the total ribonucleotide molecules in the
pharmaceutical preparation.
In some embodiments of an one of the pharmaceutical preparations described
herein the
pharmaceutical preparation includes less than 10 EU/kg (e.g., 9 EU/kg, 8
EU/kg, 7 EU/kg, 6 EU/kg, 5
EU/kg, 4 EU/kg, 3 EU/KG, 2 EU/kg, and 1 EU/kg) or lacks endotoxin as measured
by a Limulus
amebocyte lysate test. In some embodiments, the pharmaceutical preparation
includes a bioburden of
less than 100 CFU/100 ml or less than 10 CFU/100 ml before sterilization. In
some embodiments, the
pharmaceutical preparation is a sterile pharmaceutical preparation, e.g.,
supports growth of fewer than
100 viable microorganisms as tested under aseptic conditions. In some
embodiments, the
pharmaceutical preparation meets the standard of USP <71>. In some
embodiments, the pharmaceutical
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preparation meets the standard of USP <85>. In some embodiments, the
pharmaceutical preparation is
an intermediate pharmaceutical preparation of a final drug product.
In some embodiments, the pharmaceutical preparation is a final drug product
for administration to
a subject. In some embodiments, the pharmaceutical preparation includes a
concentration of at least 0.1
ng/mL, 0.5 ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL, 50 ng/mL, 0.1 g/mL, 0.5 g/mL,1
g/mL, 2 g/mL, 5
g/mL, 10 g/mL, 20 g/mL, 30 g/mL, 40 g/mL, 50 g/mL, 60 g/mL, 70 g/mL, 80
g/mL, 100 g/mL,
200 g/mL, 300 g/mL, 500 g/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 5 mg/mL, 10 mg/mL,
100 mg/mL, 200
mg/mL, or 500 mg/mL circular polyribonucleotide molecules. In some
embodiments, the pharmaceutical
preparation includes no more than 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20
ng/ml, 25 ng/ml, 30 ng/ml, 35
ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml,
200 ng/ml, 300 ng/ml, 400
ng/ml, 500 ng/ml, 1000 g/mL, 5000 g/mL, 10,000 g/mL, or 100,000 g/mL of
deoxyribonucleotide
molecules. In some embodiments, the pharmaceutical preparation includes a
protein contamination (e.g.
an enzyme) of less than 0.1 ng, 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng,
35 ng, 40 ng, 50 ng, 60 ng,
70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, or 500 ng of the protein
contamination per milligram
(mg) of the circular polyribonucleotide molecules. In some embodiments, the
pharmaceutical preparation
includes an A260/A280 absorbance ratio of from about 1.6 to 2.3 (e.g., 1.7,
1.8, 1.9, 2.0, 2.1, and 2.2) as
measured by a spectrophotometer.
In some embodiments, the pharmaceutical preparation is substantially free of a
process-related
impurity selected from a cell protein, a cell deoxyribonucleic acid, an
enzyme, a reagent component, a gel
component, or a chromatographic material. In some embodiments, the
pharmaceutical preparation has a
reduced level of one or more markers of an immune or inflammatory response
after purification compared
to prior to purification. In some embodiments, the circular polyribonucleotide
is a circular
polyribonucleotide described herein.
In some embodiments, the circular polyribonucleotide is produced according to
a method
including the steps of: providing a plurality of linear polyribonucleotide
molecules; circularizing the linear
polyribonucleotide molecules to provide a preparation of circular
polyribonucleotide molecules; and
processing the preparation to remove linear polyribonucleotide molecules from
the preparation. In some
embodiments, the method further includes evaluating the amount of linear
polyribonucleotide molecules
in the preparation remaining after the processing step. In some embodiments,
the method further
includes further processing the preparation, wherein further processing
includes: processing the
preparation to remove nicked polyribonucleotide molecules from the
preparation; and/or processing the
preparation to remove deoxyribonucleotide molecules from the preparation;
and/or processing the
preparation to remove protein contaminants from the preparation; and/or
processing the preparation to
remove endotoxin from the preparation.
In another aspect the disclosure provides a pharmaceutical composition
including any one of the
circular polyribonucleotides, compositions, or pharmaceutical preparations
described herein and a
pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical
composition includes
any one of the circular polyribonucleotides, compositions, or pharmaceutical
preparations described
herein and protamine or a protamine salt (e.g., protamine sulfate). In some
embodiments, the
pharmaceutical composition further includes an adjuvant. In some embodiments,
the adjuvant is an
inorganic adjuvant, a small molecule adjuvant, and oil in water emulsion, a
lipid or polymer, a peptide or
peptidoglycan, a carbohydrate or polysaccharide, a saponin, an RNA-based
adjuvant, a DNA-based
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adjuvant, a viral particle, a bacterial adjuvant, a hybrid molecule, a fungal
or oocyte microbe-associated
molecular pattern (MAMP), an inorganic nanoparticle, or a multi-component
adjuvant. In some
embodiments, the adjuvant is an inorganic adjuvant. In some embodiments, the
inorganic adjuvant is an
aluminum salt or a calcium salt. In some embodiments, the adjuvant is a small
molecule. In some
embodiments, the small molecule is imiquimod, resiquimod, or gardiquimod. In
some embodiments, the
adjuvant is an oil in water emulsion. In some embodiments, the oil in water
emulsion is Squalene, MF59,
AS03, a Montanide formulation, optionally Montanide ISA 51 or Montanide ISA
720, or Incomplete
Freunds Adjuvant an oil in water emulsion. In some embodiments, the adjuvant
is a lipid or polymer. In
some embodiments, the lipid or polymer is a polymeric nanoparticle, optionally
PLGA, PLG, PLA, PGA, or
PHB, a liposome, optionally a Virosomes or CAF01, a lipid nanoparticle or a
component thereof, a
lipopolysaccharide (LPS), optionally monophosphoryl lipid A (MPLA) or
glucopyranosyl Lipid A (GLA), a
lipopeptide, optionally Pam2 (Pam2CSK4) or Pam3 (Pam3CSK4), or a glycolipid,
optionally, trehalose
dimycolate. In some embodiments, the adjuvant is a peptide or peptidoglycan.
In some embodiments,
the peptide or peptidoglycan corresponds to all or a portion of a synthetic or
purified gram negative or
gram positive bacteria, optionally N-acetyl-muramyl-L-alanyl-D-isoglutamine
(MDP), a flagellin-fusion
protein, Mannose-binding Lectin (MBL), a cytokines, or a chemokine. In some
embodiments, the
adjuvant is a carbohydrate or polysaccharide. In some embodiments, the
carbohydrate or polysaccharide
is dextran (branched microbial polysaccharide), dextran-sulfate, Lentinan,
zymosan, Beta-glucan, Deltin,
Mannan, or Chitin. In some embodiments, the adjuvant is a saponin. In some
embodiments, the saponin
is a glycoside or a polycyclic aglycones attached to one or more sugar side
chains, optionally ISCOMS,
ISCOMS matrix, or QS-21. In some embodiments, the adjuvant is an RNA-based
adjuvant. In some
embodiments, the RNA-based adjuvant is Poly IC, Poly IC:LC, a hairpin RNAs,
optionally with a 5'PPP
containing sequence, a viral sequence, a polyU containing sequences, dsRNA, a
natural or synthetic
immunostimulatory RNA sequence, a nucleic acid analogs, optionally cyclic GMP-
AMP or a cyclic
dinucleotide such as cyclic di-GMP, or an immunostimulatory base analog,
optionally a 08-substitued or
an N7,08-disubstituted guanine ribonucleotide. In some embodiments, the
adjuvant is a DNA-based
adjuvant. In some embodiments, the DNA-based adjuvant is a CpG, dsDNA, or a
natural or synthetic
immunostimulatory DNA sequence. In some embodiments, the adjuvant is a viral
particle. In some
embodiments, the viral particle is a virosome, optionally, a phospholipid cell
membrane bilayer. In some
embodiments, the adjuvant is a bacterial adjuvant. In some embodiments, the
bacterial adjuvant is
flagellin, LPS, or a bacterial toxin, optionally an enterotoxin, a heat-labile
toxin, or a Cholera toxin. In
some embodiments, the adjuvant is a hybrid molecule. In some embodiments, the
adjuvant is CpG
conjugated to Imiquimod. In some embodiments, the adjuvant is a fungal or
oocyte microbe-associated
molecular pattern (MAMP). In some embodiments, the fungal or oocyte MAMP is
chitin or beta-glucan.
In some embodiments, the adjuvant is an inorganic nanoparticle. In some
embodiments, the inorganic
nanoparticle is a gold nanorod, a silica-based nanoparticle, optionally a
mesoporous silica nanoparticle
(MSN). In some embodiments, the adjuvant is a multi-component adjuvant. In
some embodiments, the
multi-component adjuvant is AS01, A503, A504, Complete Freunds Adjuvant, or
CAF01.
In another aspect, the disclosure provides a pharmaceutical composition
including a mixture
including any one of the circular polyribonucleotides, the compositions, or
the pharmaceutical
preparations described herein, and an alcohol, wherein the alcohol constitutes
about 0.3% v/v to about
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75% v/v (e.g., 0.5 /0 v/v, 1% v/v, 5% v/v, 10% v/v, 15% v/v, 20% v/v, 25% v/v,
30% v/v, 35% v/v, 40 /0 v/v,
45% v/v, 50% v/v, 55% v/v, 60% v/v, 65% v/v, and 70% v/v) of the mixture.
In another aspect, the disclosure provides a pharmaceutical composition
including a mixture
including a linear polyribonucleotide that includes at least one sequence
encoding a polypeptide
immunogen and an alcohol, wherein the alcohol constitutes about 0.3% v/v to
about 75% v/v (e.g., 0.5%
v/v, 1% v/v, 5% v/v, 10% v/v, 15 /0 v/v, 20% v/v, 25% v/v, 30 /0 v/v, 35% v/v,
40% v/v, 45% v/v, 50% v/v,
55% v/v, 60% v/v, 65% v/v, and 70% v/v) of the mixture.
In some embodiments, the linear polyribonucleotide includes a plurality of
sequences, each
sequence encoding a polypeptide immunogen, wherein at least two (e.g., at
least three, at least four, at
least five, at least six, at least seven, at least eight, and at least nine)
of the polypeptide immunogens
have at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least
96%, at least 97%, at least 98%, and at least 99%) amino acid sequence
identity. In some embodiments,
the at least two polypeptide immunogens have at least 95% (e.g., 96%, at least
97%, at least 98%, and at
least 99%) amino acid sequence identity. In some embodiments, the at least two
polypeptide
immunogens have less than 100% amino acid sequence identity. In some
embodiments, each of the
polypeptide immunogens includes one or more epitopes that identifies a target.
In some embodiments,
the target is a pathogen. In some embodiments, the pathogen is a virus, a
bacterium, a fungus, or a
parasite. In some embodiments, the pathogen is a virus and each polypeptide
immunogen includes one
or more epitopes (e.g., two, three, four, five, six, seven, eight, nine, ten,
or more) corresponding to a viral
immunogen. In some embodiments, the pathogen is a bacterium and each
polypeptide immunogen
includes one or more (e.g., two, three, four, five, six, seven, eight, nine,
ten, or more) epitopes
corresponding to a bacterial immunogen. In some embodiments, the target is a
cancer cell. In some
embodiments, polypeptide immunogen includes one or more (e.g., two, three,
four, five, six, seven, eight,
nine, ten, or more) epitopes corresponding to a tumor antigen. In some
embodiments, the target is a
toxin or an allergen. In some embodiments, the target is associated with a
disease, disorder, or
condition. In some embodiments, the disease, disorder, or condition is a viral
infection. In some
embodiments, the disease, disorder, or condition is a bacterial infection. In
some embodiments, the
disease, disorder, or condition is a cancer.
In some embodiments, the linear polyribonucleotide includes a plurality of
sequences, each
sequence encoding a polypeptide immunogen, wherein at least two (e.g., at
least three, at least four, at
least five, at least six, at least seven, at least eight, and at least nine)
of the polypeptide immunogens
identify different proteins, wherein each of the different proteins identifies
the same target. In some
embodiments, each of the polypeptide immunogens identifies a different
protein. In some embodiments,
the target is a pathogen. In some embodiments, the pathogen is a virus, a
bacterium, a fungus, or a
parasite. In some embodiments, the pathogen is a virus and each of the
different proteins is a viral
protein associated with the virus. In some embodiments, the pathogen is a
bacterium and each of the
different proteins is a bacterial protein associated with the bacteria. In
some embodiments the target is a
cancer cell. In some embodiments, each of the different proteins is a
different tumor antigen associate
with the cancer cell. In some embodiments, the target is an allergen or a
toxin.
In some embodiments, the linear polyribonucleotide includes a plurality of
sequences, each
sequence encoding a polypeptide immunogen, wherein at least two (e.g., at
least three, at least four, at
least five, at least six, at least seven, at least eight, ant at least nine)
of the polypeptide immunogens
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identify different targets. In some embodiments, each of the polypeptide
immunogens identifies a
different target. In some embodiments, each target is a different pathogen. In
some embodiments, each
target is, independently, a cancer cell, a virus, a bacterium, a fungus, or a
parasite. In some
embodiments, each target is a different virus. In some embodiments, each
target is a different bacterium.
In some embodiments, the targets include a virus and a bacterium.
In some embodiments, the linear polyribonucleotide includes at least three, at
least four, at least
five, at least six, at least seven, at least eight, or at least nine
sequences, each sequence encoding a
polypeptide immunogen. In some embodiments, the linear polyribonucleotide
includes between two and
three, between two and five (e.g., three and four), or between five and ten
sequences (e.g., six, seven,
eight, and nine sequences), each sequence encoding a polypeptide immunogen.
In some embodiments, the linear polyribonucleotide includes further includes
at least one
sequence (e.g., at least two, at least three, at least four, at least five, at
least six, at least seven, at least,
eight, at least nine, or at least ten sequences) encoding an adjuvant. In some
embodiments, the at least
one (e.g., at least two, at least three, at least four, at least five, at
least six, at least seven, at least, eight,
at least nine, or at least ten sequences) sequence encoding the adjuvant is
operably linked to an !RES.
In some embodiments, the linear polyribonucleotide includes a sequence
encoding a polypeptide
immunogen and a sequence encoding an adjuvant or a sequence that is an innate
immune system
stimulator. In some embodiments, the sequence encoding the polypeptide
immunogen is operably linked
to an !RES. In some embodiments, the sequence encoding the adjuvant is
operably linked to an !RES.
In some embodiments, the adjuvant is a polypeptide. In some embodiments, the
adjuvant is a cytokine, a
chemokine, a costimulatory molecule, an innate immune stimulator, a signaling
molecule, a transcriptional
activator, a cytokine receptor, a bacterial component, or a component of the
innate immune system. In
some embodiments, the cytokine is a pro-inflammatory cytokine. In some
embodiments, the pro-
inflammatory cytokine is selected from GM-CSF, IL-1 alpha, IL-1 beta, TGF-
beta, TNF-alpha, and TNF-
beta. In some embodiments, cytokine is a Th-1 inducing cytokine. In some
embodiments, the Th-1
inducing cytokine is selected from IFN-gamma, IL-2, IL-12, IL-15, and IL-18.
In some embodiments, the
cytokine is a Th-2 inducing cytokine. In some embodiments, the Th-2 inducing
cytokine is selected from
IFN-gamma, IL-4, IL-5, IL-6, IL-10, and IL-13. In some embodiments, the
chemokine is selected from
MCP-1, MIP-1alpha, MIP-1beta, Rantes, and TCA-3. In some embodiments, the
costimulatory molecule
is selected from CD80, 0D86, CD4O-L, CD70, and 0D27. In some embodiments, the
innate immune
stimulator is selected from STING (e.g., constitutively active STING
(caSTING), such as STINGv155m,
STINGR284m, STINGR284MN147L/N154SN155M or STINGV147L/N154S/V155M\
) TLR3, TLR4, TLR9, TLR7, TLR8,
TLR7, RIG-I/DDX58, and MDA-5/IFIH1. In some embodiments, the innate immune
stimulator is a
constitutively active mutant. In some embodiments, the signaling molecule is
selected from STING (e.g.,
caSTING, such as STINGv155m, STINGR284m, STINGR284.mA14.7uN154.sN155m, or
STING V147UN 154SN155M
) TRIF,
TRAM, MyD88, IPS1, ASC, MAVS, MAPKs, IKK-alpha, IKK complex, TBK1, B-catenin,
and caspase 1.
In some embodiments, the transcriptional activator is selected from AP1, NF-
kappa B, IRF3, IRF7, IRF1,
and IRF5. In some embodiments, the cytokine receptor is selected from IL-
2betaR, IFN-gammaR, and
IL-6R. In some embodiments, the bacterial component is selected from flagellin
and MBL.
In some embodiments, the linear polyribonucleotide includes a sequence that is
an innate
immune system stimulator. In some embodiments, the sequence that is an innate
immune system
stimulator is a GU-rich motif, an AU-rich motif, a structured region including
dsRNA, or an aptamer.
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In some embodiments, the alcohol constitutes about 0.3% v/v to about 60% v/v
(e.g., 0.5% v/v,
1% v/v, 5 /0 v/v, 10 /0 v/v, 15 /0 v/v, 20 /0 v/v, 25 /0 v/v, 30 /0 v/v, 35 /0
v/v, 40 /0 v/v, 45 /0 v/v, 50 /0 v/v, and
55% v/v) , about 0.3% v/v to about 50% v/v (e.g., 0.5% v/v, 1% v/v, 5% v/v,
10% v/v, 15% v/v, 20% v/v,
25 /0 v/v, 30% v/v, 35% v/v, 40 /0 v/v, and 45% v/v), about 0.3% v/v to about
40% v/v (e.g., 0.5 /0 v/v, 1%
v/v, 5% v/v, 10% v/v, 15% v/v, 20% v/v, 25% v/v, 30% v/v, and 35% v/v), about
0.3% v/v to about 20%
v/v, about 0.3% v/v to about 15% v/v (e.g., 0.5% v/v, 1% v/v, 5% v/v, 10% v/v,
and 15% v/v), about 0.3%
v/v to about 10% v/v (e.g., 0.5% v/v, 1% v/v, 5% v/v, and 10% v/v), about 0.3%
v/v to about 5% v/v (e.g.,
0.5% v/v, 1% v/v, 2% v/v, 3% v/v, and 4% v/v), about 0.3% v/v to about 1% v/v
(e.g., 0.4% v/v, 0.5% v/v,
0.6% v/v, 0.7% v/v, 0.8% v/v, and 0.9% v/v) or about 0.3% v/v to about 0.5%
v/v of the mixture. In some
embodiments, the alcohol constitutes about 0.5% v/v to about 75% v/v, about 1%
v/v to about 75% v/v,
about 5% v/v to about 75% v/v, about 10% v/v to about 75% v/v, about 15% v/v
to about 75% v/v, about
20% v/v to about 75% v/v, about 30% v/v to about 75% v/v, about 40% v/v to
about 75% v/v, about 50%
v/v to about 75% v/v, about 60% v/v to about 75% v/v, or about 70% v/v to
about 75% v/v of the mixture.
In some embodiments, the alcohol is selected from methanol, ethanol,
isopropanol,
phenoxyethanol, triethanolamine, phenethyl alcohol, butanol, pentanol, cetyl
alcohol, ethylene glycol,
propylene glycol, denatured alcohol, benzyl alcohol, specially denatured
alcohol, glycol, stearyl alcohol,
cetearyl alcohol, menthol, polyethylene glycols (PEG)-400, ethoxylated fatty
acids, and
hydroxyethylcellulose. In some embodiments, the alcohol is ethanol.
In some embodiments, the pharmaceutical composition is formulated for topical
administration.
In some embodiments, the pharmaceutical composition is a liquid, gel, lotion,
paste, cream, foam, or
stick. In some embodiments, the pharmaceutical composition has a pH of about 6-
8 (e.g., of about 6.5-
7.5, or of about 7). In some embodiments, the pharmaceutical composition has a
viscosity that is about
the same as water.
In some embodiments, the pharmaceutical composition is substantially free of
hydrophobic or
lipophilic groups. In some embodiments, the pharmaceutical composition is
substantially free of
hydrocarbons. In some embodiments, the pharmaceutical composition is
substantially free of cationic
liposomes. In some embodiments, the pharmaceutical composition is
substantially free of fatty acids,
lipids, liposomes, cholesterol, or any combination thereof. In some
embodiments, the composition further
including an adjuvant. In some embodiments, the adjuvant is an inorganic
adjuvant, a small molecule
adjuvant, and oil in water emulsion, a lipid or polymer, a peptide or
peptidoglycan, a carbohydrate or
polysaccharide, a saponin, an RNA-based adjuvant, a DNA-based adjuvant, a
viral particle, a bacterial
adjuvant, a hybrid molecule, a fungal or oocyte microbe-associated molecular
pattern (MAMP), an
inorganic nanoparticle, or a multi-component adjuvant. In some embodiments,
the composition further
includes a cell-penetrating agent.
In another aspect, the disclosure provides, a method of delivering a
polyribonucleotide to a
subject including topically applying a circular polyribonucleotide described
herein, an immunogenic
composition described herein, a pharmaceutical preparation descried herein to
a surface area of the
subject.
In another aspect, the disclosure provides, a method of delivering a
polyribonucleotide to a
subject including: (a) applying a sterilizing agent to a surface area of the
subject; and (b) applying a
composition including a circular polyribonucleotide described herein, a
immunogenic composition
described herein, a pharmaceutical preparation descried herein, or the
pharmaceutical composition
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described herein and a diluent to the surface area of the subject. In some
embodiments, the composition
of (b) does not comprise a carrier. In some embodiments, the sterilizing agent
is an alcohol, UV light,
laser light, or heat.
In another aspect, the disclosure provides a method of delivering a
polyribonucleotide to a
subject including: (a) applying an alcohol to a surface area of the subject;
and (b) applying a composition
including a circular polyribonucleotide described herein, a immunogenic
composition described herein, a
pharmaceutical preparation descried herein, or the pharmaceutical composition
described herein and a
diluent to the surface area of the subject. In some embodiments, the alcohol
is selected from the group
consisting of: methanol, ethanol, isopropanol, phenoxyethanol,
triethanolamine, phenethyl alcohol,
butanol, pentanol, cetyl alcohol, ethylene glycol, propylene glycol, denatured
alcohol, benzyl alcohol,
specially denatured alcohol, glycol, stearyl alcohol, cetearyl alcohol,
menthol, polyethylene glycols (PEG)-
400, ethoxylated fatty acids, and hydroxyethylcellulose. In some embodiments,
the alcohol is ethanol.
In some embodiments (e.g., in some embodiments of topical delivery described
herein), the
surface area of the subject is selected from a surface area of the skin, oral
cavity, nasal cavity,
gastrointestinal tract, respiratory tract, or any combination thereof.
In another aspect the disclosure provides a method of treating or preventing a
disease, disorder,
or condition in a subject, the method including administering to the subject
any one of the circular
polyribonucleotides, the compositions, pharmaceutical preparations, or the
pharmaceutical compositions
described herein. In some embodiments, the disease, disorder, or condition is
a viral infection, a bacterial
infection, or a fungal infection. In some embodiments, the disease, disorder,
or condition is a cancer. In
some embodiments, the disease, disorder, or condition is associated with
exposure to an allergen. In
some embodiments, disease, disorder, or condition is associated with exposure
to a toxin.
In another aspect, the disclosure provides a method of inducing an immune
response in a
subject, the method including administering to the subject any one of the
circular polyribonucleotides, the
compositions, pharmaceutical preparations, or pharmaceutical compositions
described herein. In some
embodiments, the subject is a mammal. In some embodiments, the subject is a
human. In some
embodiments, the method is a non-human mammal. In some embodiments, the non-
human mammal is a
cow, a sheep, a goat, a pig, a dog, a horse, or a cat. In some embodiments,
the subject is a bird. In
some embodiments, the bird is a hen, a rooster, a turkey, or a parrot. In some
embodiments, the method
further includes administering an adjuvant to the subject. In some
embodiments, the adjuvant is a
separate molecular entity from the circular polyribonucleotide, linear
polyribonucleotide, or preparation or
composition thereof. In some embodiments, the adjuvant is an inorganic
adjuvant, a small molecule
adjuvant, and oil in water emulsion, a lipid or polymer, a peptide or
peptidoglycan, a carbohydrate or
polysaccharide, a saponin, an RNA-based adjuvant, a DNA-based adjuvant, a
viral particle, a bacterial
adjuvant, a hybrid molecule, a fungal or oocyte microbe-associated molecular
pattern (MAMP), an
inorganic nanoparticle, or a multi-component adjuvant. In some embodiments,
the adjuvant is a
polypeptide adjuvant. In some embodiments, the polypeptide adjuvant is a
cytokine, a chemokine, a
costimulatory molecule, an innate immune stimulator, a signaling molecule, a
transcriptional activator, a
cytokine receptor, a bacterial component, or a component of the innate immune
system. In some
embodiments, the adjuvant is an innate immune system stimulator. In some
embodiments, the innate
immune system stimulator is selected from an RNA including a GU-rich motif, an
AU-rich motif, a
structured region including dsRNA, or an aptamer.
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In some embodiments, any one of the circular polyribonucleotides,
compositions, pharmaceutical
preparations, or pharmaceutical compositions described herein is administered
to the subject as a single
dose. In some embodiments, any one of the circular polyribonucleotides,
compositions, pharmaceutical
preparations, or pharmaceutical compositions described herein is administered
to the subject two or more
times, three or more times, four or more times, or five or more times. In some
embodiments,
administration of any one of the circular polyribonucleotides, compositions,
pharmaceutical preparations,
or pharmaceutical compositions described herein occurs about weekly, about
every two weeks, about
every three weeks, about every month, about every two months, about every
three months, about every
four months, about every five months, about every six months, about every
year, about every two years,
about every three years, about every four years, about every five years, or
about every ten years.
In some embodiments, the method further comprises administering to the subject
a polypeptide
immunogen (e.g., a protein subunit comprising a polypeptide immunogen). In
some embodiments the
polypeptide immunogen corresponds to (e.g., shares 90%, 95%, 96%, 97%, 98%, or
100% amino acid
sequence identity with a polypeptide immunogen encoded by a sequence of the
circular
polyribonucleotide. In some embodiments, the polypeptide immunogen is
administered to the subject
after administering any one of the circular polyribonucleotides, immunogenic
compositions,
pharmaceutical preparations, or pharmaceutical compositions described herein.
In some embodiments,
the polypeptide immunogen maintains or enhances an immune response in the
subject against the
polypeptide immunogen.
In another aspect, the disclosure provides a method of maintaining or
enhancing an immune
response in a subject comprising (i) administering to the subject a circular
polyribonucleotide encoding a
polypeptide immunogen and (ii) administering to the subject the polypeptide
immunogen, wherein step (ii)
occurs between 1 week and 6 months (e.g., between 1 month and 5 months, 2
months and 3 months, 2
weeks and 3 months, or 3 months and 6 months) after step (i), and wherein
administration of the
polypeptide immunogen of step (ii) maintains or enhances the immune response
in the subject against
the polypeptide immunogen. In some embodiments, the polypeptide immunogen
comprises one or more
epitopes that identifies a target. In some embodiments, the target is a
pathogen. In some embodiments,
the target is a cancer cell, an allergen, or a toxin.
Numbered Embodiments:
[1] A circular polyribonucleotide comprising a plurality of sequences, each
sequence encoding a
polypeptide immunogen, wherein at least two of the polypeptide immunogens have
at least 90% amino
acid sequence identity.
[2] The circular polyribonucleotide of paragraph [1], wherein the at least two
polypeptide
immunogens have at least 95% amino acid sequence identity.
[3] The circular polyribonucleotide of paragraph [1] or [2], wherein the at
least two polypeptide
immunogens have less than 100% amino acid sequence identity.
[4] The circular polyribonucleotide of paragraph [1], wherein each of the
polypeptide
immunogens have at least 90% amino acid sequence identity.
[5] The circular polyribonucleotide of paragraph [4], wherein each of the
polypeptide
immunogens have at least 95% amino acid sequence identity.
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[6] The circular polyribonucleotide of paragraph [4] or [5], wherein each of
the polypeptide
immunogens have less than 100% amino acid sequence identity.
[7] The circular polyribonucleotide of any one of paragraphs [1] to [6],
wherein each of the
polypeptide immunogens comprises one or more epitopes that identifies a
target.
[8] The circular polyribonucleotide of paragraph [7], wherein the target is a
pathogen.
[9] The circular polyribonucleotide of paragraph [8], wherein the pathogen is
a virus, a bacterium,
a fungus, or a parasite.
[10] The circular polyribonucleotide of paragraph [9], wherein the pathogen is
a virus and each
polypeptide immunogen comprises one or more epitopes corresponding to a viral
immunogen.
[11] The circular polyribonucleotide of paragraph [9], wherein the pathogen is
a bacterium and
each polypeptide immunogen comprises one or more epitopes corresponding to a
bacterial immunogen.
[12] The circular polyribonucleotide of paragraph [7], wherein the target is a
cancer cell.
[13] The circular polyribonucleotide of paragraph [12], wherein each
polypeptide immunogen
comprises one or more epitopes corresponding to a tumor antigen.
[14] The circular polyribonucleotide of paragraph [7], wherein the target is a
toxin or an allergen.
[15] The circular polyribonucleotide of any one of paragraphs [7] to [14],
wherein the target is
associated with a disease, disorder, or condition.
[16] The circular polyribonucleotide of paragraph [15], wherein the disease,
disorder, or condition
is a viral infection.
[17] The circular polyribonucleotide of paragraph [15], wherein the disease,
disorder, or condition
is a bacterial infection.
[18] The circular polyribonucleotide of paragraph [15], wherein the disease,
disorder, or condition
is a cancer.
[19] A circular polyribonucleotide comprising a plurality of sequences, each
sequence encoding a
polypeptide immunogen, wherein at least two of the polypeptide immunogens
identify different proteins,
wherein each of the different proteins identifies the same target.
[20] The circular polyribonucleotide of paragraph [19], wherein each of the
polypeptide
immunogens identifies a different protein.
[21] The circular polyribonucleotide of paragraph [19] or [20], wherein the
target is a pathogen.
[22] The circular polyribonucleotide of paragraph [21], wherein the pathogen a
virus, a bacterium,
a fungus, or a parasite.
[23] The circular polyribonucleotide of paragraph [22], wherein the pathogen
is a virus and each
of the different proteins is a viral protein associated with the virus.
[24] The circular polyribonucleotide of paragraph [22], wherein the pathogen
is a bacterium and
each of the different proteins is a bacterial protein associated with the
bacteria.
[25] The circular polyribonucleotide of paragraph [19] or [20], wherein the
target is a cancer cell
[26] The circular polyribonucleotide of paragraph [25], wherein and each of
the different proteins
is a different tumor antigen associate with the cancer cell.
[27] The circular polyribonucleotide of paragraph [19] or [20], wherein the
target is an allergen or
a toxin.
[28] A circular polyribonucleotide comprising a plurality of sequences, each
sequence encoding a
polypeptide immunogen, wherein at least two of the polypeptide immunogens
identify different targets.
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[29] The circular polyribonucleotide of paragraph [28], wherein each of the
polypeptide
immunogens identifies a different target.
[30] The circular polyribonucleotide of paragraph [28] or [29], wherein each
target is a different
pathogen.
[31] The circular polyribonucleotide of paragraph [30], wherein each target
is, independently, a
cancer cell, a virus, a bacterium, a fungus, or a parasite.
[32] The circular polyribonucleotide of paragraph [31], wherein each target is
a different virus.
[33] The circular polyribonucleotide of paragraph [31], wherein each target is
a different
bacterium.
[34] The circular polyribonucleotide of paragraph [31], wherein the targets
include a virus and a
bacterium.
[35] The circular polyribonucleotide of any one of paragraphs [1] to [34],
wherein the circular
polyribonucleotide comprises between 500 and 20,000 ribonucleotides.
[36] The circular polyribonucleotide of paragraph [35], wherein the circular
polyribonucleotide
comprises between 500 and 10,000 ribonucleotides.
[37] The circular polyribonucleotide of paragraph [36], wherein the circular
polyribonucleotide
comprises between 500 and 5,000 ribonucleotides.
[38] The circular polyribonucleotide of any one of paragraphs [1] to [34],
wherein the circular
polyribonucleotide comprises at least 500 ribonucleotides.
[39] The circular polyribonucleotide of paragraph [38], wherein the circular
polyribonucleotide
comprises at least 1,000 ribonucleotides.
[40] The circular polyribonucleotide of paragraph [39], wherein the circular
polyribonucleotide
comprises at least 5,000 ribonucleotides.
[41] The circular polyribonucleotide of any one of paragraphs [1] to [40],
wherein the circular
polyribonucleotide comprises at least three, at least four, at least five, at
least six, at least seven, at least
eight, or at least nine sequences, each sequence encoding a polypeptide
immunogen.
[42] The circular polyribonucleotide of any one of paragraphs [1] to [40],
wherein the circular
polyribonucleotide comprises between two and three, between two and five, or
between five and ten
sequences, each sequence encoding a polypeptide immunogen.
[43] The circular polyribonucleotide of any one of paragraphs [1] to [40],
wherein at least one
sequence encoding a polypeptide immunogen further encodes a signal sequence.
[44] The circular polyribonucleotide of any one of paragraphs [1] to [40],
wherein each sequence
encoding a polypeptide immunogen further encodes a signal sequence.
[45] The circular polyribonucleotide of any one of paragraphs [1] to [44],
wherein each of the
sequences encoding each of the polypeptide immunogens is operably linked to an
internal ribosomal
entry site (IRES).
[46] The circular polyribonucleotide of paragraph [45], wherein the circular
polyribonucleotide
comprises a single !RES.
[47] The circular polyribonucleotide of paragraph [46], wherein each of the
polypeptide
immunogens is encoded by a single open-reading frame operably linked to the
single IRES, wherein the
expression of the open reading frame produces a polypeptide comprising the
amino acid sequence of
each the polypeptide immunogens.
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[48] The circular polyribonucleotide of paragraph [47], wherein the
polypeptide immunogens are
each separated by a polypeptide linker.
[49] The circular polyribonucleotide of paragraph [47], wherein the
polypeptide immunogens are
each separated by a cleavage domain.
[50] The circular polyribonucleotide of paragraph [49], wherein each stagger
element a 2A self-
cleaving peptide.
[51] The circular polyribonucleotide of paragraph [45], wherein the circular
polyribonucleotide
comprises a plurality of IRESs.
[52] The circular polyribonucleotide of paragraph [51], wherein each IRES is
operably linked to
an open reading frame comprising a sequence encoding a polypeptide immunogen.
[53] The circular polyribonucleotide of any one of paragraphs [1] to [52],
wherein the circular
polyribonucleotide further comprises at least one sequence encoding an
adjuvant.
[54] The circular polyribonucleotide of paragraph [53], wherein the at least
one sequence
encoding the adjuvant is operably linked to an !RES.
[55] The circular polyribonucleotide of paragraph [53] or [54], wherein the
adjuvant is a
polypeptide.
[56] The circular polyribonucleotide of paragraph [55], wherein the adjuvant
is a cytokine, a
chemokine, a costimulatory molecule, an innate immune stimulator, a signaling
molecule, a transcriptional
activator, a cytokine receptor, a bacterial component, or a component of the
innate immune system.
[57] The circular polyribonucleotide of paragraph [56], wherein the cytokine
is a pro-inflammatory
cytokine.
[58] The circular polyribonucleotide of paragraph [57], wherein the pro-
inflammatory cytokine is
selected from GM-CSF, IL-1 alpha, IL-1 beta, TGF-beta, TNF-alpha, and TNF-
beta.
[59] The circular polyribonucleotide of paragraph [57], wherein the cytokine
is a Th-1 inducing
cytokine.
[60] The circular polyribonucleotide of paragraph [59], wherein the Th-1
inducing cytokine is
selected from IFN-gamma, IL-2, IL-12, IL-15, and IL-18.
[61] The circular polyribonucleotide of paragraph [56], wherein the cytokine
is a Th-2 inducing
cytokine.
[62] The circular polyribonucleotide of paragraph [61], wherein the Th-2
inducing cytokine is
selected from IFN-gamma, IL-4, IL-5, IL-6, IL-10, and IL-13.
[63] The circular polyribonucleotide of paragraph [56], wherein the chemokine
is selected from
MCP-1, MIP-1alpha, MIP-1beta, Rantes, and TCA-3.
[64] The circular polyribonucleotide of paragraph [56], wherein the
costimulatory molecule is
selected from CD80, 0D86, CD4O-L, CD70, and 0D27.
[65] The circular polyribonucleotide of paragraph [56], wherein the innate
immune stimulator is
selected from STING (e.g., caSTING), TLR3, TLR4, TLR9, TLR7, TLR8, TLR7, RIG-
I/DDX58, and MDA-
5/1F1H1.
[66] The circular polyribonucleotide of paragraph [65], wherein the innate
immune stimulator is a
constitutively active mutant.
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[67] The circular polyribonucleotide of paragraph [567], wherein the signaling
molecule is
selected from STING (e.g., caSTING), TRIF, TRAM, MyD88, PS1, ASC, MAVS, MAPKs,
IKK-alpha, IKK
complex, TBK1, B-catenin, and caspase 1.
[68] The circular polyribonucleotide of paragraph [56], wherein the
transcriptional activator is
selected from AP1, NF-kappa B, IRF3, IRF7, IRF1, and IRF5.
[69] The circular polyribonucleotide of paragraph [56], wherein the cytokine
receptor is selected
from IL-2betaR, IFN-gammaR, and IL-6R.
[70] The circular polyribonucleotide of paragraph [56], wherein the bacterial
component is
selected from flagellin and MBL.
[71] The circular polyribonucleotide of any one of paragraphs [53] to [70],
wherein at least
sequence encoding the adjuvant further encodes a signal sequence.
[72] The circular polyribonucleotide of any one of paragraphs [1] to [70],
wherein the circular
polyribonucleotide further comprises a sequence that is an innate immune
system stimulator.
[73] The circular polyribonucleotide of paragraph [72], wherein the sequence
that is an innate
immune system stimulator is a GU-rich motif, an AU-rich motif, a structured
region comprising dsRNA, or
an aptamer.
[74] A circular polyribonucleotide comprising (a) a sequence encoding a
polypeptide immunogen
and (b) a sequence encoding an adjuvant or a sequence that is an innate immune
system stimulator.
[75] The circular polyribonucleotide of paragraph [74], wherein the sequence
encoding the
polypeptide immunogen is operably linked to an !RES.
[76] The circular polyribonucleotide of paragraph [74] or [75], wherein the
sequence encoding the
adjuvant is operably linked to an !RES.
[77] The circular polyribonucleotide of any one of paragraphs [74] to [76],
wherein the adjuvant is
a polypeptide.
[78] The circular polyribonucleotide of paragraph [77], wherein the
polypeptide immunogen and
the adjuvant polypeptide are encoded by a single open-reading frame operably
linked to the single IRES,
wherein the expression of the open reading frame produces a polypeptide
comprising the amino acid
sequence of each of the polypeptide immunogen and the adjuvant polypeptide.
[79] The circular polyribonucleotide of paragraph [78], wherein the
polypeptide immunogen and
the adjuvant polypeptide are each separated by a polypeptide linker.
[80] The circular polyribonucleotide of paragraph [78], wherein the
polypeptide immunogen and
the adjuvant polypeptide are each separated by a cleavage domain.
[81] The circular polyribonucleotide of paragraph [80], wherein each stagger
element is a 2A self-
cleaving peptide.
[82] The circular polyribonucleotide of paragraph [77], wherein the circular
polyribonucleotide
comprises a plurality of IRESs.
[83] The circular polyribonucleotide of paragraph [82], wherein a first IRES
is operably linked to
the sequence encoding the polypeptide immunogen and a second IRES is operably
linked to the
sequence encoding the adjuvant polypeptide.
[84] The circular polyribonucleotide of ant one of paragraphs [74] to [83],
wherein the expression
of the sequence encoding the polypeptide immunogen is 2-fold greater than the
expression of the
sequence encoding the adjuvant.
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[85] The circular polyribonucleotide of ant one of paragraphs [74] to [84],
wherein the expression
of the sequence encoding the polypeptide immunogen is 5-fold, 10-fold, or 100-
fold greater than the
expression of the sequence encoding the adjuvant.
[86] The circular polyribonucleotide of any one of paragraphs [74] to [85],
wherein the adjuvant is
a cytokine, a chemokine, a costimulatory molecule, an innate immune
stimulator, a signaling molecule, a
transcriptional activator, a cytokine receptor, a bacterial component, or a
component of the innate
immune system.
[87] The circular polyribonucleotide of paragraph [86], wherein the cytokine
is a pro-inflammatory
cytokine.
[88] The circular polyribonucleotide of paragraph [87], wherein the pro-
inflammatory cytokine is
selected from GM-CSF, IL-1 alpha, IL-1 beta, TGF-beta, TNF-alpha, and TNF-
beta.
[89] The circular polyribonucleotide of paragraph [86], wherein the cytokine
is a Th-1 inducing
cytokine.
[90] The circular polyribonucleotide of paragraph [89], wherein the Th-1
inducing cytokine is
selected from IFN-gamma, IL-2, IL-12, IL-15, and IL-18.
[91] The circular polyribonucleotide of paragraph [86], wherein the cytokine
is a Th-2 inducing
cytokine.
[92] The circular polyribonucleotide of paragraph [91], wherein the Th-2
inducing cytokine is
selected from IFN-gamma, IL-4, IL-5, IL-6, IL-10, and IL-13.
[93] The circular polyribonucleotide of paragraph [86], wherein the chemokine
is selected from
MCP-1, MIP-1alpha, MIP-1beta, Rantes, and TCA-3.
[94] The circular polyribonucleotide of paragraph [93], wherein the
costimulatory molecule is
selected from CD80, 0D86, CD4O-L, CD70, and 0D27.
[95] The circular polyribonucleotide of paragraph [86], wherein the innate
immune stimulator is
selected from STING (e.g., caSTING), TLR3, TLR4, TLR9, TLR7, TLR8, TLR7, RIG-
I/DDX58, and MDA-
5/1F1H1.
[96] The circular polyribonucleotide of paragraph [95], wherein the innate
immune stimulator is a
constitutively active mutant.
[97] The circular polyribonucleotide of paragraph [86], wherein the signaling
molecule is selected
from STING (e.g., caSTING), TRIF, TRAM, MyD88, IPS1, ASC, MAVS, MAPKs, IKK-
alpha, IKK complex,
TBK1, B-catenin, and caspase 1.
[98] The circular polyribonucleotide of paragraph [86], wherein the
transcriptional activator is
selected from AP1, NF-kappa B, IRF3, IRF7, IRF1, and IRF5.
[99] The circular polyribonucleotide of paragraph [86], wherein the cytokine
receptor is selected
from IL-2betaR, IFN-gammaR, and IL-6R.
[100] The circular polyribonucleotide of paragraph [86], wherein the bacterial
component is
selected from flagellin and MBL.
[101] The circular polyribonucleotide of any one of paragraphs [74] to [100],
wherein the
sequence encoding the adjuvant further encodes a signal sequence.
[102] The circular polyribonucleotide of any one of paragraphs [74] to [101],
wherein the
sequence encoding the polypeptide immunogen further encodes a signal sequence.
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[103] The circular polyribonucleotide of any one of paragraph [74] to [101],
wherein the sequence
encoding the polypeptide immunogen does not encode a signal sequence.
[104] The circular polyribonucleotide of paragraph [74] or [75], wherein the
sequence that is an
innate immune system stimulator is selected from a GU-rich motif, an AU-rich
motif, a structured region
comprising dsRNA, or an aptamer.
[105] The circular polyribonucleotide of any one of paragraphs [1] to [104],
wherein the circular
polyribonucleotide comprises a first polyribonucleotide with a 5' end and a 3'
end, wherein 5' end and 3'
end are each hybridized to a second polynucleotide, there by linking the 5'
end and the 3' end of the first
polyribonucleotide to form a circular polyribonucleotide.
[106] The circular polyribonucleotide of paragraph [105], wherein the circular
polyribonucleotide
is produced by splint ligation.
[107] The circular polyribonucleotide of any one of paragraphs [1] to [104],
wherein the circular
polyribonucleotide is produced by providing a linear polyribonucleotide having
a 3' end and a 5' end,
wherein the 3' end and the 5' end each comprise a portion of an intron, and
wherein the intron potion of
the 3' end and the intron portion of the 5' end catalyze a splicing reaction
thereby covalently conjugating
the 5' end and the 3' end to produce a circular polyribonucleotide.
[108] The circular polyribonucleotide of paragraph [107], wherein the intron
is a Group I self-
splicing intron.
[109] An immunogenic composition comprising a plurality of circular
polyribonucleotides, each
comprising a sequence encoding a polypeptide immunogen.
[110] The immunogenic composition of paragraph [109], wherein each of the
plurality of circular
polyribonucleotides is a circular polyribonucleotide described by any one of
paragraphs [1] to [108].
[111] The immunogenic composition of paragraph [109], wherein the composition
comprises (a)
at least a first circular polyribonucleotide comprising a sequence encoding a
first polypeptide immunogen
and (b) at least a second circular polyribonucleotide comprising a sequence
encoding a second
polypeptide immunogen, wherein the first and second polypeptide immunogens
have at least 90% amino
acid sequence identity.
[112] The immunogenic composition of paragraph [111], wherein the first and
second
polypeptide immunogens have at least 95% amino acid sequence identity.
[113] The immunogenic composition of paragraph [111] or [112], wherein the
first and second
polypeptide immunogens have less than 100% amino acid sequence identity.
[114] The immunogenic composition of any one of paragraphs [111] to [113],
wherein the each of
the polypeptide immunogens comprises one or more epitopes that identifies a
target.
[115] The immunogenic composition of paragraph [114], wherein the target is a
pathogen.
[116] The immunogenic composition of paragraph [115], wherein the target is a
cancer cell, an
allergen, or a toxin.
[117] The immunogenic composition of paragraph [109], wherein the composition
comprises (a)
at least a first circular polyribonucleotide comprising a sequence encoding a
first polypeptide immunogen
and (b) at least a second circular polyribonucleotide comprising a sequence
encoding a second
polypeptide immunogen, wherein the first and second polypeptide immunogens
identify different proteins,
wherein each different protein identifies the same target.
[118] The immunogenic composition of paragraph [117], wherein the target is a
pathogen.
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[119] The immunogenic composition of paragraph [118], wherein the pathogen is
a virus, a
bacterium, a fungus, or a parasite.
[120] The immunogenic composition of paragraph [119], wherein the pathogen is
a cancer cell,
an allergen, or a toxin.
[121] The immunogenic composition of paragraph [109], wherein the composition
comprises (a)
at least a first circular polyribonucleotide comprising a sequence encoding a
first polypeptide immunogen
and (b) at least a second circular polyribonucleotide comprising a sequence
encoding a second
polypeptide immunogen, wherein the first polypeptide immunogens identifies a
first target and the second
polypeptide immunogen identifies a second target.
[122] The immunogenic composition of paragraph [121], wherein each target is a
pathogen.
[123] The immunogenic composition of paragraph [121] or [122], wherein each
target is,
independently, a cancer cell, a virus, a bacterium, a fungus, a parasite, a
toxin, or an allergen.
[124] The immunogenic composition of any one of paragraphs [109] to [123],
wherein each
polypeptide immunogen is operably linked to an !RES.
[125] An immunogenic composition comprising a plurality of circular
polyribonucleotides, wherein
the composition comprises (a) at least a first circular polyribonucleotide
comprising a sequence encoding
a polypeptide immunogen and (b) at least a second circular polyribonucleotide
comprising a sequence
encoding an adjuvant or a sequence that is an innate immune system stimulator.
[126] The immunogenic composition of paragraph [125], wherein the adjuvant is
a polypeptide.
[127] The immunogenic composition of paragraph [126], wherein the adjuvant is
a cytokine, a
chemokine, a costimulatory molecule, an innate immune stimulator, a signaling
molecule, a transcriptional
activator, a cytokine receptor, a bacterial component, or a component of the
innate immune system.
[128] The immunogenic composition of paragraph [127], wherein the cytokine is
a pro-
inflammatory cytokine.
[129] The immunogenic composition of paragraph [128], wherein the pro-
inflammatory cytokine
is selected from GM-CSF, IL-1 alpha, IL-1 beta, TGF-beta, TNF-alpha, and TNF-
beta.
[130] The immunogenic composition of paragraph [127], wherein the cytokine is
a Th-1 inducing
cytokine.
[131] The immunogenic composition of paragraph [130], wherein the Th-1
inducing cytokine is
selected from IFN-gamma, IL-2, IL-12, IL-15, and IL-18.
[132] The immunogenic composition of paragraph [127], wherein the cytokine is
a Th-2 inducing
cytokine.
[133] The immunogenic composition of paragraph [132], wherein the Th-2
inducing cytokine is
selected from IFN-gamma, IL-4, IL-5, IL-6, IL-10, and IL-13.
[134] The immunogenic composition of paragraph [127], wherein the chemokine is
selected from
MCP-1, MIP-1alpha, MIP-1beta, Rantes, and TCA-3.
[135] The immunogenic composition of paragraph [134], wherein the
costimulatory molecule is
selected from CD80, 0D86, CD4O-L, CD70, and 0D27.
[136] The immunogenic composition of paragraph [127], wherein the innate
immune stimulator is
selected from STING (e.g., caSTING), TLR3, TLR4, TLR9, TLR7, TLR8, TLR7, RIG-
I/DDX58, and MDA-
5/1F1H1.
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[137] The immunogenic composition of paragraph [136], wherein the innate
immune stimulator is
a constitutively active mutant.
[138] The immunogenic composition of paragraph [127], wherein the signaling
molecule is
selected from STING (e.g., caSTING), TRIF, TRAM, MyD88, PS1, ASC, MAVS, MAPKs,
IKK-alpha, IKK
complex, TBK1, B-catenin, and caspase 1.
[139] The immunogenic composition of paragraph [127], wherein the
transcriptional activator is
selected from AP1, NF-kappa B, IRF3, IRF7, IRF1, and IRF5.
[140] The immunogenic composition of paragraph [127], wherein the cytokine
receptor is
selected from IL-2betaR, IFN-gammaR, and IL-6R.
[141] The immunogenic composition of paragraph [127], wherein the bacterial
component is
selected from flagellin and MBL.
[142] The immunogenic composition of any one of paragraphs [125] to [141],
wherein the
polypeptide immunogen and the adjuvant are each operably linked to an !RES.
[143] The immunogenic composition of paragraph [125], wherein the sequence
that is an innate
immune system stimulator is a GU-rich motif, an AU-rich motif, a structured
region comprising dsRNA, or
an aptamer.
[144] The immunogenic composition of any one of paragraphs [1] to [143],
wherein the circular
polyribonucleotide comprises a first polyribonucleotide with a 5' end and a 3'
end, wherein 5' end and 3'
end are each hybridized to a second ribonucleotide, there by linking the 5'
end and the 3' end of the first
polyribonucleotide to form a circular polyribonucleotide.
[145] The circular polyribonucleotide of paragraphs [1] to [144], wherein the
circular
polyribonucleotide is produced by splint ligation.
[146] The immunogenic composition of any one of paragraphs [1] to [143],
wherein the circular
polyribonucleotide is produced by providing a linear polyribonucleotide having
a 3' end and a 5' end,
.. wherein the 3' end and the 5' end each comprise a portion of an intron, and
wherein the intron potion of
the 3' end and the intron portion of the 5' end catalyze a splicing reaction
thereby covalently conjugating
the 5' end and the 3' end to produce a circular polyribonucleotide.
[147] The immunogenic composition of paragraph [146], wherein the intron is a
Group I self-
splicing intron.
[148] A pharmaceutical preparation of a circular polyribonucleotide, wherein
the circular
polyribonucleotide comprises a sequence encoding a polypeptide immunogen, and
wherein the circular
polyribonucleotide constitutes at least 25% (w/w) of total ribonucleotide
molecules in the pharmaceutical
preparation.
[149] The pharmaceutical preparation of paragraph [148], wherein the circular
polyribonucleotide
constitutes at least 30 /0(w/w), 40 /0(w/w), 50% (w/w), 60% (w/w), 70% (w/w),
75% (w/w), 80% (w/w),
85% (w/w), 90% (w/w), 91% (w/w), 92% (w/w), 93% (w/w), 94% (w/w), 95% (w/w),
96% (w/w), 97%
(w/w), 98% (w/w) or 99% (w/w) of total ribonucleotide molecules in the
pharmaceutical preparation.
[150] The pharmaceutical preparation of any one of paragraphs [148] or [149],
wherein the
pharmaceutical preparation comprises no more than 75% (w/w), 60% (w/w), 50%
(w/w), 40% (w/w), 30%
(w/w), 20% (w/w), 15% (w/w), 10% (w/w), 5% (w/w), or 1% (w/w) linear
polyribonucleotide molecules of
the total ribonucleotide molecules in the pharmaceutical preparation.
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[151] The pharmaceutical preparation of paragraph [150], wherein the
pharmaceutical
preparation comprises no more than 0.5% (w/w) linear polyribonucleotide
molecules of the total
ribonucleotide molecules in the pharmaceutical preparation.
[152] The pharmaceutical preparation of any one of paragraphs [148] to [151],
wherein the
preparation comprises no more than 15% (w/w) nicked polyribonucleotide
molecules of the total
ribonucleotide molecules in the pharmaceutical preparation.
[153] The pharmaceutical preparation of any one of paragraphs [148] to [152],
wherein the
preparation comprises no more than 10% (w/w), 9% (w/w), 8% (w/w), 7% (w/w), 6%
(w/w), 5% (w/w),
4% (w/w), 3% (w/w), 2% (w/w), 1% (w/w), or 0.5% (w/w) nicked
polyribonucleotide molecules of the total
ribonucleotide molecules in the pharmaceutical preparation.
[154] The pharmaceutical preparation of any one of paragraph [148] or [149],
wherein the
pharmaceutical preparation comprises at least 1% (w/w) linear
polyribonucleotide.
[155] The pharmaceutical composition of paragraph [154], wherein the
pharmaceutical
preparation comprises between 30%-40% (w/w) circular polyribonucleotide and
between 1%-10% (w/w),
10%-20% (w/w), 20%-30% (w/w), 30%-40% (w/w), 40%-50% (w/w), 50%-60% (w/w), or
60%-70% (w/w)
linear polyribonucleotide.
[156]. The pharmaceutical composition of paragraph [154], wherein the
pharmaceutical
preparation comprises between 40%-60% (w/w) circular polyribonucleotide and
between 1%-10% (w/w),
10%-20% (w/w), 20%-30% (w/w), 30%-40% (w/w), 40%-50% (w/w), 50%-60% (w/w)
linear
polyribonucleotide.
[157] The pharmaceutical composition of paragraph [154], wherein the
pharmaceutical
preparation comprises between 60%-80% (w/w) circular polyribonucleotide and
between 1%-10% (w/w),
10%-20% (w/w), 20%-30% (w/w), 30%-40% (w/w) linear polyribonucleotide.
[158] A pharmaceutical preparation of a circular polyribonucleotide, wherein
the circular
polyribonucleotide comprises a sequence encoding a polypeptide immunogen, and
wherein the
pharmaceutical preparation comprises no more than 75% (w/w) linear
polyribonucleotide molecules of
the total ribonucleotide molecules in the pharmaceutical preparation.
[159] The pharmaceutical preparation of paragraph [158], wherein the
pharmaceutical
preparation comprises no more than 60% (w/w), 50% (w/w), 40% (w/w), 30% (w/w),
20% (w/w), 15%
(w/w), 10% (w/w), 5% (w/w), 1% (w/w), or 0.5% (w/w) linear polyribonucleotide
molecules of the total
ribonucleotide molecules in the pharmaceutical preparation.
[160] The pharmaceutical preparation of paragraph [158] or [159], wherein the
preparation
comprises no more than 15% (w/w) nicked polyribonucleotide molecules of the
total ribonucleotide
molecules in the pharmaceutical preparation.
[161] The pharmaceutical preparation of any one of paragraphs [158] to [160],
wherein the
preparation comprises no more than 10% (w/w), 9% (w/w), 8% (w/w), 7% (w/w), 6%
(w/w), 5% (w/w),
4% (w/w), 3% (w/w), 2% (w/w), 1% (w/w), or 0.5% (w/w) nicked
polyribonucleotide molecules of the total
ribonucleotide molecules in the pharmaceutical preparation.
[162] A pharmaceutical preparation of a circular polyribonucleotide, wherein
the circular
polyribonucleotide comprises a sequence encoding a polypeptide immunogen, and
wherein the
pharmaceutical preparation comprises no more than 15% (w/w) nicked
polyribonucleotide molecules of
the total ribonucleotide molecules in the pharmaceutical preparation.
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[163] The pharmaceutical preparation of paragraph [162], wherein the
preparation comprises no
more than 10% (w/w), 9% (w/w), 8% (w/w), 7% (w/w), 6% (w/w), 5% (w/w), 4%
(w/w), 3% (w/w), 2%
(w/w), 1% (w/w), or 0.5% (w/w) nicked polyribonucleotide molecules of the
total ribonucleotide
molecules in the pharmaceutical preparation.
[164] The pharmaceutical preparation of any one of paragraphs [148] to [163],
wherein the
pharmaceutical preparation comprises less than 10 EU/kg or lacks endotoxin as
measured by a Limulus
amebocyte lysate test.
[165] The pharmaceutical preparation of any one of paragraphs [148] to [163],
wherein the
pharmaceutical preparation comprises a bioburden of less than 100 CFU/100 ml
or less than 10 CFU/100
ml before sterilization.
[166] The pharmaceutical preparation of any one of paragraphs [148] to [165],
wherein the
pharmaceutical preparation is a sterile pharmaceutical preparation, e.g.,
supports growth of fewer than
100 viable microorganisms as tested under aseptic conditions.
[167] The pharmaceutical preparation of any one of paragraphs [148] to [166],
wherein the
pharmaceutical preparation meets the standard of USP <71>.
[168] The pharmaceutical preparation of any one of paragraphs [148] to [67],
wherein the
pharmaceutical preparation meets the standard of USP <85>.
[169] The pharmaceutical preparation of any one of paragraphs [148] to [168],
wherein the
pharmaceutical preparation is an intermediate pharmaceutical preparation of a
final drug product.
[170] The pharmaceutical preparation of any one of paragraphs [148] to [169],
wherein the
pharmaceutical preparation is a final drug product for administration to a
subject.
[171] The pharmaceutical preparation of any one of paragraphs [148] to [170],
wherein the
pharmaceutical preparation comprises a concentration of at least 0.1 ng/mL,
0.5 ng/mL, 1 ng/mL, 5
ng/mL, 10 ng/mL, 50 ng/mL, 0.1 g/mL, 0.5 g/mL,1 g/mL, 2 g/mL, 5 g/mL, 10
g/mL, 20 g/mL, 30
g/mL, 40 g/mL, 50 g/mL, 60 g/mL, 70 g/mL, 80 g/mL, 100 g/mL, 200 g/mL,
300 g/mL, 500
g/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 5 mg/mL, 10 mg/mL, 100 mg/mL, 200 mg/mL, or
500 mg/mL
circular polyribonucleotide molecules.
[172] The pharmaceutical preparation of any one of paragraphs [148] to [171],
wherein the
pharmaceutical preparation comprises no more than 1 ng/ml, 5 ng/ml, 10 ng/ml,
15 ng/ml, 20 ng/ml, 25
ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml,
90 ng/ml, 100 ng/ml, 200
ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 1000 g/mL, 5000 g/mL, 10,000 g/mL,
or 100,000 g/mL of
deoxyribonucleotide molecules.
[173] The pharmaceutical preparation of any one of paragraphs [148] to [172],
wherein the
pharmaceutical preparation comprises a protein contamination (e.g. an enzyme)
of less than 0.1 ng, 1 ng,
5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80
ng, 90 ng, 100 ng, 200 ng,
300 ng, 400 ng, or 500 ng of the protein contamination per milligram (mg) of
the circular
polyribonucleotide molecules.
[174] The pharmaceutical preparation of any one of paragraphs [148] to [173],
wherein the
pharmaceutical preparation comprises an A260/A280 absorbance ratio of from
about 1.6 to 2.3 as
measured by a spectrophotometer.
[175] The pharmaceutical preparation of any one of paragraphs [148] to [174],
wherein the
pharmaceutical preparation is substantially free of a process-related impurity
selected from a cell protein,
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a cell deoxyribonucleic acid, an enzyme, a reagent component, a gel component,
or a chromatographic
material.
[176] The pharmaceutical preparation of any one of paragraphs [148] to [175],
wherein the
pharmaceutical preparation has a reduced level of one or more markers of an
immune or inflammatory
response after purification compared to prior to purification.
[177] The pharmaceutical preparation of any one of paragraphs [148] to [176],
wherein the
circular polyribonucleotide is a circular polyribonucleotide of any one of
paragraphs [1] to [108].
[178] The pharmaceutical preparation of any one of paragraphs [148] to [177],
wherein the
circular polyribonucleotide is produced according to a method comprising the
steps of:
(a) providing a plurality of linear polyribonucleotide molecules;
b) circularizing the linear polyribonucleotide molecules to provide a
preparation of circular
polyribonucleotide molecules; and
c) processing the preparation to remove linear polyribonucleotide molecules
from the preparation.
[179] The pharmaceutical preparation of paragraph [178], wherein the method
further comprises
d) evaluating the amount of linear polyribonucleotide molecules in the
preparation remaining after the
processing step.
[180] The pharmaceutical preparation of paragraph [178] or [179], wherein the
method further
comprises e) further processing the preparation, wherein further processing
comprises:
- processing the preparation to remove nicked polyribonucleotide molecules
from the preparation;
and/or
- processing the preparation to remove deoxyribonucleotide molecules from
the preparation;
and/or
- processing the preparation to remove protein contaminants from the
preparation; and/or
- processing the preparation to remove endotoxin from the preparation.
[181] A pharmaceutical composition comprising the circular polyribonucleotide
of any one of
paragraphs [1] to [108], the immunogenic composition of any one of paragraphs
[109] to [147], or the
pharmaceutical preparation of any one of paragraphs [148] to [180], and a
pharmaceutically acceptable
excipient.
[182] The pharmaceutical composition of paragraph [181], further comprising an
adjuvant.
[183] The pharmaceutical composition of paragraph [182], wherein the adjuvant
is an inorganic
adjuvant, a small molecule adjuvant, and oil in water emulsion, a lipid or
polymer, a peptide or
peptidoglycan, a carbohydrate or polysaccharide, a saponin, an RNA-based
adjuvant, a DNA-based
adjuvant, a viral particle, a bacterial adjuvant, a hybrid molecule, a fungal
or oocyte microbe-associated
molecular pattern (MAMP), an inorganic nanoparticle, or a multi-component
adjuvant.
[184] The pharmaceutical composition of paragraph [183], wherein the adjuvant
is an inorganic
adjuvant.
[185] The pharmaceutical composition of paragraph [184], wherein the inorganic
adjuvant is an
aluminum salt or a calcium salt.
[186] The pharmaceutical composition of paragraph [183], wherein the adjuvant
is a small
molecule.
[187] The pharmaceutical composition of paragraph [183], wherein the small
molecule is
imiquimod, resiquimod, or gardiquimod.
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[188] The pharmaceutical composition of paragraph [183], wherein the adjuvant
is an oil in water
emulsion.
[189] The pharmaceutical composition of paragraph [188], wherein the oil in
water emulsion is
Squalene, MF59, AS03, a Montanide formulation, optionally Montanide ISA 51 or
Montanide ISA 720, or
Incomplete Freunds Adjuvant an oil in water emulsion.
[190] The pharmaceutical composition of paragraph [183], wherein the adjuvant
is a lipid or
polymer.
[191] The pharmaceutical composition of paragraph [190], wherein the lipid or
polymer is a
polymeric nanoparticle, optionally PLGA, PLG, PLA, PGA, or PHB, a liposome,
optionally a Virosomes or
CAF01, a lipid nanoparticle or a component thereof, a lipopolysaccharide
(LPS), optionally
monophosphoryl lipid A (MPLA) or glucopyranosyl Lipid A (GLA), a lipopeptide,
optionally Pam2
(Pam2CSK4) or Pam3 (Pam3CSK4), or a glycolipid, optionally, trehalose
dimycolate.
[192] The pharmaceutical composition of paragraph [187], wherein the adjuvant
is a peptide or
peptidoglycan.
[193] The pharmaceutical composition of paragraph [192], wherein the peptide
of peptidoglycan
corresponds to all or a portion of a synthetic or purified gram negative or
gram positive bacteria, optionally
N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), a flagellin-fusion protein,
Mannose-binding Lectin
(MBL), a cytokines, or a chemokine.
[194] The pharmaceutical composition of paragraph [183], wherein the adjuvant
is a
carbohydrate or polysaccharide.
[195] The pharmaceutical composition of paragraph [194], wherein the
carbohydrate or
polysaccharide is dextran (branched microbial polysaccharide), dextran-
sulfate, Lentinan, zymosan, Beta-
glucan, Deltin, Mannan, or Chitin.
[196] The pharmaceutical composition of paragraph [183], wherein the adjuvant
is a saponin.
[197] The pharmaceutical composition of paragraph [196], wherein the saponin
is a glycoside or
a polycyclic aglycones attached to one or more sugar side chains, optionally
ISCOMS, ISCOMS matrix,
or QS-21.
[198] The pharmaceutical composition of paragraph [183], wherein the adjuvant
is an RNA-
based adjuvant.
[199] The pharmaceutical composition of paragraph [198], wherein the RNA-based
adjuvant is
Poly IC, Poly IC:LC, a hairpin RNAs, optionally with a 5'PPP containing
sequence, a viral sequence, a
polyU containing sequences, dsRNA, a natural or synthetic immunostimulatory
RNA sequence, a nucleic
acids analogs, optionally cyclic GMP-AMP or a cyclic dinucleotide such as
cyclic di-GMP, or an
immunostimulatory base analog, optionally a 08-substitued or an N7,08-
disubstituted guanine
ribonucleotide.
[200] The pharmaceutical composition of paragraph [183], wherein the adjuvant
is a DNA-based
adjuvant.
[201] The pharmaceutical composition of paragraph [200], wherein the DNA-based
adjuvant is a
CpG, dsDNA, or a natural or synthetic immunostimulatory DNA sequence.
[202] The pharmaceutical composition of paragraph [183], wherein the adjuvant
is a viral particle.
[203] The pharmaceutical composition of paragraph [202], wherein the viral
particle is a
virosome, optionally, a phospholipid cell membrane bilayer.
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[204] The pharmaceutical composition of paragraph [183], wherein the adjuvant
is a bacterial
adjuvant.
[205] The pharmaceutical composition of paragraph [204], wherein the bacterial
adjuvant is
flagellin, LPS, or a bacterial toxin, optionally an enterotoxins, a heat-
labile toxin, or a Cholera toxin
[206] The pharmaceutical composition of paragraph [183], wherein the adjuvant
is a hybrid
molecule.
[207] The pharmaceutical composition of paragraph [206], wherein the adjuvant
is CpG
conjugated to lmiquimod.
[208] The pharmaceutical composition of paragraph [183], wherein the adjuvant
is a fungal or
oocyte microbe-associated molecular pattern (MAMP).
[209] The pharmaceutical composition of paragraph [208], wherein the fungal or
oocyte MAMP is
chitin or beta-glucan.
[210] The pharmaceutical composition of paragraph [183], wherein the adjuvant
is an inorganic
nanoparticle.
[211] The pharmaceutical composition of paragraph [210], wherein the inorganic
nanoparticle is
a gold nanorod, a silica-based nanoparticle, optionally a mesoporous silica
nanoparticles (MSN).
[212] The pharmaceutical composition of paragraph [183], wherein the adjuvant
is a multi-
component adjuvant.
[213] The pharmaceutical composition of paragraph [212], wherein the multi-
component adjuvant
is AS01, AS03, AS04, Complete Freunds Adjuvant, or CAF01.
[214] A pharmaceutical composition comprising a mixture comprising (a) a
polyribonucleotide
comprising a sequence encoding a polypeptide immunogen and (b) an alcohol,
wherein the alcohol
constitutes about 0.3% v/v to about 75% v/v of the mixture.
[215] The pharmaceutical composition of paragraph [214], wherein the
polyribonucleotide is a
circular polyribonucleotide.
[216] A pharmaceutical composition comprising a mixture comprising (a) the
circular
polyribonucleotide of any one of paragraphs [1] to [108], the immunogenic
composition of any one of
paragraphs [109] to [147], or the pharmaceutical preparation of any one of
paragraphs [148] to [180], and
(b) an alcohol, wherein the alcohol constitutes about 0.3% v/v to about 75%
v/v of the mixture.
[217] The pharmaceutical composition of paragraph [214], wherein the
polyribonucleotide is a
linear polyribonucleotide.
[218] The pharmaceutical composition of paragraph [217], wherein the linear
polyribonucleotide
comprises a plurality of sequences, each sequence encoding a polypeptide
immunogen, wherein at least
two of the polypeptide immunogens have at least 90% amino acid sequence
identity.
[219] The pharmaceutical composition of paragraph [218], wherein the at least
two polypeptide
immunogens have less than 100% amino acid sequence identity.
[220] The pharmaceutical composition of paragraph [217], wherein the linear
polyribonucleotide
comprises a plurality of sequences, each sequence encoding a polypeptide
immunogen, wherein at least
two of the polypeptide immunogens identify different proteins, wherein each of
the different proteins
identifies the same target.
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[221] The pharmaceutical composition of paragraph [217], wherein the linear
polyribonucleotide
comprises a plurality of sequences, each sequence encoding a polypeptide
immunogen, wherein at least
two of the polypeptide immunogens identify different targets.
[222] The pharmaceutical composition of any one of paragraphs [217] to [221],
wherein the linear
polyribonucleotide comprises at least three, at least four, at least five, at
least six, at least seven, at least
eight, or at least nine sequences, each sequence encoding a polypeptide
immunogen.
[223] The pharmaceutical composition of paragraph [215], wherein the circular
polyribonucleotide further comprises at least one sequence encoding an
adjuvant.
[224] The pharmaceutical composition of paragraph [223], wherein the adjuvant
is a polypeptide.
[225] The pharmaceutical composition of paragraph [224], wherein the adjuvant
is a cytokine, a
chemokine, a costimulatory molecule, an innate immune stimulator, a signaling
molecule, a transcriptional
activator, a cytokine receptor, a bacterial component, or a component of the
innate immune system.
[226] The pharmaceutical composition of any one of paragraphs [215] or [223]
to [225], wherein
the circular polyribonucleotide further comprises a sequence that is an innate
immune system stimulator.
[227] The pharmaceutical composition of paragraph [226], wherein the sequence
that is an
innate immune system stimulator is a GU-rich motif, an AU-rich motif, a
structured region comprising
dsRNA, or an aptamer.
[228] The pharmaceutical composition of any one of paragraphs [214] to [227],
wherein the
alcohol constitutes about 0.3% v/v to about 60% v/v, about 0.3% v/v to about
50% v/v, about 0.3% v/v to
about 40% v/v about 0.3% v/v to about 20% v/v, about 0.3% v/v to about 15%
v/v, about 0.3% v/v to
about 10% v/v, about 0.3% v/v to about 5% v/v, about 0.3% v/v to about 1% v/v,
or about 0.3% v/v to
about 0.5% v/v of the mixture.
[229] The pharmaceutical composition of any one of paragraphs [214] to [227],
wherein the
alcohol constitutes about 0.5% v/v to about 75% v/v, about 1% v/v to about 75%
v/v, about 5% v/v to
about 75% v/v, about 10% v/v to about 75% v/v, about 15% v/v to about 75% v/v,
about 20% v/v to about
75% v/v, about 30% v/v to about 75% v/v, about 40% v/v to about 75% v/v, about
50% v/v to about 75%
v/v, about 60% v/v to about 75% v/v, or about 70% v/v to about 75% v/v of the
mixture.
[230] The method of any one of paragraphs [214] to [229], wherein the alcohol
is selected from
methanol, ethanol, isopropanol, phenoxyethanol, triethanolamine, phenethyl
alcohol, butanol, pentanol,
cetyl alcohol, ethylene glycol, propylene glycol, denatured alcohol, benzyl
alcohol, specially denatured
alcohol, glycol, stearyl alcohol, cetearyl alcohol, menthol, polyethylene
glycols (PEG)-400, ethoxylated
fatty acids, and hydroxyethylcellulose.
[231] The method of paragraph [230], wherein the alcohol is ethanol.
[232] The pharmaceutical composition of any one of paragraphs [214] to [231],
wherein the
pharmaceutical composition is formulated for topical administration.
[233] The pharmaceutical composition of any one of paragraphs [214] to [232],
wherein the
pharmaceutical composition is a liquid, gel, lotion, paste, cream, foam, or
stick.
[234] The pharmaceutical composition of any one of paragraphs [214] to [233],
wherein the
pharmaceutical composition has a pH of about 6-8.
[235] The pharmaceutical composition of any one of paragraphs [214] to [234],
wherein the
pharmaceutical composition has a viscosity that is about the same as water.
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[236] The pharmaceutical composition of any one of paragraphs [214] to [235],
wherein the
pharmaceutical composition is substantially free of hydrophobic or lipophilic
groups.
[237] The pharmaceutical composition of any one of paragraphs [214] to [236],
wherein the
pharmaceutical composition is substantially free of hydrocarbons.
[238] The pharmaceutical composition of any one of paragraphs [214] to [237],
wherein the
pharmaceutical composition is substantially free of cationic liposomes.
[239] The pharmaceutical composition of any one of paragraphs [214] to [238],
wherein the
pharmaceutical composition is substantially free of fatty acids, lipids,
liposomes, cholesterol, or any
combination thereof.
[240] The pharmaceutical composition of any one of paragraphs [214] to [239],
the composition
further comprising an adjuvant.
[241] The pharmaceutical composition of paragraph [240], wherein the adjuvant
is an inorganic
adjuvant, a small molecule adjuvant, and oil in water emulsion, a lipid or
polymer, a peptide or
peptidoglycan, a carbohydrate or polysaccharide, a saponin, an RNA-based
adjuvant, a DNA-based
adjuvant, a viral particle, a bacterial adjuvant, a hybrid molecule, a fungal
or oocyte microbe-associated
molecular pattern (MAMP), an inorganic nanoparticle, or a multi-component
adjuvant.
[242] A method of delivering a polyribonucleotide to a subject comprising
topically applying the
pharmaceutical composition of any one of paragraphs [214] to [241] to a
surface area of the subject.
[243] A method of delivering a polyribonucleotide to a subject comprising:
a) applying a sterilizing agent to a surface area of the subject; and
b) applying a composition comprising the circular polyribonucleotide of any
one of paragraphs [1]
to [108], the immunogenic composition of any one of paragraphs [109] to -
[147], the pharmaceutical
preparation of any one of paragraphs [148] to [180], or the pharmaceutical
composition of any one of
paragraphs [181] to [242] and a diluent to the surface area of the subject.
[244] The method of paragraph [243], wherein the composition of (b) does not
comprise a
carrier.
[245] The method of paragraph [243] or [244], wherein the sterilizing agent is
an alcohol, UV
light, laser light, or heat.
[246] A method of delivering a polyribonucleotide to a subject comprising
a) applying an alcohol to a surface area of the subject; and
b) applying a composition comprising the circular polyribonucleotide of any
one of paragraphs [1]
to [108], the immunogenic composition of any one of paragraphs [109] to [147],
the pharmaceutical
preparation of any one of paragraphs [148] to [180], or the pharmaceutical
composition of any one of
paragraphs [181] to [241] and a diluent to the surface area of the subject.
[247] The method of paragraph [246], wherein the alcohol is selected from the
group consisting
of: methanol, ethanol, isopropanol, phenoxyethanol, triethanolamine, phenethyl
alcohol, butanol,
pentanol, cetyl alcohol, ethylene glycol, propylene glycol, denatured alcohol,
benzyl alcohol, specially
denatured alcohol, glycol, stearyl alcohol, cetearyl alcohol, menthol,
polyethylene glycols (PEG)-400,
ethoxylated fatty acids, and hydroxyethylcellulose.
[248] The method of paragraph [247], wherein the alcohol is ethanol.
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[249] The method of any one of paragraphs [243] to [248], wherein the surface
area of the
subject is selected from a surface area of the skin, oral cavity, nasal
cavity, gastrointestinal tract,
respiratory tract, or any combination thereof.
[250] A lipid nanoparticle (LNP) comprising the circular polyribonucleotide of
any one of
paragraphs [1] to [108] or the immunogenic composition of any one of
paragraphs [109] to [147].
[251] The LNP of paragraph [250], comprising an ionizable lipid.
[252] The LNP of paragraph [250], comprising a cationic lipid.
[253] The LNP of paragraph [252], wherein the cationic lipid has a structure
according to:
(i)
-
0 (ii), or
0
o (iii).
[254] The LNP of any one of paragraphs [250] to [253], further comprising one
or more neutral
lipid, e.g., DSPC, DPPC, DMPC, DOPC, POPC, DOPE, SM, a steroid, e.g.,
cholesterol, and/or one or
more polymer conjugated lipid, e.g., a pegylated lipid, e.g., PEG-DAG, PEG-PE,
PEG-S-DAG, PEG-cer
or a PEG dialkyoxypropylcarbamate.
[255] A method of treating or preventing a disease, disorder, or condition in
a subject, the
method comprising administering to the subject the circular polyribonucleotide
of any one of paragraphs
[1] to [108], the immunogenic composition of any one of paragraphs [109] to
[147], the pharmaceutical
preparation of any one of paragraphs [148] to [180], the pharmaceutical
composition of any one of
paragraphs [181] to [241], or the LNP of any one of paragraphs [250] to [254].
[256] The method of paragraph [255], wherein the disease, disorder, or
condition is a viral
infection, a bacterial infection, or a fungal infection.
[257] The method of paragraph [255], wherein the disease, disorder, or
condition is a cancer.
[258] The method of paragraph [255], wherein the disease, disorder, or
condition is associated
with exposure to an allergen.
[259] The method of paragraph [255], wherein the disease, disorder, or
condition is associated
with exposure to a toxin.
[260] A method of inducing an immune response in a subject, the method
comprising
administering to the subject the circular polyribonucleotide of any one of
paragraphs [1] to [108], the
immunogenic composition of any one of paragraphs [109] to [147], the
pharmaceutical preparation of any
one of paragraphs [148] to [180], the pharmaceutical composition of any one of
paragraphs [181] to [241],
or the LNP of any one of paragraphs [250] to [254].
[261] The method of any one of paragraphs [255] to [260], wherein the subject
is a mammal.
[262] The method of paragraph [261], wherein the subject is a human.
[263] The method of paragraph [261], wherein the method is a non-human mammal.
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[264] The method of paragraph [261], wherein in the non-human mammal is a cow,
a sheep, a
goat, a pig, a dog, a horse, or a cat.
[265] The method of any one of paragraphs [255] to [264], wherein the subject
is a bird.
[266] The method of paragraph [265], wherein in the bird is a hen, a rooster,
a turkey, or a parrot.
[267] The method of any one of paragraphs [255] to [266], wherein the method
further comprises
administering an adjuvant to the subject.
[268] The method of paragraph [267], wherein the adjuvant is an inorganic
adjuvant, a small
molecule adjuvant, and oil in water emulsion, a lipid or polymer, a peptide or
peptidoglycan, a
carbohydrate or polysaccharide, a saponin, an RNA-based adjuvant, a DNA-based
adjuvant, a viral
particle, a bacterial adjuvant, a hybrid molecule, a fungal or oocyte microbe-
associated molecular pattern
(MAMP), an inorganic nanoparticle, or a multi-component adjuvant.
[269] The method of paragraph [267], wherein the adjuvant is a polypeptide
adjuvant.
[270] The method of paragraph [269], wherein the polypeptide adjuvant is a
cytokine, a
chemokine, a costimulatory molecule, an innate immune stimulator, a signaling
molecule, a transcriptional
activator, a cytokine receptor, a bacterial component, or a component of the
innate immune system.
[271] The method of paragraph [267], wherein the adjuvant is an innate immune
system
stimulator.
[272] The method of paragraph [271], wherein the innate immune system
stimulator is selected
from an RNA including a GU-rich motif, an AU-rich motif, a structured region
comprising dsRNA, or an
aptamer.
[273] The method of any one of paragraphs [255] to [272], wherein the circular
polyribonucleotide
of any one of paragraphs [1] to [110], the immunogenic composition of any one
of paragraphs [111] to
[151], the pharmaceutical preparation of any one of paragraphs [152] to [184],
the pharmaceutical
composition of any one of paragraphs [185] to [245], or the LNP of any one of
paragraphs [250] to [254] is
administered to the subject as a single dose.
[274] The method of any one of paragraphs [255] to [272], wherein the circular
polyribonucleotide of any one of paragraphs [1] to [108], the immunogenic
composition of any one of
paragraphs [109] to [147], the pharmaceutical preparation of any one of
paragraphs [148] to [180], the
pharmaceutical composition of any one of paragraphs [181] to [241], or the LNP
of any one of paragraphs
[250] to [254] is administered to the subject two or more times, three or more
times, four or more times, or
five or more times.
[275] The method of paragraph [274], wherein administration of the circular
polyribonucleotide of
any one of paragraphs [1] to [108], the immunogenic composition of any one of
paragraphs [109] to [147],
the pharmaceutical preparation of any one of paragraphs [148] to [180], the
pharmaceutical composition
of any one of paragraphs [181] to [241], or the LNP of any one of paragraphs
[250] to [254] occurs about
weekly, about every two weeks, about every three weeks, about every month,
about every two months,
about every three months, about every four months, about every five months,
about every six months,
about every year, about every two years, about every three years, about every
four years, about every
five years, or about every ten years.
[276] The method any one of paragraphs [255] to [275], wherein the method
further comprises
administering to the subject a polypeptide immunogen (e.g., a protein subunit
comprising a polypeptide
immunogen).
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[277] The method of paragraph [276] wherein the polypeptide immunogen
corresponds to (e.g.,
shares 90%, 95%, 96%, 97%, 98%, or 100% amino acid sequence identity with a
polypeptide immunogen
encoded by a sequence of the circular polyribonucleotide.
[278] The method of paragraph [276], wherein the polypeptide immunogen is
administered to the
subject after administering the circular polyribonucleotide of any one of
paragraphs [1] to [108], the
immunogenic composition of any one of paragraphs [109] to [147], the
pharmaceutical preparation of any
one of paragraphs [148] to [180], the pharmaceutical composition of any one of
paragraphs [181] to [241],
or the LNP of any one of paragraphs [250] to [254].
[279] The method of any one of paragraphs [276] to [278], wherein the
polypeptide immunogen
maintains or enhances an immune response in the subject against the
polypeptide immunogen.
[280] A method of maintaining or enhancing an immune response in a subject,
the method
comprising (i) administering to the subject a circular polyribonucleotide
encoding a polypeptide
immunogen and (ii) administering to the subject the polypeptide immunogen,
wherein step (ii) occurs
between 1 week and 6 months after step (i), and wherein administration of the
polypeptide immunogen of
step (ii) maintains or enhances the immune response in the subject against the
polypeptide immunogen.
[281] The method of paragraph [280], wherein the polypeptide immunogen
comprises one or
more epitopes that identifies a target.
[282] The method of paragraph [281], wherein the target is a pathogen.
[283] The method of paragraph [281], wherein the target is a cancer cell, an
allergen, or a toxin.
Definitions
The present disclosure will be described with respect to particular
embodiments and with
reference to certain figures, but the disclosure is not limited thereto but
only by the claims. Terms as set
forth hereinafter are generally to be understood in their common sense unless
indicated otherwise.
As used herein, the term "adaptive immune response" means either a humoral or
cell-mediated
immune response. For purposes of the present disclosure, a "humoral immune
response" refers to an
immune response mediated by antibody molecules, while a "cellular immune
response" is one mediated
by T-lymphocytes and/or other white blood cells.
As used herein, the term "adjuvant" refers to a composition (e.g., a compound,
polypeptide,
nucleic acid, or lipid) that increases an immune response, for example,
increases a specific immune
response against an immunogen. Increasing an immune response includes
intensification or broadening
the specificity of either or both antibody and cellular immune responses.
The term "alcohol" means any organic compound in which the hydroxyl functional
group (¨OH) is
bound to a carbon. In some embodiments, an alcohol includes a hydroxyl bound
to a saturated carbon,
which is in turn bound to other hydrogen or carbon atoms. An alcohol as
discussed herein can include,
but is not limited to, monohydric alcohols, polyhydric alcohols, unsaturated
aliphatic alcohols, and alicyclic
alcohols. In some cases, an alcohol can refer to ethanol. In some cases, an
alcohol can include, but is
not limited to, methanol, ethanol, isopropanol, phenoxyethanol,
triethanolamine, phenethyl alcohol,
butanol, pentanol, cetyl alcohol, ethylene glycol, propylene glycol, denatured
alcohol, benzyl alcohol,
specially denatured alcohol, glycol, stearyl alcohol, cetearyl alcohol,
menthol, polyethylene glycols (PEG)-
400, ethoxylated fatty acids, and hydroxyethylcellulose.
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As used herein, the term "associated with" a disease, disorder, or condition
refers to a
relationship, either causative or correlative, between an entity and the
occurrence or severity of a
disease, disorder, or condition in a subject. For example, if a target is
associated with a disease,
disorder, or condition, the target may be the causative agent of the disease,
disorder, or condition. For
example, a virus may be the causative agent in a viral infection, bacteria may
be the causative agent in a
bacterial infection, a fungus may be the causative agent in a fungal
infection, or a parasite may be the
causative agent in a parasitic infection, a cancer cell may be the causative
agent of a cancer, a toxin may
be the causative agent of toxicity, or an allergen may the causative agent of
an allergic reaction. The
target associated with a disease, disorder, or condition may also or
alternately be correlated with an
increased likelihood of occurrence or an increase severity of a disease
disorder, or condition.
As used herein, the term "carrier" means a compound, composition, reagent, or
molecule that
facilitates the transport or delivery of a composition (e.g., a linear or a
circular polyribonucleotide) into a
subject, a tissue, or a cell. Non-limiting examples of carriers include
carbohydrate carriers (e.g., an
anhydride-modified phytoglycogen or glycogen-type material), nanoparticles
(e.g., a nanoparticle that
encapsulates or is covalently linked binds to the circular or linear
polyribonucleotide), liposomes,
fusosomes, ex vivo differentiated reticulocytes, exosomes, protein carriers
(e.g., a protein covalently
linked to the polyribonucleotide), or cationic carriers (e.g., a cationic
lipopolymer or transfection reagent).
As used herein, the term "cell-penetrating agent" means an agent that, when
contacted to a cell,
facilitates entry into the cell. In some cases, a cell-penetrating agent
facilitates direct penetration of the
cell membrane, for instance, via direct electrostatic interaction with
negatively charged phospholipids of
the cell membrane, or transient pore formation by inducing configurational
changes in membrane proteins
or the phospholipid bilayer. In some cases, a cell-penetrating agent
facilitates endocytosis-mediated
translocation into the cell. For example, under certain situation, the cell-
penetrating agent can stimulate
the cell to undergo the endocytosis process, by which the cell membrane can
fold inward into the cell. In
certain embodiments, a cell-penetrating agent helps form a transitory
structure that transports across the
cell membrane. Without wishing to be bound to a particular theory, a cell-
penetrating agent as provided
herein can increase the permeability of the cell membrane or increase
internalization of a molecule into
the cell, as a result of which, delivery into the cell can be more efficient
when the cell is contacted with the
cell-penetrating agent simultaneously as compared to otherwise identical
delivery without the cell-
penetrating agent.
As used herein, the terms "circRNA" or "circular polyribonucleotide" or
"circular RNA" or "circular
polyribonucleotide molecule" are used interchangeably and mean a
polyribonucleotide molecule that has
a structure having no free ends (i.e., no free 3' and/or 5' ends), for example
a polyribonucleotide molecule
that forms a circular or end-less structure through covalent (e.g., covalently-
closed) or non-covalent
bonds.
As used herein, the term "circularization efficiency" is a measurement of
resultant circular
polyribonucleotide versus its non-circular starting material.
As used herein, the terms "circRNA preparation" or "circular
polyribonucleotide preparation" or
"circular RNA preparation" are used interchangeably and mean a composition
including circRNA
molecules and a diluent, carrier, first adjuvant, or a combination thereof.
The wording "compound, composition, product, etc. for treating, modulating,
etc." is to be
understood to refer a compound, composition, product, etc. per se which is
suitable for the indicated
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purposes of treating, modulating, etc. The wording "compound, composition,
product, etc. for treating,
modulating, etc." additionally discloses that, as a preferred embodiment, such
compound, composition,
product, etc. is for use in treating, modulating, etc.
The wording "compound, composition, product, etc. for use in ..." or "use of a
compound,
composition, product, etc. in the manufacture of a medicament, pharmaceutical
composition, veterinary
composition, diagnostic composition, etc. for ..." indicates that such
compounds, compositions, products,
etc. are to be used in therapeutic methods which may be practiced on the human
or animal body. They
are considered as an equivalent disclosure of embodiments and claims
pertaining to methods of
treatment, etc. If an embodiment or a claim thus refers to "a compound for use
in treating a human or
animal being suspected to suffer from a disease", this is considered to be
also a disclosure of a "use of a
compound in the manufacture of a medicament for treating a human or animal
being suspected to suffer
from a disease" or a "method of treatment by administering a compound to a
human or animal being
suspected to suffer from a disease".
The term "diluent" means a vehicle including an inactive solvent in which a
composition described
herein (e.g., a composition including a circular or linear polyribonucleotide)
may be diluted or dissolved.
A diluent can be an RNA solubilizing agent, a buffer, an isotonic agent, or a
mixture thereof. A diluent
can be a liquid diluent or a solid diluent. Non-limiting examples of liquid
diluents include water or other
solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl
alcohol, ethyl carbonate,
ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-
butylene glycol, dimethylformamide,
oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of
sorbitan, and 1,3-butanediol. Non-
limiting examples of solid diluents include calcium carbonate, sodium
carbonate, calcium phosphate,
dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium
phosphate lactose, sucrose,
cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol,
sodium chloride, dry starch,
cornstarch, or powdered sugar.
As used herein, the terms "disease," "disorder," and "condition" each refer to
a state of sub-
optimal health, for example, a state that is or would typically be diagnosed
or treated by a medical
professional.
As used herein, the term "epitope" refers to a portion or the whole of an
immunogen that is
recognized, targeted, or bound by an antibody or T cell receptor. An epitope
can be a linear epitope, for
example, a contiguous sequence of nucleic acids or amino acids. An epitope can
be a conformational
epitope, for example, an epitope that contains amino acids that form an
epitope in the folded
conformation of the protein. A conformational epitope can contain non-
contiguous amino acids from a
primary amino acid sequence. As another example, a conformational epitope
includes nucleic acids that
form an epitope in the folded conformation of an immunogenic sequence based on
its secondary
structure or tertiary structure.
As used herein, the term "encryptogen" is a nucleic acid sequence or structure
of the circular
polyribonucleotide that aids in reducing, evading, and/or avoiding detection
by an immune cell and/or
reduces induction of an immune response against the circular or linear
polyribonucleotide.
As used herein, the term "expression sequence" is a nucleic acid sequence that
encodes a
product, e.g., a peptide or polypeptide, or a regulatory nucleic acid. An
exemplary expression sequence
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that codes for a peptide or polypeptide can include a plurality of nucleotide
triads, each of which can code
for an amino acid and is termed as a "codon".
As used herein, the terms "identify" or "identifies" refer to indicating,
establishing, or recognizing
the identity of an entity. For example, an immunogen or an epitope thereof may
identify a target, meaning
that the target includes the immunogen or epitope thereof, that the immunogen
or epitope thereof is
derived from the target, and/or the immunogen or epitope thereof shares a high
degree of similarity with a
portion or the whole of the target. Recognition or binding of an antibody or a
T cell receptor to an
immunogen or an epitope thereof can identify a target. Where an immunogen or
an epitope thereof
identifies a target, the immunogen or epitope thereof distinguishes the target
from one or more other
targets. Likewise, a polypeptide immunogen can identify a protein. Otherwise
put, the polypeptide
immunogen is a component of, a portion of, is derived from, or shares a high
degree of similarity to the
protein or a portion of the protein, in particular to an epitope of a protein.
As used herein, the term "innate immune system stimulator," refers to a
substance that induces
an innate immunological response, in part, by inducing expression of one or
more genes involved in
innate immunity, including, but not limited to, a type I interferon (e.g.,
IFNa, INF6, and/or IFNy), a pro-
inflammatory cytokine (e.g., IL-1, IL-12, IL-18, TNF-a, and/or GM-CSF),
retinoic-acid inducible gene-I
(RIG-I, also known as DDX58), melanoma-differentiation-associated gene 5
(MDA5, also known as
IFIH1), 2'-5 ' oligoadenylate synthase 1 (OAS 1), OAS-like protein (OASL),
and/or protein kinase R
(PKR). An innate immune system stimulator may act as an adjuvant, e.g., when
administered in
combination with or formulated with, a ribonucleotide that encodes an
immunogen. An innate immune
system stimulator may be a separate molecule entity (e.g., not encoded by or
incorporated as a sequence
in a polyribonucleotide), for example, STING (e.g., caSTING), TLR3, TLR4,
TLR9, TLR7, TLR8, TLR7,
RIG-I/DDX58, and MDA-5/IFIH1 or a constitutively active mutant thereof. An
innate immune system
stimulator may be encoded by (e.g., expressed from) a polyribonucleotide. A
polyribonucleotide may
alternately or further include a ribonucleotide sequence that acts as an
innate immune system stimulator
(e.g., GU-rich motif, an AU-rich motif, a structured region comprising dsRNA,
or an aptamer).
As used herein, the term "impurity" is an undesired substance present in a
composition, e.g., a
pharmaceutical composition as described herein. In some embodiments, an
impurity is a process-related
impurity. In some embodiments, an impurity is a product-related substance
other than the desired
product in the final composition, e.g., other than the active drug ingredient,
e.g., circular or linear
polyribonucleotide, as described herein. As used herein, the term "process-
related impurity" is a
substance used, present, or generated in the manufacturing of a composition,
preparation, or product that
is undesired in the final composition, preparation, or product other than the
linear polyribonucleotides
described herein. In some embodiments, the process-related impurity is an
enzyme used in the synthesis
or circularization of polyribonucleotides. As used herein, the term "product-
related substance" is a
substance or byproduct produced during the synthesis of a composition,
preparation, or product, or any
intermediate thereof. In some embodiments, the product-related substance is
deoxyribonucleotide
fragments. In some embodiments, the product-related substance is
deoxyribonucleotide monomers. In
some embodiments, the product-related substance is one or more of: derivatives
or fragments of
polyribonucleotides described herein, e.g., fragments of 10, 9, 8, 7, 6, 5, or
4 ribonucleic acids,
monoribonucleic acids, diribonucleic acids, or triribonucleic acids.
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As used herein, the term "immunogen," refers to an any molecule or molecular
structure that
includes one or more epitopes recognized, targeted, or bound by an antibody or
a T cell receptor. In
particular, an immunogen induces an immune response in a subject (e.g., is
immunogenic as defined
herein). An immunogen is capable of inducing an immune response in a subject,
wherein the immune
response refers to a series of molecular, cellular, and organismal events that
are induced when an
immunogen is encountered by the immune system. The immune response may be
humoral and/or
cellular immune response. These may include the production of antibodies and
the expansion of B- and
T-cells. To determine whether an immune response has occurred and to follow
its course, the immunized
subject can be monitored for the appearance of immune reactants directed at
the specific immunogen.
Immune responses to most immunogen induce the production of both specific
antibodies and specific
effector T cells. In some embodiments, the immunogen is foreign to a host. In
some embodiments, the
immunogen is not foreign to a host. An immunogen may include all or a portion
of a polypeptide, a
polysaccharide, a polynucleotide, or a lipid. An immunogen may also be a mixed
polypeptide,
polysaccharide, polynucleotide, and/or lipid. For example, an immunogen maybe
a polypeptide that has
been translationally modified. A "polypeptide immunogen" refers to an
immunogen that includes a
polypeptide. A polypeptide immunogen may also include one or more post-
translational modifications,
and/or may form a complex with one or more additional molecules, and/or may
adopt a tertiary or
quaternary structure, each of which may determine or affect the immunogenicity
of the polypeptide.
As used herein, the term "immunogenic" is a potential to induce a response to
a substance in a
particular immune response assay above a pre-determined threshold. The assay
can be, e.g.,
expression of certain inflammatory markers, production of antibodies, or an
assay for immunogenicity as
described herein. In some embodiments, an immune response may be induced when
an immune system
of an organism or a certain type of immune cells are exposed to an immunogen.
An immunogenic response may be assessed may evaluating the antibodies in the
plasma or
serum of a subject using a total antibody assay, a confirmatory test,
titration and isotyping of the
antibodies, and neutralizing antibody assessment. A total antibody assay
measures the all the antibodies
generated as part of the immune response in the serum or plasma of a subject
that has been
administered the immunogen. The most commonly used test to detect antibodies
is an ELISA (enzyme-
linked immunosorbent assay), which detects antibodies in the tested serum that
bind to the antibody of
interest, including IgM, IgD, IgG, IgA, and IgE. An immunogenic response can
be further assessed by a
confirmatory assay. Following a total antibody assessment, a confirmatory
assay may be used to confirm
the results of the total antibody assay. A competition assay may be used to
confirm that antibody is
specifically binding to target and that the positive finding in the screening
assay is not a result of non-
specific interactions of the test serum or detection reagent with other
materials in the assay.
An immunogenic response can be assessed by isotyping and titration. An
isotyping assay may
be used to assess only the relevant antibody isotypes. For example, the
expected isotypes may be IgM
and IgG which may be specifically detected and quantified by isotyping and
titration, and then compared
to the total antibodies present.
An immunogenic response can be assessed by a neutralizing antibody assay
(nAb). A
neutralizing antibody assay (nAb) may be used to determine if the antibodies
produced in response to the
immunogen neutralized the immunogen thereby inhibiting the immunogen from
having an effect on the
target and leading to abnormal pharmacokinetic behaviors. An nAb assay is
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where the target cells are incubated with the antibody. A variety of cell
based nAb assays may be used
including but not limited to Cell Proliferation, Viability, Antibody-Dependent
Cell-Mediated Cytotoxicity
(ADCC), Complement-Dependent Cytotoxicity (CDC), Cytopathic Effect Inhibition
(CPE), Apoptosis,
Ligand Stimulated Cell Signaling, Enzyme Activity, Reporter Gene Assays,
Protein Secretion, Metabolic
Activity, Stress and Mitochondrial Function. Detection readouts include
Absorbance, Fluorescence,
Luminescence, Chemiluminescence, or Flow Cytometry .A ligand-binding assay may
also be used to
measure the binding affinity of an immunogen and an antibody in vitro to
evaluate neutralization efficacy.
Furthermore, induction of a cellular immune response may be assessed by
measuring T cell
activation in a subject using cellular markers on T cells obtained from the
subject. A blood sample, lymph
node biopsy, or tissue sample can be collected from a subject and T cells from
the sample evaluated for
one or more (e.g., 2, 3,4 or more) activation markers: CD25, CD71, CD26, CD27,
CD28, CD30, CD154,
CD4OL, CD134, CD69, CD62L or CD44. T cell activation can also be assessed
using the same methods
in an in vivo animal model. This assay can also be performed by adding an
immunogen to T cells in vitro
(e.g., T cells obtained from a subject, animal model, repository, or
commercial source) and measuring the
aforementioned markers to evaluate T cell activation. Similar approaches can
be used to assess the
effect of an on activation of other immune cells, such as eosinophils
(markers: CD35, CD11b, CD66,
CD69 and CD81), dendritic cells (makers: IL-8, MHC class II, CD40, CD80, CD83,
and CD86), basophils
(CD63, CD13, CD4, and CD203c), and neutrophils (CD11b, CD35, CD66b and CD63).
These markers
can be assessed using flow cytometry, immunohistochemistry, in situ
hybridization, and other assays that
allow for measurement of cellular markers. Comparing results from before and
after administration of an
immunogen can be used to determine its effect.
As used herein, the term "inducing an immune response" refers to initiating,
amplifying, or
sustaining an immune response by a subject. Inducing an immune response may
refer to an adaptive
immune response or an innate immune response. The induction of an immune
response may be
measured as discussed above.
As used herein, the term "linear counterpart" is a polyribonucleotide molecule
(and its fragments)
having the same or similar nucleotide sequence (e.g., 100%, 95%, 90%, 85%,
80%, 75%, or any
percentage therebetween sequence identity) as a circular polyribonucleotide
and having two free ends
(i.e., the uncircularized version (and its fragments) of the circularized
polyribonucleotide). In some
embodiments, the linear counterpart (e.g., a pre-circularized version) is a
polyribonucleotide molecule
(and its fragments) having the same or similar nucleotide sequence (e.g.,
100%, 95%, 90%, 85%, 80%,
75%, or any percentage therebetween sequence identity) and same or similar
nucleic acid modifications
as a circular polyribonucleotide and having two free ends (i.e., the
uncircularized version (and its
fragments) of the circularized polyribonucleotide). In some embodiments, the
linear counterpart is a
polyribonucleotide molecule (and its fragments) having the same or similar
nucleotide sequence (e.g.,
100%, 95%, 90%, 85%, 80%, 75%, or any percentage therebetween sequence
identity) and different or
no nucleic acid modifications as a circular polyribonucleotide and having two
free ends (i.e., the
uncircularized version (and its fragments) of the circularized
polyribonucleotide). In some embodiments, a
fragment of the polyribonucleotide molecule that is the linear counterpart is
any portion of linear
counterpart polyribonucleotide molecule that is shorter than the linear
counterpart polyribonucleotide
molecule. In some embodiments, the linear counterpart further includes a 5'
cap. In some embodiments,
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the linear counterpart further includes a poly adenosine tail. In some
embodiments, the linear counterpart
further includes a 3' UTR. In some embodiments, the linear counterpart further
includes a 5' UTR.
As used herein, the terms "linear RNA" or "linear polyribonucleotide" or
"linear polyribonucleotide
molecule" are used interchangeably and mean polyribonucleotide molecule having
a 5' and 3' end. One
or both of the 5' and 3' ends may be free ends or joined to another moiety.
Linear RNA includes RNA
that has not undergone circularization (e.g., is pre-circularized) and can be
used as a starting material for
circularization through, for example, splint ligation, or chemical, enzymatic,
ribozyme- or splicing-
catalyzed circularization methods.
As used herein, the term "mixture" means a material made of two or more
different substances
that are mixed. In some cases, a mixture described herein can be a homogenous
mixture of the two or
more different substances, e.g., the mixture can have the same proportions of
its components (e.g., the
two or more substances) throughout any given sample of the mixture. In some
cases, a mixture as
provided herein can be a heterogeneous mixture of the two or more different
substances, e.g., the
proportions of the components of the mixture (e.g., the two or more
substances) can vary throughout the
mixture. In some cases, a mixture is a liquid solution, e.g., the mixture is
present in liquid phase. In
some instances, a liquid solution can be regarded as comprising a liquid
solvent and a solute. Mixing a
solute in a liquid solvent can be termed as "dissolution" process. In some
cases, a liquid solution is a
liquid-in-liquid solution (e.g., a liquid solute dissolved in a liquid
solvent), a solid-in-liquid solution (e.g., a
solid solute dissolved in a liquid solvent), or a gas-in-liquid solution
(e.g., a solid solute dissolved in a
liquid solvent). In some cases, there is more than one solvent and/or more
than one solute. In some
cases, a mixture is a colloid, liquid suspension, or emulsion. In some cases,
a mixture is a solid mixture,
e.g., the mixture is present in solid phase.
As used herein, the term "modified ribonucleotide" means a nucleotide with at
least one
modification to the sugar, the nucleobase, or the internucleoside linkage.
As used herein, the term "naked delivery" means a formulation for delivery to
a cell without the
aid of a carrier and without covalent modification to a moiety that aids in
delivery to a cell. A naked
delivery formulation is free from any transfection reagents, cationic
carriers, carbohydrate carriers,
nanoparticle carriers, or protein carriers. For example, naked delivery
formulation of a circular or linear
polyribonucleotide is a formulation that includes a circular or linear
polyribonucleotide without covalent
modification and is free from a carrier.
As used herein, the terms "nicked RNA" or "nicked linear polyribonucleotide"
or "nicked linear
polyribonucleotide molecule" are used interchangeably and mean a
polyribonucleotide molecule having a
5' and 3' end that results from nicking or degradation of a circular RNA.
As used herein, the term "non-circular RNA" means total nicked RNA and linear
RNA.
The terms "obtainable by", "producible by" or the like are used to indicate
that a claim or
embodiment refers to compound, composition, product, etc. per se, i.e. that
the compound, composition,
product, etc. can be obtained or produced by a method which is described for
manufacture of the
compound, composition, product, etc., but that the compound, composition,
product, etc. may be obtained
or produced by other methods than the described one as well. The terms
"obtained by", "produced by" or
the like indicate that the compound, composition, product, is obtained or
produced by a recited specific
method. It is to be understood that the terms "obtainable by", "producible by"
and the like also disclose
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the terms "obtained by", "produced by" and the like as a preferred embodiment
of "obtainable by",
"producible by" and the like.
As used herein, the term "pathogen" refers to an infectious agent, which
causes disease or
disease symptoms in a subject, for example, by directly infecting the subject,
by producing agents that
cause disease or disease symptoms in the subject, and/or by eliciting an
immune response in the subject.
As used herein, pathogens include, but are not limited to bacteria, protozoa,
parasites, fungi, nematodes,
insects, viroids, and viruses, or any combination thereof, wherein each
pathogen is capable, either by
itself or in concert with another pathogen, of eliciting disease or symptoms a
subject.
As used herein, the term "payload" means any molecule delivered by the
polyribonucleotide as
disclosed herein. In some cases, a payload is a nucleic acid, a protein, a
chemical, a ribonucleoprotein,
or any combination thereof. In some cases, a payload is a nucleic acid
sequence directly contained
within the polyribonucleotide as disclosed herein. In some cases, a payload is
attached to or associated
with the polyribonucleotide as disclosed herein, for instance via
complementary hybridization, or via
protein-nucleic acid interactions. In certain cases, the payload is a protein
encoded by a nucleic acid
sequence contained within, attached to, or associated with the
polyribonucleotide. In some cases, the
"attachment" means covalent bond or non-covalent interaction between two
molecules. In some cases,
the "association" when used in the context of the interaction between a
payload and a polyribonucleotide
means that the payload is indirectly linked to the polyribonucleotide via one
or more other molecules in
between. In some cases, the attachment or association can be transient. In
some cases, a payload is
attached to or associated with the polyribonucleotide under one condition but
not under another condition,
for instance, depending on the ambient pH condition or the presence or absence
of a stimulus or a
binding partner.
The term "pharmaceutical composition" is intended to also disclose that the
circular or linear
polyribonucleotide included within a pharmaceutical composition can be used
for the treatment of the
human or animal body by therapy. It is thus meant to be equivalent to the "a
circular or linear
polyribonucleotide for use in therapy".
The term "polynucleotide" as used herein means a molecule comprising one or
more nucleic acid
subunits, or nucleotides, and can be used interchangeably with "nucleic acid"
or "oligonucleotide". A
polynucleotide can include one or more nucleotides selected from adenosine
(A), cytosine (C), guanine
(G), thymine (T) and uracil (U), or variants thereof. A nucleotide can include
a nucleoside and at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (P03) groups. A nucleotide can
include a nucleobase, a five-
carbon sugar (either ribose or deoxyribose), and one or more phosphate groups.
Ribonucleotides are
nucleotides in which the sugar is ribose. Polyribonucleotides or ribonucleic
acids, or RNA, can refer to
macromolecules that include multiple ribonucleotides that are polymerized via
phosphodiester bonds.
Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose.
Polydeoxyribonucleotides or deoxyribonucleic acids, or DNA, means
macromolecules that
include multiple deoxyribonucleotides that are polymerized via phosphodiester
bonds. A nucleotide can
be a nucleoside monophosphate or a nucleoside polyphosphate. A nucleotide
means a
deoxyribonucleoside polyphosphate, such as, e.g., a deoxyribonucleoside
triphosphate (dNTP), which
can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine
triphosphate (dCTP),
deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP) and
deoxythymidine triphosphate
(dTTP) dNTPs, that include detectable tags, such as luminescent tags or
markers (e.g., fluorophores). A
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nucleotide can include any subunit that can be incorporated into a growing
nucleic acid strand. Such
subunit can be an A, C, G, T, or U, or any other subunit that is specific to
one or more complementary A,
C, G, T or U, or complementary to a purine (i.e., A or G, or variant thereof)
or a pyrimidine (i.e., C, T or U,
or variant thereof). In some examples, a polynucleotide is deoxyribonucleic
acid (DNA), ribonucleic acid
(RNA), or derivatives or variants thereof. In some cases, a polynucleotide is
a short interfering RNA
(siRNA), a microRNA (miRNA), a plasmid DNA (pDNA), a short hairpin RNA
(shRNA), small nuclear RNA
(snRNA), messenger RNA (mRNA), precursor mRNA (pre-mRNA), antisense RNA
(asRNA), to name a
few, and encompasses both the nucleotide sequence and any structural
embodiments thereof, such as
single-stranded, double-stranded, triple-stranded, helical, hairpin, etc. In
some cases, a polynucleotide
molecule is circular. A polynucleotide can have various lengths. A nucleic
acid molecule can have a
length of at least about 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100
bases, 200 bases, 300
bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb,
50 kb, or more. A
polynucleotide can be isolated from a cell or a tissue. As embodied herein,
the polynucleotide sequences
may include isolated and purified DNA/RNA molecules, synthetic DNA/RNA
molecules, and synthetic
DNA/RNA analogs.
Polynucleotides, e.g., polyribonucleotides or polydeoxyribonucleotides, may
include one or more
nucleotide variants, including nonstandard nucleotide(s), non-natural
nucleotide(s), nucleotide analog(s)
and/or modified nucleotides. Examples of modified nucleotides include, but are
not limited to
diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-
acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethy1-2-
thiouridine, 5-
carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine, N6-
isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-
methyladenine, 2-
methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-
methylguanine, 5-
methylaminomethyluracil, 5-methoxyaminomethy1-2-thiouracil, beta-D-
mannosylqueosine, 5-
methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-
isopentenyladenine, uracil-5-oxyacetic
acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-
thiouracil, 2-thiouracil, 4-
thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-
oxyacetic acid(v), 5-methy1-2-
thiouracil, 3-(3-amino- 3- N-2-carboxypropyl) uracil, (acp3)w, 2,6-
diaminopurine and the like. In some
cases, nucleotides may include modifications in their phosphate moieties,
including modifications to a
triphosphate moiety. Non-limiting examples of such modifications include
phosphate chains of greater
length (e.g., a phosphate chain having, 4, 5, 6, 7, 8, 9, 10 or more phosphate
moieties) and modifications
with thiol moieties (e.g., alpha-thiotriphosphate and beta-thiotriphosphates).
Nucleic acid molecules may
also be modified at the base moiety (e.g., at one or more atoms that typically
are available to form a
hydrogen bond with a complementary nucleotide and/or at one or more atoms that
are not typically
capable of forming a hydrogen bond with a complementary nucleotide), sugar
moiety or phosphate
backbone. Nucleic acid molecules may also contain amine -modified groups, such
as amino ally 1-dUTP
(aa-dUTP) and aminohexhylacrylamide-dCTP (aha-dCTP) to allow covalent
attachment of amine reactive
moieties, such as N-hydroxysuccinimide esters (NHS). Alternatives to standard
DNA base pairs or RNA
base pairs in the oligonucleotides of the present disclosure can provide
higher density in bits per cubic
mm, higher safety (resistant to accidental or purposeful synthesis of natural
toxins), easier discrimination
in photo-programmed polymerases, or lower secondary structure. Such
alternative base pairs compatible
with natural and mutant polymerases for de novo and/or amplification synthesis
are described in Betz K,
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Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P,
Romesberg FE, Marx A.
Nat. Chem. Biol. 2012 Jul;8(7):612-4, which is herein incorporated by
reference for all purposes.
As used herein, "polypeptide" means a polymer of amino acid residues (natural
or unnatural)
linked together most often by peptide bonds. The term, as used herein, refers
to proteins, polypeptides,
and peptides of any size, structure, or function. Polypeptides can include
gene products, naturally
occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs,
fragments and other
equivalents, variants, and analogs of the foregoing. A polypeptide can be a
single molecule or may be a
multi- molecular complex such as a dimer, trimer, or tetramer. They can also
comprise single chain or
multichain polypeptides such as antibodies or insulin and can be associated or
linked. Most commonly
disulfide linkages are found in multichain polypeptides. The term polypeptide
can also apply to amino
acid polymers in which one or more amino acid residues are an artificial
chemical analogue of a
corresponding naturally occurring amino acid.
As used herein, the term "prevent," means to reduce the likelihood of
developing a disease,
disorder, or condition, or alternatively, to reduce the severity of a
subsequently developed disease or
disorder. A therapeutic agent can be administered to a subject who is at
increased risk of developing a
disease or disorder relative to a member of the general population in order to
prevent the development of,
or lessen the severity of, the disease or condition. A therapeutic agent can
be administered as a
prophylactic, e.g., before development of any symptom or manifestation of a
disease or disorder.
As used herein, the phrase "quasi-helical structure" is a higher order
structure of the circular
polyribonucleotide, wherein at least a portion of the circular
polyribonucleotide folds into a helical
structure.
As used herein, the phrase "quasi-double-stranded secondary structure" is a
higher order
structure of the circular polyribonucleotide, wherein at least a portion of
the circular polyribonucleotide
creates an internal double strand.
As used herein, the term "regulatory element" is a moiety, such as a nucleic
acid sequence, that
modifies expression of an expression sequence within the circular or linear
polyribonucleotide.
As used herein, the term "repetitive nucleotide sequence" is a repetitive
nucleic acid sequence
within a stretch of DNA or RNA or throughout a genome. In some embodiments,
the repetitive nucleotide
sequence includes poly CA or poly TG (UG) sequences. In some embodiments, the
repetitive nucleotide
sequence includes repeated sequences in the Alu family of introns.
As used herein, the term "replication element" is a sequence and/or motif
useful for replication or
that initiate transcription of the circular polyribonucleotide.
As used herein, the term "surface area" of a subject body means any area of a
subject that is or
has a potential to be exposed to an exterior environment subject body. A
surface area of a subject body,
e.g., a mammal body, e.g., a human body, can include skin, surface areas of
oral cavity, nasal cavity, ear
cavity, gastrointestinal tract, respiratory tract, vaginal, cervical, inter
uterine, urinary tract, and eye. In
some cases, a surface area of a subject body can often refer to the outer area
under which epithelial cells
are lined up. Skin, for example, can be one type of surface area as discussed
herein and can be
composed of epidermis and dermis, the former of which forms the outermost
layers of kin and can include
organized assembly of epithelial cells among many other types of cells.
As used herein, the term "stagger element" is a moiety, such as a nucleotide
sequence, that
induces ribosomal pausing during translation. In some embodiments, the stagger
element is a non-
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conserved sequence of amino-acids with a strong alpha-helical propensity
followed by the consensus
sequence -D(V/I)ExNPG P, where x= any amino acid. In some embodiments, the
stagger element may
include a chemical moiety, such as glycerol, a non-nucleic acid linking
moiety, a chemical modification, a
modified nucleic acid, or any combination thereof.
As used herein, the term "substantially free" is the level of a component in a
composition,
preparation, or product, or any intermediate thereof that is lower than the
level required to induce a
biological, chemical, physical, and/or pharmacological effect. In some
embodiments, a composition,
preparation, or product is substantially free of a component if the level of
the component is detectable
only in trace amounts or the level is less than the level detectable by a
relevant detection technique (e.g.,
chromatography (using a column, using a paper, using a gel, using HPLC, using
UHPLC, etc., or by IC,
by SEC, by reverse phase, by anion exchange, by mixed mode, etc.) or
electrophoresis (UREA PAGE,
chip-based, polyacrylamide gel, RNA, capillary, c-IEF, etc.) with or without
pre or post separation
derivatization methodologies using detection techniques based on mass
spectrometry, UV-visible,
fluorescence, light scattering, refractive index, or that use silver or dye
stains or radioactive decay for
detection. Alternatively, whether a composition, preparation, or product is
substantially free of a
component may be determined without the use of separation technologies by mass
spectrometry, by
microscopy, by circular dichroism (CD) spectroscopy, by UV or UV-vis
spectrophotometry, by fluorometry
(e.g., Qubit), by RNase H analysis, by surface plasmon resonance (SPR), or by
methods that utilize silver
or dye stains or radioactive decay for detection).
As used herein, the term "substantially resistant" is one that has at least
50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% resistance to an effector
as compared to a
reference.
As used herein, the term "sterilizing agent" means any agent that is
bacteriostatic, bactericidal,
and/or actively kills microorganisms, inactivates microorganisms, or prevents
microorganisms from
growing. A sterilizing agent that kills microorganisms can be antimicrobial
and/or antiseptic. In some
embodiments, the sterilizing agent is a liquid, such as an alcohol, iodine, or
hydrogen peroxide. In some
embodiments, the sterilizing agent, is UV light or a laser light. In some
embodiments, the sterilizing agent
is heat delivered electrically or through other means (e.g., vapor, contact).
As used herein, the term "stoichiometric translation" is a substantially
equivalent production of
expression products translated from the circular or linear polyribonucleotide.
For example, for a circular
or linear polyribonucleotide having two expression sequences, stoichiometric
translation of the circular or
linear polyribonucleotide means that the expression products of the two
expression sequences have
substantially equivalent amounts, e.g., amount difference between the two
expression sequences (e.g.,
molar difference) can be about 0, or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%, 10%, 15%, or
20%, or any percentage therebetween.
As used herein, the term "systemic delivery" or "systemic administration"
means a route of
administration of pharmaceutical compositions or other substances into the
circulatory system (e.g., blood
or lymphoid system). The systemic administration can include oral
administration, parenteral
administration, intranasal administration, sublingual administration, rectal
administration, transdermal
administration, or any combinations thereof. As used herein, the term "non-
systemic delivery" or "non-
systemic administration" can refer to any other routes of administration than
systemic delivery of
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pharmaceutical compositions or other substances, e.g., the delivered
substances do not enter the
circulation systems (e.g., blood and lymphoid system) of the subject body.
As used herein, the term "sequence identity" is determined by alignment of two
peptide or two
nucleotide sequences using a global or local alignment algorithm. Sequences
may then be referred to as
"substantially identical" or "essentially similar" when they (when optimally
aligned by for example the
programs GAP or BESTFIT using default parameters) share at least a certain
minimal percentage of
sequence identity. GAP uses the Needleman and Wunsch global alignment
algorithm to align two
sequences over their entire length, maximizing the number of matches and
minimizes the number of
gaps. Generally, the GAP default parameters are used, with a gap creation
penalty = 50 (nucleotides) / 8
(proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For
nucleotides the default scoring
matrix used is nwsgapdna and for proteins the default scoring matrix is
Blosum62 (Henikoff & Henikoff,
1992, PNAS 89, 915-919). Sequence alignments and scores for percentage
sequence identity may be
determined using computer programs, such as the GCG Wisconsin Package, Version
10.3, available
from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or
EmbossWin version 2.10.0
(using the program "needle"). Alternatively or additionally, percent identity
may be determined by
searching against databases, using algorithms such as FASTA, BLAST, etc.
Sequence identity refers to
the sequence identity over the entire length of the sequence.
A "signal sequence" refers to a polypeptide sequence, e.g., between 10 and 30
amino acids in
length, that is present at the N-terminus of a polypeptide sequence of a
nascent protein which targets the
polypeptide sequence to the secretory pathway.
As used herein, the term "target" refers to any entity that includes one or
more epitopes. For
example, a target may be a chemical moiety, a portion of a molecule, a
molecule (e.g., an allergen or a
toxin), a macromolecule (e.g., a polypeptide, a nucleic acid, or
carbohydrate), a post-translational
modification state of a macromolecule (e.g., a macromolecule that is
phosphorylated, glycosylated,
acylated, alkylated, and the like), a higher-order macromolecular structure
(e.g., a complex of two or more
polypeptides), a cell (e.g., a cancer cell), a portion of a cell (e.g., a
tumor antigen), a receptor on the
surface of a cell, a pathogen (e.g., a virus or a portion or a virus; a
bacterium or a portion of a bacterium;
a fungus or a portion of a fungus; or a parasite or a portion of a parasite),
or a tissue-type.
As used herein, the term "treat," or "treating," refers to a therapeutic
treatment of a disease or
disorder (e.g., an infectious disease, a cancer, a toxicity, or an allergic
reaction) in a subject. The effect
of treatment can include reversing, alleviating, reducing severity of, curing,
inhibiting the progression of,
reducing the likelihood of recurrence of the disease or one or more symptoms
or manifestations of the
disease or disorder, stabilizing (i.e., not worsening) the state of the
disease or disorder, and/or preventing
the spread of the disease or disorder as compared to the state and/or the
condition of the disease or
disorder in the absence of the therapeutic treatment.
As used herein, the term "termination element" is a moiety, such as a nucleic
acid sequence, that
terminates translation of the expression sequence in the circular or linear
polyribonucleotide.
As used herein, the term "topical delivery" means delivery of a substance to
skin or an epithelial
layer accessible though non-invasive means, e.g., the intestinal and other
gastrointestinal (GI) epithelia or
the vaginal epithelium. Topical delivery of a pharmaceutical composition can
have a local
pharmacodynamic effect on the subject, e.g., the topically delivered
pharmaceutical composition has a
pharmacodynamic effect at or proximate to the particular part of the body
(e.g. skin) where the
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pharmaceutical composition is delivered. In some other embodiments, topical
delivery of a
pharmaceutical composition as discussed herein is used only to refer to the
delivery mode (locally to,
e.g., a specific surface area), whereas the pharmaceutical composition can
have either a local or
systemic pharmacodynamic effect. For instance, the pharmaceutical composition
can either stay local at
or proximate to the administration site, or can enter a circulation system
(e.g., blood or lymphoid system)
of the subject body, through which the pharmaceutical composition can be
transported to remote parts of
the body that are typically not reachable by the pharmaceutical composition
via routes other than the
circulation systems.
As used herein, the term "total ribonucleotide molecules" means the total
amount of any
ribonucleotide molecules, including linear polyribonucleotide molecules,
circular polyribonucleotide
molecules, monomeric ribonucleotides, other polyribonucleotide molecules,
fragments thereof, and
modified variations thereof, as measured by total mass of the ribonucleotide
molecules
As used herein, the term "translation efficiency" is a rate or amount of
protein or peptide
production from a ribonucleotide transcript. In some embodiments, translation
efficiency can be
expressed as amount of protein or peptide produced per given amount of
transcript that codes for the
protein or peptide, e.g., in a given period of time, e.g., in a given
translation system, e.g., an in vitro
translation system like rabbit reticulocyte lysate, or an in vivo translation
system like a eukaryotic cell or a
prokaryotic cell.
As used herein, the term "translation initiation sequence" is a nucleic acid
sequence that initiates
translation of an expression sequence in the circular or linear
polyribonucleotide.
Brief Description of the Drawings
FIGs. 1A-1B are schematics of exemplary circular RNAs that include two
expression sequences,
where each expression sequence encodes either an immunogen or an adjuvant.
FIG. 1A depicts a
circular polyribonucleotide that includes two open reading frames (ORFs), each
ORF encoding an
expression sequence, where each ORF is operably linked to an !RES. FIG. 1B
depicts a circular
polyribonucleotide that includes two expression sequences separated by a 2A
sequence, all operably
linked to an !RES.
FIG. 2 shows a schematic of a circular RNA that includes an ORF that encodes
an immunogen
and a polynucleotide adjuvant sequence (e.g., a nucleotide sequence that
stimulates the innate immune
system).
FIG. 3 shows a schematic of a plurality of polyribonucleotides, where each
polynucleotide
includes an ORF that encodes either an immunogen or an adjuvant.
FIG. 4 shows an RBD immunogen encoded by a circular RNA was detected in BJ
Fibroblasts and
HeLa cells and was not detected in BJ Fibroblasts and HeLa cells with the
vehicle control.
FIG. 5 shows that sustainable anti-RBD antibody response was attained
following administration
of a circular RNA encoding a SARS-CoV-2 RBD immunogen, formulated with a
cationic polymer (e.g.,
protamine), in a mouse model.
FIG. 6 shows that an anti-Spike response was attained following administration
of a circular RNA
encoding a SARS-CoV-2 RBD antigen, formulated with a cationic polymer (e.g.,
protamine), in a mouse
model.
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FIG. 7 shows anti-RBD IgG2a and IgG1 isotype levels that were obtained after
administration of a
circular RNA encoding a SARS-CoV-2 RBD immunogen, formulated with a cationic
polymer (e.g.
protamine), in a mouse model.
FIG. 8 shows protein expression from circular RNA in vivo for prolonged
periods of time after
intramuscular injection of circular RNA preparations (Trans-IT formulated,
protamine formulated,
unformulated), protamine vehicle only, and uninjected control mice.
FIG. 9 shows protein expression from circular RNA in vivo for prolonged
periods of time after
simultaneous intramuscular delivery of AddavaxTM adjuvant with (i)
unformulated circular RNA
preparations (left graph), (ii) circular RNA formulated with TransIT (middle
graph), and (iii) circular RNA
formulated with protamine (right graph). In each case, AddavaxTM adjuvant was
delivered as an individual
injection at 0 and 24 h.
FIG. 10 shows protein expression from circular RNA in vivo for prolonged
periods of time after
intradermal delivery of (i) circular RNA formulated with protamine, (ii)
circular RNA formulated with
protamine, with an injection of AddavaxTM adjuvant at 24 hours, (iii)
protamine vehicle only, and (iv) an
uninjected control mice.
FIG. 11 shows the binding of probes to circular and linear RNA and subsequent
degradation of
the RNA by RNase H. Circular RNA is detected as a single cleaved linear band
compared to linear and
concatemeric RNA, which is detected as multiple bands. Degradation was
detected by running samples
on a denaturing polyacrylamide gel and comparing degradation bands with or
without addition of RNase
H.
FIG. 12 shows linear RNA content quantified on a denaturing polyacrylamide gel
by comparing
linear RNA band intensity to a linear RNA standard.
FIG. 13 shows the quantification of RNA extracted from different bands of a
denaturing
polyacrylamide gel.
FIG. 14 shows Gaussia Luciferase activity in cells at 6, 24, 48, 72, 96, and
120 hours post-
transfection in experiments using circular RNA of 84% purity, circular RNA of
71% purity, and vehicle
only.
FIG. 15 shows cells transfected with the gel purified circular RNA preparation
only showed
minimal expression of innate immune genes such as RIG-I, MDA-5, OAS, and IFN-
B compared to cells
transfected with both the combined circular RNA and linear counterpart RNA,
which exhibited
upregulation of these innate immune genes in a dose dependent manner.
FIG. 16 is an image showing a protein blot of expression products from
circular RNA or linear
RNA with a stagger element.
FIG. 17 shows generation of exemplary circular RNA by self-splicing.
FIG. 18 illustrates an example in which both exemplary linear and circular RNA
were delivered
topically to the ear skin of mice and their RNA levels in the ear tissue were
examined over the 6 hours to
3 days after delivery period.
FIGs. 19A and 19B are plots summarizing qPCR results from ear punches of mice
6 hours, 1
day, 3 days, or 12 days after topical delivery of linear or circular RNAs with
the aid of Boost (ethanol).
FIG. 19A shows results from qPCR assays using primers that for detection of
both the linear and circular
RNAs. FIG. 19B shows results from qPCR assays using primers for detection of
the circular RNA, but
not the linear RNA.
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FIG. 20 shows fluorescent images (B/VV) from topical administration of circRNA-
Cy5 results in
RNA delivery to tissue.
FIG. 21 shows quantification of fluorescent images from topical administration
of circRNA-Cy5
results in RNA delivery to tissue.
FIG. 22 shows topical administration of circular RNA results in RNA delivery
to tissue at day 1
and day 4 after administration when tissue is wiped with an ethanol wipe prior
to application.
FIG. 23 shows topical administration of circular RNA results in RNA delivery
to tissue at day 1
and day 4 after administration when the tissue is wiped with an isopropyl
alcohol wipe prior to application.
FIG. 24 shows topical administration of circRNA results in RNA delivery to
tissue when circRNA is
administered with 10% ethanol.
FIG. 25 is an image showing a protein blot of expression products from
circular RNA or linear
RNA.
FIG. 26 shows experimental data demonstrating increased persistence of Gaussia
luciferase
expression in mice following redosing with a circular polyribonucleotide
("Endless") as compared to a
linear polyribonucleotide counterpart ("Linear").
FIG. 27 shows experimental data demonstrating increased persistence of Gaussia
luciferase
expression in mice following staggered dosing with a circular
polyribonucleotide ("Endless 3 doses") as
compared to staggered dosing a linear polyribonucleotide counterpart ("Linear
3 doses"), or a single dose
with the circular polyribonucleotide ("Endless"), or a single dose with a
linear polyribonucleotide
counterpart ("Linear").
FIG. 28 shows experimental data demonstrating increased persistence of Gaussia
luciferase
expression in mice following a single dose of a circular polyribonucleotide
("Endless RNA") as compared
to a single dose of a linear polyribonucleotide counterpart ("Linear RNA"),
staggered dosing with a linear
polyribonucleotide counterpart ("3 doses Linear RNA") as compared to a single
dose ("Linear RNA"), or
staggered dosing with a circular polyribonucleotide ("3 doses Endless RNA") as
compared a single dose
("Endless RNA").
FIG. 29 shows circular polyribonucleotide administered intramuscularly,
without a carrier,
expressed protein in vivo for prolonged periods of time, with levels of
protein activity in the plasma at
multiple days post injection.
FIG. 30 shows circular polyribonucleotide administered intravenously,
expressed protein in vivo
for prolonged periods of time, with levels of protein activity in the plasma
at multiple days post injection
and could be re-dosed at least 5 times.
FIG. 31A shows multi-immunogen expression from a circular polyribonucleotide.
RBD
immunogen expression was detected from circular RNAs encoding a SARSs-CoV-2
RBD immunogen
and a GLuc polypeptide.
FIG. 31B shows multi-immunogen expression from a circular polyribonucleotide.
GLuc activity
was detected from circular RNAs encoding a SARSs-CoV-2 RBD immunogen and a
GLuc polypeptide.
FIG. 32A shows immunogenicity of multiple immunogens from circular RNAs in
mouse model.
Mice were vaccinated with a first circular RNA encoding a SARS-CoV-2 RBD
immunogen and a second
circular RNA encoding a GLuc polypeptide. Anti-RBD antibodies were obtained at
17 days after injection.
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FIG. 32B shows immunogenicity of multiple immunogens from circular RNAs in
mouse model.
Mice were vaccinated with a first circular RNA encoding a SARS-CoV-2 RBD
immunogen and a second
circular RNA encoding a GLuc polypeptide. GLuc activity was detected at 2 days
after injection.
FIG. 33A shows immunogenicity of multiple immunogens from circular RNAs in
mouse model.
.. Mice were vaccinated with a first circular RNA encoding a SARS-CoV-2 RBD
immunogen and a second
circular RNA encoding Influenza hemagglutinin (HA) immunogen. Anti-RBD
antibodies were obtained at
17 days after injection.
FIG. 33B shows immunogenicity of multiple immunogens from circular RNAs in
mouse model.
Mice were vaccinated with a first circular RNA encoding a SARS-CoV-2 RBD
immunogen and a second
circular RNA encoding Influenza hemagglutinin (HA) immunogen. Anti-HA
antibodies were obtained at
17 days after injection.
FIG. 34A shows immunogenicity of multiple immunogens from circular RNAs in
mouse model.
Mice were vaccinated with a first circular RNA encoding a SARS-CoV-2 Spike
immunogen and a second
circular RNA encoding Influenza hemagglutinin (HA) immunogen. Anti-RBD (domain
of Spike) antibodies
were obtained at 17 days after injection.
FIG. 34B shows immunogenicity of multiple immunogens from circular RNAs in
mouse model.
Mice were vaccinated with a first circular RNA encoding a SARS-CoV-2 Spike
immunogen and a second
circular RNA encoding Influenza hemagglutinin (HA) immunogen. Anti-HA
antibodies were obtained at
17 days after injection.
FIG. 35 shows an anti-HA antibody response in mice administered circular RNA
encoding
multiple immunogens. Mice were administered a circular RNA encoding: a SARS-
CoV-2 RBD
immunogen, a SARS-CoV-2 Spike immunogen, an Influenza HA immunogen, a SARS-CoV-
2 RBD
immunogen and an Influenza HA immunogen, a SARS-CoV-2 RBD immunogen and a GLuc
polypeptide,
or a SARS-CoV-2 RBD immunogen and a SARS-CoV-2 Spike immunogen. A
hemagglutination inhibition
assay (HAI) was used to measure anti-Influenza HA antibodies. FIG. 24 shows
HAI titer in samples that
were administered circular RNA preparations encoding the Influenza HA
immunogen when it was
administered alone or when administered in combination with SARS-CoV-2
immunogens, e.g., RBD or
Spike.
FIG. 36 shows Gaussia luciferase (Gluc) activity in mice at 2 days post-prime
with: (i) a circular
RNA encoding a Gluc polypeptide, or (ii) a combination of a first circular RNA
encoding a Gluc
polypeptide and a second circular RNA encoding a SARS-CoV-2 RBD immunogen, or
(iii) a circular RNA
encoding both a Gluc polypeptide and a SARS-CoV-2 RBD immunogen.
FIG. 37A shows anti-RBD antibody response in mice at 14 days post-prime with:
(i) a circular
RNA encoding a SARS-CoV-2 RBD immunogen, or (ii) a combination of a first
circular RNA encoding a
GLuc polypeptide and a second circular RNA encoding a SARS-CoV-2 RBD
immunogen, or (iii) a circular
RNA encoding both a Gluc polypeptide and a SARS-CoV-2 RBD immunogen.
FIG. 37B shows anti-RBD neutralizing antibodies, as evaluated by a Plaque
Reduction
Neutralization Test (PRNT), in mice administered: (i) a circular RNA encoding
a SARS-CoV-2 RBD
immunogen, or (ii) a combination of a first circular RNA encoding a GLuc
polypeptide and a second
circular RNA encoding a SARS-CoV-2 RBD immunogen, or (iii) a circular RNA
encoding both a Gluc
polypeptide and a SARS-CoV-2 RBD immunogen.
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FIG. 38 shows a circular RNA encoding a Gluc polypeptide and a SARS-CoV-2 RBD
immunogen
primes RBD-specific T cell responses as determined by ELISpot assay.
FIG. 39 shows IL-12, measured using an IL-12 specific ELISA, was expressed
from circular RNA
in mammalian cells. A circular RNA encoding a SARS-CoV-2 RBD immunogen was
included as a
negative control.
FIG. 40A shows IL-12 expression was detected in serum at 2 days after
injection with a circular
RNA preparation including a first circular RNA encoding IL-12 and a second
circular RNA encoding a
SARS-CoV-2 RBD immunogen, in a mouse model. Injection with PBS or with a
preparation including
only the circular RNA encoding a SARS-CoV-2 RBD immunogen were included as
controls.
FIG. 40B shows an increase in serum IFN-y (directly downstream of IL12
signaling) was detected
in serum at 2 days after injection with a circular RNA preparation including a
first circular RNA encoding
IL-12 and a second circular RNA encoding a SARS-CoV-2 RBD immunogen, in a
mouse model. Injection
with PBS or with a preparation including only the circular RNA encoding a SARS-
CoV-2 RBD immunogen
were included as controls.
FIG. 41A shows that administration of a circular RNA preparation including a
first circular RNA
encoding IL-12 and a second circular RNA encoding a SARS-CoV-2 RBD immunogen
increased the
number of SARS-CoV-2 RBD specific CD4 T cells. Administration of PBS or a
preparation including only
the circular RNA encoding a SARS-CoV-2 RBD immunogen were included as
controls. Asterisks denotes
statistical significance as determined by a two-way RM ANOVA protected Tukey's
post hoc test.
FIG. 41B shows that administration of a circular RNA preparation including a
first circular RNA
encoding IL-12 and a second circular RNA encoding a SARS-CoV-2 RBD immunogen
produced no
change in the number of RBD specific CD8 T cells. Administration of PBS or a
preparation including only
the circular RNA encoding a SARS-CoV-2 RBD immunogen were included as
controls.
FIG. 41C shows that administration of a circular RNA preparation including a
first circular RNA
encoding IL-12 and a second circular RNA encoding SARS-CoV-2 RBD immunogen
increased the
amount of IFN-y production by CD4 T cells. Administration of PBS or a
preparation including only the
circular RNA encoding a SARS-CoV-2 RBD immunogen were included as controls.
Asterisks denotes
statistical significance as determined by unpaired t-test.
FIG. 410 shows that administration of a circular RNA preparation including a
first circular RNA
encoding IL-12 and a second circular RNA encoding a SARS-CoV-2 RBD immunogen
increased the
amount of IFN-y production by CD8 T cells. Administration of PBS or a
preparation including only the
circular RNA encoding a SARS-CoV-2 RBD immunogen were included as controls.
Asterisks denotes
statistical significance as determined by unpaired t-test.
FIG. 42 shows that a circular RNA expressing Ovalbumin (OVA) combined with
circular RNA
expressing IL12 induces a stronger CD8 T cell response when compared to the
control (circular RNA
expressing OVA only). This demonstrates that circular RNA expressing IL12 acts
as an adjuvant.
Detailed Description
This disclosure provides compositions and pharmaceutical preparations of
circular or linear
polyribonucleotides encoding one or more polypeptide immunogens and uses
thereof. In particular, the
disclosure provides circular or linear polyribonucleotides encoding multiple
immunogens, circular or linear
polyribonucleotides encoding at least one immunogen and further encoding at
least one adjuvant, and
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immunogenic compositions including multiple circular or linear
polyribonucleotides. This disclosure
further features pharmaceutical compositions and preparations including one or
more circular or linear
polyribonucleotides encoding one or more immunogens. Compositions and
pharmaceutical preparations
of circular or linear polyribonucleotides described herein may induce an
immune response in a subject
upon administration. Compositions and pharmaceutical preparations of circular
or linear
polyribonucleotides described herein may be used to treat or prevent a
disease, disorder, or condition in a
subject.
Polyribonucleotides
The polyribonucleotide includes the elements as described below as well as the
in addition to one
or more immunogens as described herein. In particular embodiments, the
polyribonucleotide is a circular
polyribonucleotide.
In some embodiments, the polyribonucleotide (e.g., the circular
polyribonucleotide) is at least
about 20 nucleotides, at least about 30 nucleotides, at least about 40
nucleotides, at least about 50
nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at
least about 200 nucleotides,
at least about 300 nucleotides, at least about 400 nucleotides, at least about
500 nucleotides, at least
about 1,000 nucleotides, at least about 2,000 nucleotides, at least about
5,000 nucleotides, at least about
6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000
nucleotides, at least about 9,000
nucleotides, at least about 10,000 nucleotides, at least about 12,000
nucleotides, at least about 14,000
nucleotides, at least about 15,000 nucleotides, at least about 16,000
nucleotides, at least about 17,000
nucleotides, at least about 18,000 nucleotides, at least about 19,000
nucleotides, or at least about 20,000
nucleotides.
In some embodiments, the polyribonucleotide (e.g. the circular
polyribonucleotide) may be of a
sufficient size to accommodate a binding site for a ribosome. In some
embodiments, the maximum size
of a circular polyribonucleotide can be as large as is within the technical
constraints of producing a
circular polyribonucleotide, and/or using the circular polyribonucleotide.
Without wishing to be bound by
any particular theory, it is possible that multiple segments of RNA may be
produced from DNA and their 5'
and 3 free ends annealed to produce a "string" of RNA, which ultimately may be
circularized when only
one 5' and one 3' free end remains. In some embodiments, the maximum size of a
circular
polyribonucleotide may be limited by the ability of packaging and delivering
the RNA to a target. In some
embodiments, the size of a circular polyribonucleotide is a length sufficient
to encode useful polypeptides,
such as immunogens or an epitopes thereof of the disclosure, and thus, lengths
of at least 20,000
nucleotides, at least 15,000 nucleotides, at least 10,000 nucleotides, at
least 7,500 nucleotides, or at
least 5,000 nucleotides, at least 4,000 nucleotides, at least 3,000
nucleotides, at least 2,000 nucleotides,
at least 1,000 nucleotides, at least 500 nucleotides, at least 400
nucleotides, at least 300 nucleotides, at
least 200 nucleotides, at least 100 nucleotides, or at least 70 nucleotides,
may be useful. In some
embodiments, the maximum size of the circular polyribonucleotide is a length
sufficient to encode one or
more immunogens (e.g., two or more, three or more, four or more, and five or
more). In some
embodiments, the maximum size of the circular polyribonucleotide is a length
sufficient to encode
between two and five (e.g., three, four, and five) immunogens. In some
embodiments, the maximum size
of the circular polyribonucleotide is a length sufficient to encode an
immunogen and an adjuvant. In some
embodiments, the maximum size of the circular polyribonucleotide is a length
sufficient to encode one or
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more immunogens (e.g., two or more, three or more, and four or more) and one
or more adjuvants (e.g.,
two or more, three or more, and four or more).
Circular polyribonucleotide elements
In some embodiments, the circular polyribonucleotide includes one or more of
the elements as
described herein in addition to including a sequence encoding an immunogen. In
some embodiments,
the circular polyribonucleotide lacks a poly-A sequence, lacks a free 3' end,
lacks an RNA polymerase
recognition motif, or any combination thereof. In some embodiments, the
circular polyribonucleotide
includes any feature or any combination of features as disclosed in
International Patent Publication No.
W02019/118919, which is hereby incorporated by reference in its entirety.
lmmunogens
The circular or linear polyribonucleotides described herein includes at least
one sequence
encoding an immunogen and optionally further includes at least one sequence
encoding an adjuvant. An
immunogen includes one or more epitopes that is recognized, targeted, or bound
by a given antibody or T
cell receptor. An epitope can be a linear epitope, for example, a contiguous
sequence of nucleic acids or
amino acids. An epitope can be a conformational epitope, for example, an
epitope that contains amino
acids that form an epitope in the folded conformation of the protein. A
conformational epitope can contain
non-contiguous amino acids from a primary amino acid sequence. As another
example, a conformational
epitope includes nucleic acids that form an epitope in the folded conformation
of an immunogenic
sequence based on its secondary structure or tertiary structure.
In some embodiments, an immunogen includes all or a part of a protein, a
peptide, a
glycoprotein, a lipoprotein, a phosphoprotein, a ribonucleoprotein, a
carbohydrate (e.g., a
polysaccharide), a lipid (e.g., a phospholipid or triglyceride), or a nucleic
acid (e.g., DNA, RNA).
In other embodiments, an immunogen includes a protein immunogen or epitope
(e.g., a peptide
immunogen or peptide epitope from a protein, glycoprotein, lipoprotein,
phosphoprotein, or
ribonucleoprotein). An immunogen can include an amino acid, a sugar, a lipid,
a phosphoryl, or a sulfonyl
group, or a combination thereof.
In a particular embodiment, the immunogen is a polypeptide immunogen.
A polypeptide immunogen may include a post-translational modification, for
example,
glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation,
acetylation, amidation,
hydroxylation, sulfation, or lipidation.
In some embodiments, an immunogen includes an epitope including at least 4, at
least 5, at least
6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12,
at least 13, at least 14, at least 15,
at least 16, at least 17, at least 18, at least. 19, at least 20, at least 21,
at least 22, at least 23, at least 24,
at least 25, at least 26, at least 27, at least 28, at least 29, or at least
30 amino acids, or more. In some
embodiments, an epitope includes or contains at most 4, at most 5, at most 6,
at most 7, at most 8, at
most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most
15, at most 16, at most 17, at
most 18, at most. 19, at most 20, at most 21, at most 22, at most 23, at most
24, at most 25, at most 26,
at most 27, at most 28, at most 29, or at most 30 amino acids, or less. In
some embodiments, an epitope
includes or contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, or 30 amino acids. In some embodiments, an epitope contains 5
amino acids. In some
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embodiments, an epitope contains 6 amino acids. In some embodiments, an
epitope contains 7 amino
acids. In some embodiments, an epitope contains 8 amino acids. In some
embodiments, an epitope can
be about 8 to about 11 amino acids. In some embodiments, an epitope can be
about 9 to about 22 amino
acids.
The immunogens may include immunogens recognized by B cells, immunogens
recognized by T
cells, or a combination thereof. In some embodiments, the immunogens include
immunogens recognized
by B cells. In some embodiments, the immunogens are immunogens recognized by B
cells. In some
embodiments, the immunogens include immunogens recognized by T cells. In some
embodiments, the
immunogens are immunogens recognized by T cells.
The epitopes may include epitopes recognized by B cells, epitopes recognized
by T cells, or a
combination thereof. In some embodiments, the epitopes include epitopes
recognized by B cells. In
some embodiments, the epitopes are epitopes recognized by B cells. In some
embodiments, the
epitopes include epitopes recognized by T cells. In some embodiments, the
epitopes are epitopes
recognized by T cells.
Techniques for identifying immunogens and epitopes in silico have been
disclosed, for example,
in Sanchez-Trincado JL, et al. (Fundamentals and methods for T-and B-cell
epitope prediction, J.
Immunol. Res., 2017:2680160. doi: 10.1155/2017/2680160 (2017)); Grifoni, A, et
al. (A Sequence
Homology and Bioinformatic Approach Can Predict Candidate Targets for Immune
Responses to SARS-
CoV-2, Cell Host Microbe, 27(4):671-680 (2020)); Russi RC et al. (In silico
prediction of epitopes
recognized by T cells and B cells in PmpD: First step towards to the design of
a Chlamydia trachomatis
vaccine, Biomedical J., 41(2)1 09-117 (2018)); Baruah V, et al.
(Immunoinformatics-aided identification of
T cell and B cell epitopes in the surface glycoprotein of 2019-nCoV, J. Med.
Virol., 92(5), doi:
10.1002/jmv.25698 (2020)); each of which is incorporated herein by reference
in its entirety.
In some embodiments, an immunogen includes a polynucleotide. In some
embodiments, an
immunogen is a polynucleotide. In some embodiments, an immunogen includes an
RNA. In some
embodiments, an immunogen is an RNA. In some embodiments, an immunogen
includes a DNA. In
some embodiments, an immunogen is a DNA. In some embodiments, the
polynucleotide is encoded in
the circular or linear polyribonucleotide.
A circular or linear polyribonucleotide of the disclosure includes or encodes
any number of
immunogens. In a particular embodiment, a circular or linear
polyribonucleotide includes or encodes at
least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least
7, at least 8, at least 9, at least 10, at
least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at
least 60, at least 70, at least 80, at
least 90, at least 100, at least 120, at least 140, at least 160, at least
180, at least 200, at least 250, at
least 300, at least 350, at least 400, at least 450, at least 500, or more of
immunogens.
In some embodiments, a circular or linear polyribonucleotide includes or
encodes, for example, at
most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at
most 8, at most 9, at most 10,
at most 15, at most 20, at most 25, at most 30, at most 40, at most 50, at
most 60, at most 70, at most 80,
at most 90, at most 100, at most 120, at most 140, at most 160, at most 180,
at most 200, at most 250, at
most 300, at most 350, at most 400, at most 450, at most 500, or less
immunogens.
In some embodiments, a circular or linear polyribonucleotide includes or
encodes about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200, 250, 300, 350, 400,
450, or 500 of immunogens.
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In some embodiments, the circular or linear polyribonucleotide encodes a
plurality of
immunogens. In some embodiments, a circular or linear polyribonucleotide
includes or encodes between
1 and 100 immunogens. In some embodiments, a circular or linear
polyribonucleotide includes or
encodes between 1 and 50 immunogens. In some embodiments, a circular or linear
polyribonucleotide
includes or encodes between 1 and 10 immunogens; for example, a circular or
linear polyribonucleotide
encodes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 immunogens. In some embodiments, a
circular or linear
polyribonucleotide includes or encodes 2 immunogens. In some embodiments, a
circular or linear
polyribonucleotide includes or encodes 3 immunogens. In some embodiments, a
circular or linear
polyribonucleotide includes or encodes 4 immunogens. In some embodiments, a
circular or linear
polyribonucleotide includes or encodes 5 immunogens.
In some embodiments, the plurality of immunogens each identify the same
target. Otherwise put,
a single target may include each of the plurality of immunogens, each of the
plurality of immunogens may
be derived from the same target, and/or each of the plurality of immunogens
may share a high degree of
similarity with a portion or the whole of the target. For example, a target
may be a cell and each of the
immunogens may correspond to a protein of that cell. For example, the target
may a particular cancer
cell and each of the immunogens may correspond to a tumor antigen associate
with that cancer.
Accordingly, in some embodiments, each of the plurality of immunogens are
derived from different
proteins from the same target.
In some embodiments, the plurality of immunogens are derived from different
targets. In some
embodiments, the plurality of immunogens may be derived various capsid
proteins of a given virus. For
example, the one immunogen may be derived from Orthopoxvirus, another
immunogen may be derived
Hepadnavirus, and a third immunogen may be derived Flavivirus. For example, a
polyribonucleotide may
encode multiple immunogens, where each immunogen is derived from yellow fever
virus, Chikungunya
virus, Zika, Hepatitis A, or Hepatitis B. A polyribonucleotide may encode an
immunogen from each of
yellow fever virus, Chikungunya virus, Zika, Hepatitis A, and Hepatitis B. A
polyribonucleotide may
encode multiple immunogens, where each immunogen is derived from Japanese
encephalitis,
Chikungunya virus, Zika, Hepatitis A, or Hepatitis B. A polyribonucleotide may
encode an immunogen
from each of Japanese encephalitis, Chikungunya virus, Zika, Hepatitis A, and
Hepatitis B. A
polyribonucleotide may encode multiple immunogens, where each immunogen is
derived from SARS-
CoV2, a poxvirus, respiratory syncytial virus, or human papilloma virus. A
polyribonucleotide may encode
an immunogen from each of SARS-CoV2, a poxvirus, respiratory syncytial virus,
and human papilloma
virus. A polyribonucleotide may encode multiple immunogens, where each
immunogen is derived from a
herpes virus (CMV, EBV, or VZV). A polyribonucleotide may encode an immunogen
from each of the
following herpes viruses: CMV, EBV, or VZV. A polyribonucleotide may encode
multiple immunogens,
where each immunogen is derived Singles or West Nile Virus. A
polyribonucleotide may encode an
immunogen from each of Shingles and West Nile Virus.
In some embodiments, the plurality of immunogens have at least 90%, 91%, 92%,
93%, 94%,
95%, 96%, 97%, 98%, or 99% sequence identity. In some embodiments, the
plurality of immunogens
also have less than 100% sequence identity. This may be indicative of
immunogens related to one
another by genetic drift, as such, a single circular or linear
polyribonucleotide composition or
immunogenic composition may be able to induce an immune response against a
target that exists in
various mutational states in a population or may induce an immune response
against multiple targets
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having the same immunogen where the immunogen is related by genetic drift. For
example, the
immunogens may be related to one another by genetic drift of a target virus.
In some embodiments, the
plurality of immunogens may be derived from receptor-binding domains (RBD)
from unique but related
viruses.
In some embodiments, a circular or linear polyribonucleotide encodes variants
of an immunogen.
Variants can be naturally-occurring variants (for example, variants identified
in sequence data from
different viral genera, species, isolates, or quasispecies), or can be
derivative sequences as disclosed
herein that have been generated in silico (for example, immunogens or epitopes
with one or more amino
acid insertions, deletions, substitutions, or a combination thereof compared
to a wild-type immunogen or
epitope).
An immunogen is from, for example, a virus, such as a viral surface protein, a
viral membrane
protein, a viral envelope protein, a viral capsid protein, a viral
nucleocapsid protein, a viral spike protein, a
viral entry protein, a viral membrane fusion protein, a viral structural
protein, a viral non-structural protein,
a viral regulatory protein, a viral accessory protein, a secreted viral
protein, a viral polymerase protein, a
viral DNA polymerase, a viral RNA polymerase, a viral protease, a viral
glycoprotein, a viral fusogen, a
viral helical capsid protein, a viral icosahedral capsid protein, a viral
matrix protein, a viral replicase, a
viral transcription factor, or a viral enzyme.
In some embodiments, the immunogen is from one of these viruses:
Orthomyxovirus: Useful immunogens can be from an influenza A, B or C virus,
such as the
hemagglutinin, neuraminidase, or matrix M2 proteins. Where the immunogen is an
influenza A virus
hemagglutinin it may be from any subtype e.g. HI, H2, H3, H4, H5, H6, H7, H8,
H9, H10, HI I, H12, H13,
H14, H15, or H16.
Paramyxoviridae viruses: Viral immunogens include, but are not limited to,
those derived from
Pneumoviruses (e.g. respiratory syncytial virus (RSV)), Rubulaviruses (e.g.
mumps virus),
Paramyxoviruses (e.g. parainfluenza virus), Metapneumoviruses and
Morbilliviruses (e.g. measles virus),
Henipaviruses (e.g. Nipah virus).
Poxviridae: Viral immunogens include, but are not limited to, those derived
from Orthopoxvirus
such as Variola vera, including but not limited to, Variola major and Variola
minor.
Picornavirus: Viral immunogens include, but are not limited to, those derived
from Picornaviruses,
such as Enteroviruses, Rhinoviruses, Heparnavirus, Cardioviruses and
Aphthoviruses. In one
embodiment, the enterovirus is a poliovirus e.g. a type 1, type 2 and/or type
3 poliovirus. In another
embodiment, the enterovirus is an EV71 enterovirus. In another embodiment, the
enterovirus is a
coxsackie A or B virus.
Bunyavirus: Viral immunogens include, but are not limited to, those derived
from an
Orthobunyavirus, such as California encephalitis virus, a Phlebovirus, such as
Rift Valley Fever virus, or a
Nairovirus, such as Crimean-Congo hemorrhagic fever virus.
Heparnavirus: Viral immunogens include, but are not limited to, those derived
from a
Heparnavirus, such as hepatitis A virus (HAV).
Filovirus: Viral immunogens include, but are not limited to, those derived
from a filovirus, such as
an Ebola virus (including a Zaire, Ivory Coast, Reston, or Sudan ebolavirus)
or a Marburg virus.
Togavirus: Viral immunogens include, but are not limited to, those derived
from a Togavirus, such
as a Rubivirus, an Alphavirus, or an Arterivirus. This includes rubella virus.
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Flavivirus: Viral immunogens include, but are not limited to, those derived
from a Flavivirus, such
as Tick-borne encephalitis (TBE) virus, Dengue (types 1, 2, 3 or 4) virus,
Yellow Fever virus, Japanese
encephalitis virus, Kyasanur Forest Virus, West Nile encephalitis virus, St.
Louis encephalitis virus,
Russian spring-summer encephalitis virus, Powassan encephalitis virus, Zika
virus.
Pestivirus: Viral immunogens include, but are not limited to, those derived
from a Pestivirus, such
as Bovine viral diarrhea (BVDV), Classical swine fever (CSFV) or Border
disease (BDV).
Hepadnavirus: Viral immunogens include, but are not limited to, those derived
from a
Hepadnavirus, such as Hepatitis B virus. The hepatitis B virus immunogen may
be a hepatitis B virus
surface immunogen (HBsAg).
Other hepatitis viruses: Viral immunogens include, but are not limited to,
those derived from a
hepatitis C virus, delta hepatitis virus, hepatitis E virus, or hepatitis G
virus.
Rhabdovirus: Viral immunogens include, but are not limited to, those derived
from a Rhabdovirus,
such as a Lyssavirus (e.g. a Rabies virus) and Vesiculovirus (VSV).
Caliciviridae: Viral immunogens include, but are not limited to, those derived
from Calciviridae,
such as Norwalk virus (Norovirus), and Norwalk-like Viruses, such as Hawaii
Virus and Snow Mountain
Virus.
Retrovirus: Viral immunogens include, but are not limited to, those derived
from an Oncovirus, a
Lentivirus (e.g. HIV-1 or HIV-2) or a Spumavirus.
Reovirus: Viral immunogens include, but are not limited to, those derived from
an Orthoreovirus,
a Rotavirus, an Orbivirus, or a Coltivirus.
Parvovirus: Viral immunogens include, but are not limited to, those derived
from Parvovirus B19.
Bocavirus: Viral immunogens include, but are not limited to, those derived
from bocavirus.
Herpesvirus: Viral immunogens include, but are not limited to, those derived
from a human
herpesvirus, such as, by way of example only, Herpes Simplex Viruses (HSV)
(e.g. HSV types 1 and 2),
Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV),
Human Herpesvirus 6
(HHV6), Human Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8).
Papovaviruses: Viral immunogens include, but are not limited to, those derived
from
Papillomaviruses and Polyomaviruses. The (human) papillomavirus may be of
serotype 1, 2, 4, 5, 6, 8,
11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 or 65 e.g. from one
or more of serotypes 6, 11,
16 and/or 18.
Orthohantaviruses: Viral immunogens include, but are not limited to, those
derived from
hantavi ruses.
Arenavirus: Viral immunogens include, but are not limited to, those derived
from Guanarito virus,
Junin virus, Lassa virus, Lujo virus, Machupo virus, Sabia virus, or
Whitewater Arroyo virus.
Adenovirus: Viral immunogens include those derived from adenovirus serotype 36
(Ad-36).
Community acquired respiratory viruses: Viral immunogens include those derived
from
community acquired respiratory viruses.
Coronavirus: Viral immunogens include, but are not limited to, those derived
from a SARS
coronavirus (e.g., SARS-CoV-1 and SARS-CoV-2), MERS coronavirus, avian
infectious bronchitis (IBV),
Mouse hepatitis virus (MHV), and Porcine transmissible gastroenteritis virus
(TGEV). The coronavirus
immunogen may be a spike polypeptide or a receptor binding domain (RBD) of a
spike protein. The
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coronavirus immunogen may also be an envelope polypeptide, a membrane
polypeptide or a
nucleocapsid polypeptide.
In some embodiments, the immunogen is from a virus which infects fish. In some
embodiments,
the immunogen elicits an immune response against a virus which infects fish.
For example, the virus
which infects fish is selected from infectious salmon anemia virus (ISAV),
salmon pancreatic disease
virus (SPDV), infectious pancreatic necrosis virus (IPNV), channel catfish
virus (CCV), fish lymphocystis
disease virus (FLDV), infectious hematopoietic necrosis virus (IHNV), koi
herpesvirus, salmon picorna-
like virus (also known as picorna-like virus of atlantic salmon), landlocked
salmon virus (LSV), atlantic
salmon rotavirus (ASR), trout strawberry disease virus (TSD), coho salmon
tumor virus (CSTV), or viral
hemorrhagic septicemia virus (VHSV).
In some embodiments, an immunogen is from a host subject cell. For example,
antibodies that
block viral entry can be generated by using an immunogen or epitope from a
component of a host cell
that a virus uses as an entry factor.
An immunogen is from, for example, a bacteria, such as a bacterial surface
protein, a bacterial
membrane protein, a bacterial envelope protein, a bacterial inner membrane
protein, a bacterial outer
membrane protein, a bacterial periplasmic protein, a bacterial entry protein,
a bacterial membrane fusion
protein, a bacterial structural protein, a bacterial non-structural protein, a
secreted bacterial protein, a
bacterial polymerase protein, a bacterial DNA polymerase, a bacterial RNA
polymerase, a bacterial
protease, a bacterial glycoprotein, bacterial transcription factor, a
bacterial enzyme, or a bacterial toxin.
In some embodiments, the immunogen elicits an immune response from one of
these bacteria:
Streptococcus agalactiae (also known as group B streptococcus or GBS));
Streptococcus pyogenes (also
known as group A Streptococcus (GAS)); Staphylococcus aureus; Methicillin-
resistant Staphylococcus
aureus (M RSA); Staphylococcus epidermis; Treponema paffidum; Francisella
tularensis; Rickettsia
species; Yersinia pestis; Neisseria meningitidis: Immunogens include, but are
not limited to, membrane
proteins such as adhesins, autotransporters, toxins, iron acquisition
proteins, and factor H binding protein;
Streptococcus pneumoniae; Moraxella catarrhalis; Bordetella pertussis:
Immunogens include, but are not
limited to, pertussis toxin or toxoid (PT), filamentous haemagglutinin (FHA),
pertactin, and agglutinogens
2 and 3; Clostridium tetani: the typical immunogen is tetanus toxoid;
Comynebacterium diphtheriae: the
typical immunogen is diphtheria toxoid; Haemophilus influenzae; Pseudomonas
aeruginosa; Chlamydia
trachomatis; Chlamydia pneumoniae; Helicobacter pylori; Escherichia coli
(Immunogens include, but are
not limited to, immunogens derived from enterotoxigenic E. coli (ETEC),
enteroaggregative E. coli
(EAggEC), diffusely adhering E. coli (DAEC), enteropathogenic E. coli (EPEC),
extraintestinal pathogenic
E. coli (ExPEC) and/or enterohemorrhagic E. coli (EHEC)). ExPEC strains
include uropathogenic E. coli
(UPEC) and meningitis/sepsis-associated E. coli (MNEC). Also included are
Bacillus anthracis;
Clostridium perfringens or Clostridium botulinums; Legionella pneumophila;
Coxiella bumetiid; Brucella
species, such as B. abortus, B. canis, B. melitensis, B. neotomae, B. ovis, B.
suis, and B. pinnipediae.
Francisella species, such as F. novicida, F. philomiragia, and F. tularensis;
Neisseria gonorrhoeae;
Haemophilus ducreyi; Enterococcus faecalis or Enterococcus faecium;
Staphylococcus saprophyticus;
Yersinia enterocolitica; Mycobacterium tuberculosis; Listeria monocytogenes;
Vibrio cholerae; Salmonella
typhi; Borrelia burgdorferi; Porphyromonas gingivalis; and Klebsiella species.
An immunogen is from, for example, fungus, such as a fungal surface protein, a
fungal
membrane protein, a fungal envelope protein, a fungal inner membrane protein,
a fungal outer membrane
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protein, a fungal periplasmic protein, a fungal entry protein, a fungal
membrane fusion protein, a fungal
structural protein, a fungal non-structural protein, a secreted fungal
protein, a fungal polymerase protein,
a fungal DNA polymerase, a fungal RNA polymerase, a fungal protease, a fungal
glycoprotein, fungal
transcription factor, a fungal enzyme, or a fungal toxin.
In some embodiments, the fungal immunogen is derived from Dermatophytes,
including:
Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum
distortum,
Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton
concentricum,
Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum,
Trichophyton megnini,
Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum,
Trichophyton
schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum
var. album, var. discoides,
var. ochraceum, Trichophyton violaceum, and/or Trichophyton faviforme; or from
Aspergillus fumigatus,
Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus
terreus, Aspergillus sydowi,
Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus,
Candida albicans, Candida
enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida
parapsilosis, Candida
stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida
pseudotropicalis, Candida
guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces
dermatidis, Cryptococcus
neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella
pneumoniae, Microsporidia,
Encephalitozoon spp., Septata intestinalis and Enterocytozoon bieneusi; the
less common are Brachiola
spp, Microsporidium spp., Nosema spp., Pleistophora spp., Trachipleistophora
spp., Vittaforma spp
Paracoccidioides brasiliensis, Pneumocystis carinii Pythiumn insidiosum,
Pityrosporum ovale,
Sacharomyces cerevisae, Saccharomyces boulardii, Saccharomyces pombe,
Scedosporium apiosperum,
Sporothrix schenckii, Trichosporon beige/h, Toxoplasma gondii, Peniciffium
mameffei, Malassezia spp.,
Fonsecaea spp., Wangiella spp., Sporothrix spp., Basidiobolus spp.,
Conidiobolus spp., Rhizopus spp,
Mucor spp, Absidia spp, Mortierella spp, Cunninghamella spp, Saksenaea spp.,
Altemaria spp, Curvularia
spp, Helminthosporium spp, Fusarium spp, Aspergillus spp, Peniciffium spp,
Monolinia spp, Rhizoctonia
spp, Paecilomyces spp, Pithomyces spp, and Cladosporium spp.
An immunogen is from, for example, a eukaryotic parasite surface protein,
eukaryotic parasite
membrane protein, a eukaryotic parasite envelope protein, a eukaryotic
parasite entry protein, a
eukaryotic parasite membrane fusion protein, a eukaryotic parasite structural
protein, a eukaryotic
parasite non-structural protein, a secreted eukaryotic parasite protein, a
eukaryotic parasite polymerase
protein, a eukaryotic parasite DNA polymerase, a eukaryotic parasite RNA
polymerase, a eukaryotic
parasite protease, a eukaryotic parasite glycoprotein, eukaryotic parasite
transcription factor, a eukaryotic
parasite enzyme, or a eukaryotic parasite toxin.
In some embodiments, the immunogen elicits an immune response against a
parasite from the
Plasmodium genus, such as P. falciparum, P. vivax, P. malariae, or P. ovale.
In some embodiments, the
immunogen elicits an immune response against a parasite from the Caligidae
family, particularly those
from the Lepeophtheirus and Caligus genera, e.g., sea lice such as
Lepeophtheirus salmonis or Caligus
rogercresseyi. In some embodiments, the immunogen elicits an immune response
against the parasite
Toxoplasma
In some embodiments, the immunogens are cancer immunogens (e.g., neoepitopes).
For
example, an immunogen is a neoantigen and/or neoepitope that is associated
with acute leukemia,
astrocytomas, biliary cancer (cholangiocarcinoma), bone cancer, breast cancer,
brain stem glioma,
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bronchioloalveolar cell lung cancer, cancer of the adrenal gland, cancer of
the anal region, cancer of the
bladder, cancer of the endocrine system, cancer of the esophagus, cancer of
the head or neck, cancer of
the kidney, cancer of the parathyroid gland, cancer of the penis, cancer of
the pleural/peritoneal
membranes, cancer of the salivary gland, cancer of the small intestine, cancer
of the thyroid gland,
cancer of the ureter, cancer of the urethra, carcinoma of the cervix,
carcinoma of the endometrium,
carcinoma of the fallopian tubes, carcinoma of the renal pelvis, carcinoma of
the vagina, carcinoma of the
vulva, cervical cancer, chronic leukemia, colon cancer, colorectal cancer,
cutaneous melanoma,
ependymoma , epidermoid tumors, Ewings sarcoma, gastric cancer, glioblastoma,
glioblastoma
multiforme, glioma, hematologic malignancies, hepatocellular (liver)
carcinoma, hepatoma, Hodgkin's
Disease, intraocular melanoma, Kaposi sarcoma, lung cancer, lymphomas,
medulloblastoma, melanoma,
meningioma, mesothelioma, multiple myeloma, muscle cancer, neoplasms of the
central nervous system
(CNS), neuronal cancer, small cell lung cancer, non-small cell lung cancer,
osteosarcoma, ovarian
cancer, pancreatic cancer, pediatric malignancies, pituitary adenoma, prostate
cancer, rectal cancer,
renal cell carcinoma, sarcoma of soft tissue, schwanoma, skin cancer, spinal
axis tumors, squamous cell
carcinomas, stomach cancer, synovial sarcoma, testicular cancer, uterine
cancer, or tumors and their
metastases, including refractory versions of any of the above cancers, or any
combination thereof.
In some embodiments, the immunogen is a tumor antigen selected from: (a)
cancer-testis
antigens such as NY-ESO-1, SSX2, SCP1 as well as RAGE, BAGE, GAGE and MAGE
family
polypeptides, for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4,
MAGE-5, MAGE-6,
and MAGE- 12 (which can be used, for example, to address melanoma, lung, head
and neck, NSCLC,
breast, gastrointestinal, and bladder tumors; (b) mutated antigens, for
example, p53 (associated with
various solid tumors, e.g., colorectal, lung, head and neck cancer), p21/Ras
(associated with, e.g.,
melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with,
e.g., melanoma), MUMI
(associated with, e.g., melanoma), caspase-8 (associated with, e.g., head and
neck cancer), CIA 0205
(associated with, e.g., bladder cancer), HLA-A2-R1701, beta catenin
(associated with, e.g., melanoma),
TCR (associated with, e.g., T-cell non-Hodgkins lymphoma), BCR-abl (associated
with, e.g., chronic
myelogenous leukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLR-
FUT; (c) over-
expressed antigens, for example, Galectin 4 (associated with, e.g., colorectal
cancer), Galectin 9
(associated with, e.g., Hodgkin's disease), proteinase 3 (associated with,
e.g., chronic myelogenous
leukemia), WT 1 (associated with, e.g., various leukemias), carbonic anhydrase
(associated with, e.g.,
renal cancer), aldolase A (associated with, e.g., lung cancer), PRAME
(associated with, e.g., melanoma),
HER-2/neu (associated with, e.g., breast, colon, lung and ovarian cancer),
mammaglobin, alpha-
fetoprotein (associated with, e.g., hepatoma), KSA (associated with, e.g.,
colorectal cancer), gastrin
(associated with, e.g., pancreatic and gastric cancer), telomerase catalytic
protein, MUC-1 (associated
with, e.g., breast and ovarian cancer), G-250 (associated with, e.g., renal
cell carcinoma), p53
(associated with, e.g. , breast, colon cancer), and carcino embryonic antigen
(associated with, e.g., breast
cancer, lung cancer, and cancers of the gastrointestinal tract such as
colorectal cancer); (d) shared
antigens, for example, melanoma-melanocyte differentiation antigens such as
MART-I/Melan A, gp100,
MOIR, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase related
protein- 1/TRPI and
tyrosinase related protein-2/TRP2 (associated with, e.g., melanoma); (e)
prostate associated antigens
such as PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2, associated with e.g., prostate
cancer; (f)
immunoglobulin idiotypes (associated with myeloma and B cell lymphomas, for
example); (g)
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neoantigens. In certain embodiments, tumor immunogens include, but are not
limited to, pi 5, Horn/Mel-
40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens,
EBNA, human
papillomavirus (HPV) antigens, including E6 and E7, hepatitis B and C virus
antigens, human T-cell
lymphotropic virus antigens, TSP-180, p185erbB2, p180erbB-3, c-met, mn-23H1,
TAG-72-4, CA 19-9, CA
72-4, CAM 17.1, NuMa, K-ras, pI6, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-
HCG, BCA225,
BTAA, CA 125, CA 15-3 (CA 27.29YBCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1,
CO-029, FGF-
5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1,
RCAS1,
SDCCAG16, TA-90 (Mac-2 binding protein cyclophilin C-associated protein),
TAAL6, TAG72, TLP, TPS,
and the like.
In some embodiments, the immunogen elicits an immune response against: pollen
allergens
(tree-, herb, weed-, and grass pollen allergens); insect or arachnid allergens
(inhalant, saliva and venom
allergens, e.g. mite allergens, cockroach and midges allergens, hymenopthera
venom allergens); animal
hair and dandruff allergens (from e.g. dog, cat, horse, rat, mouse, etc.); and
food allergens (e.g. a
gliadin). Important pollen allergens from trees, grasses and herbs are such
originating from the taxonomic
orders of Fagales, Oleales, Pinales and platanaceae including, but not limited
to, birch (Betula), alder
(Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar
(Cryptomeria and Juniperus),
plane tree (Platanus), the order of Poales including grasses of the genera
Lolium, Phleum, Poa,
Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, the orders of
Asterales and Urticales
including herbs of the genera Ambrosia, Artemisia, and Parietaria. Other
important inhalation allergens
are those from house dust mites of the genus Dermatophagoides and Euroglyphus,
storage mite e.g.,
Lepidoglyphys, Glycyphagus and Tyrophagus, those from cockroaches, midges and
fleas e.g., Blatella,
Periplaneta, Chironomus, and Ctenocepphalides, and those from mammals such as
cat, dog and horse,
venom allergens including such originating from stinging or biting insects
such as those from the
taxonomic order of Hymenoptera including bees (Apidae), wasps (Vespidea), and
ants (Formicoidae).
In some embodiments, the immunogen is derived from, for example, toxin in a
venom, such as a
venom from a snake (e.g., most species of rattlesnakes (e.g., eastern
diamondback rattlesnake), species
of brown snakes (e.g., king brown snake and eastern brown snake), russel's
viper, cobras (e.g., Indian
cobra, king cobra), certain species of kraits (e.g., common krait), mambas
(e.g., black mamba), saw-
scaled viper, boomslang, dubois sea snake, species of taipans (e.g., coastal
taipan and inland taipan
snake), species of lanceheads (e.g., fer-de-lance and terciopelo),
bushmasters, copperhead,
cottonmouth, coral snakes, death adders, Belcher's sea snake, tiger snakes,
Australian black snakes),
spider (e.g., brown recluse, black widow spider, Brazilian wandering spider,
funnel-web spider, button
spider, Australian redback spider, katipo, false black widow, Chilean recluse
spider, mouse spider,
species of Macrothele, species of Sicarius, species of Hexpthalma, certain
species of tarantulas),
scorpion and other arachnids (e.g., fat-tailed scorpion, deathstalker
scorpion, Indian red scorpion, species
of Centruroides, species of Tityus such as the Brazilian yellow scorpion),
insects (e.g., species of bees,
species of wasps, certain ants such as fire ants, some species of lepidopteran
caterpillars, certain
species of centipede, remipede Xibalbanus tulumensis), fish (e.g., certain
species of catfish (e.g., striped
eel catfish and other eeltail catfishes), certain species of stingrays (e.g.,
blue-spotted stingray), lionfishes,
stonefishes, scorpionfishes, toadfishes, rabbitfishes, goblinfishes, cockatoo
waspfish, striped blenny,
stargazers, chimaeras, weevers, dogfish sharks), cnidarians (e.g., certain
species jellyfish (e.g., Irukanjdi
jellyfish and box jellyfish), hydrozoans (e.g., Portuguese Man o'War), sea
anemones, certain species of
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coral), a lizard (e.g., a gila monster, Mexican bearded lizard, certain
species of Varanus (e.g., Komodo
dragon), perentie, and lace monitor), a mammal (e.g., Southern short-tailed
shrew, duck-billed platypus,
European mole, Eurasian water shrew, Mediterranean water shrew, Northern short-
tailed shrew, Elliot's
short-tailed shrew, certain species of solenodon (e.g., Cuban solenodon,
Hispaniolan solenodon), slow
loins), mollusks (e.g., certain species of cone snail), cephalopods (e.g.,
certain species of octopus (e.g.,
blue-ringed octopus), squid, and cuttlefish), amphibians (e.g., frogs such as
poison dart frogs, Bruno's
casque-headed frog, Greening's frog, salamanders (e.g., Fire salamander,
Iberian ribbed newt).
In some embodiments, the toxin is from a plant or fungi (e.g., a mushroom).
In some embodiments, the toxin immunogen is derived from a toxin such as a
cyanotoxins,
dinotoxins, myotoxins, cytotoxins (e.g., ricin, apitoxin, mycotoxins (e.g.,
aflatoxin), ochratoxin, citrinin,
ergot alkaloid, patulin, fusarium, fumonisins, trichothecenes, cardiotoxin),
tetrodotoxin, batrachotoxin,
botulinum toxin A, tetanus toxin A, diptheria toxin, dioxin, muscarine,
bufortoxin, sarin, hemotoxins,
phototoxins, necrotoxins, nephrotoxins, and neurotoxins (e.g., calciseptine,
cobrotoxin, calcicludine,
fasciculin-I, calliotoxin).
Immunogens from any number of microorganisms or cancers can be utilized in the
circular or
linear polyribonucleotides. In some cases, the immunogens are associated with
or expressed by one
microorganism disclosed above. In some embodiments, the immunogens are
associated with or
expressed by two or more microorganisms disclosed above. In some cases, the
immunogens are
associated with or expressed by one cancer disclosed above. In some
embodiments, immunogens are
associated with or expressed by two or more cancers disclosed above. In some
embodiments, the
immunogens are derived from toxins as disclosed above. In some embodiments,
the immunogens are
from two or more toxins disclosed above.
The two or more microorganisms are related or unrelated. In some cases, two or
more
microorganisms are phylogenetically related. For example, the circular or
linear polyribonucleotides of
the disclosure include or encode immunogens from two or more viruses, two or
more members of a viral
family, two or more members of a viral class, two or more members of a viral
order, two or more members
of a viral genus, two or more members of a viral species, two or more
bacterial pathogens. In some
embodiments, the two or more microorganisms are not phylogenetically related.
In some cases, two or more microorganisms are phenotypically related. For
example, the circular
or linear polyribonucleotides of the disclosure include or encode immunogens
from two or more
respiratory pathogens, two or more select agents, two or more microorganisms
associated with severe
disease, two or more microorganisms associated with adverse outcomes in
immunocompromised
subjects, two or more microorganisms associated with adverse outcomes related
to pregnancy, two or
more microorganisms associated with hemorrhagic fever.
An immunogen of the disclosure may include a wild-type sequence. When
describing an
immunogen, the term "wild-type" refers to a sequence (e.g., a nucleic acid
sequence or an amino acid
sequence) that is naturally occurring and encoded by a genome (e.g., a viral
genome). A species (e.g.,
microorganism species) can have one wild-type sequence, or two or more wild-
type sequences (for
example, with one canonical wild-type sequence present in a reference
microorganism genome, and
additional variant wild-type sequences present that have arisen from
mutations).
When describing an immunogen, the terms "derivative" and "derived from" refers
to a sequence
(e.g., nucleic acid sequence or amino acid sequence) that differs from a wild-
type sequence by one or
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more nucleic acids or amino acids, for example, containing one or more nucleic
acid or amino acid
insertions, deletions, and/or substitutions relative to a wild-type sequence.
An immunogen derivative sequence is a sequence that has at least 60%, 70%,
75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to
a wild-type
sequence, for example, a wild-type nucleic acid, protein, immunogen, or
epitope sequence.
In some embodiments, an immunogen contains one or more amino acid insertions,
deletions,
substitutions, or a combination thereof that affect the structure of an
encoded protein. In some
embodiments, an immunogen contains one or more amino acid insertions,
deletions, substitutions, or a
combination thereof that affect the function of an encoded protein. In some
embodiments, an immunogen
contains one or more amino acid insertions, deletions, substitutions, or a
combination thereof that affect
the expression or processing of an encoded protein by a cell.
In some embodiments, an immunogen contains one or more nucleic acid
insertions, deletions,
substitutions, or a combination thereof that affect the structure of an
encoded immunogenic nucleic acid.
Amino acid insertions, deletions, substitutions, or a combination thereof can
introduce a site for a
post-translational modification (for example, introduce a glycosylation,
ubiquitination, phosphorylation,
nitrosylation, methylation, acetylation, amidation, hydroxylation, sulfation,
or lipidation site, or a sequence
that is targeted for cleavage). In some embodiments, amino acid insertions,
deletions, substitutions, or a
combination thereof remove a site for a post-translational modification (for
example, remove a
glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation,
acetylation, amidation,
hydroxylation, sulfation, or lipidation site, or a sequence that is targeted
for cleavage). In some
embodiments, amino acid insertions, deletions, substitutions, or a combination
thereof modify a site for a
post-translational modification (for example, modify a site to alter the
efficiency or characteristics of
glycosylation, ubiquitination, phosphorylation, nitrosylation, methylation,
acetylation, amidation,
hydroxylation, sulfation, or lipidation site, or cleavage).
An amino acid substitution can be a conservative or a non-conservative
substitution. A
conservative amino acid substitution can be a substitution of one amino acid
for another amino acid of
similar biochemical properties (e.g., charge, size, and/or hydrophobicity). A
non-conservative amino acid
substitution can be a substitution of one amino acid for another amino acid
with different biochemical
properties (e.g., charge, size, and/or hydrophobicity). A conservative amino
acid change can be, for
example, a substitution that has minimal effect on the secondary or tertiary
structure of a polypeptide. A
conservative amino acid change can be an amino acid change from one
hydrophilic amino acid to
another hydrophilic amino acid. Hydrophilic amino acids can include Thr (T),
Ser (S), His (H), Glu (E),
Asn (N), Gln (Q), Asp (D), Lys (K) and Arg (R). A conservative amino acid
change can be an amino acid
change from one hydrophobic amino acid to another hydrophilic amino acid.
Hydrophobic amino acids
can include Ile (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly
(G), Tyr (Y), and Pro (P). A
conservative amino acid change can be an amino acid change from one acidic
amino acid to another
acidic amino acid. Acidic amino acids can include Glu (E) and Asp (D). A
conservative amino acid
change can be an amino acid change from one basic amino acid to another basic
amino acid. Basic
amino acids can include His (H), Arg (R) and Lys (K). A conservative amino
acid change can be an
.. amino acid change from one polar amino acid to another polar amino acid.
Polar amino acids can include
Asn (N), Gin (Q), Ser (S) and Thr (T). A conservative amino acid change can be
an amino acid change
from one nonpolar amino acid to another nonpolar amino acid. Nonpolar amino
acids can include Leu
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(L), Val(V), Ile (I), Met (M), Gly (G) and Ala (A). A conservative amino acid
change can be an amino acid
change from one aromatic amino acid to another aromatic amino acid. Aromatic
amino acids can include
Phe (F), Tyr (Y) and Trp (W). A conservative amino acid change can be an amino
acid change from one
aliphatic amino acid to another aliphatic amino acid. Aliphatic amino acids
can include Ala (A), Val (V),
Leu (L) and Ile (I). In some embodiments, a conservative amino acid
substitution is an amino acid
change from one amino acid to another amino acid within one of the following
groups: Group I: ala, pro,
gly, gin, asn, ser, thr; Group II: cys, ser, tyr, thr; Group III: val, ile,
leu, met, ala, phe; Group IV: lys, arg,
his; Group V: phe, tyr, trp, his; and Group VI: asp, glu.
In some embodiments, an immunogen derivative or epitope derivative of the
disclosure includes
at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10,
at least 11, at least 12, at least 13, at least 14, at least 15, at least 16,
at least 17, at least 18, at least 19,
at least 20, at least 25, at least 30, at least 35, at least 40, at least 45,
at least 50, at least 60, at least 70,
at least 80, at least 90, or at least 100 amino acid deletions relative to a
sequence disclosed herein (e.g.,
a wild-type sequence).
In some embodiments, an immunogen derivative or epitope derivative of the
disclosure includes
at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10,
at least 11, at least 12, at least 13, at least 14, at least 15, at least 16,
at least 17, at least 18, at least 19,
at least 20, at least 25, at least 30, at least 35, at least 40, at least 45,
or at least 50 amino acid
substitutions relative to a sequence disclosed herein (e.g., a wild-type
sequence).
In some embodiments, an immunogen derivative or epitope derivative of the
disclosure includes
at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7,
at most 8, at most 9, at most
10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at
most 17, at most 18, at most
19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or
at most 50 amino acid
substitutions relative to a sequence disclosed herein (e.g., a wild-type
sequence).
In some embodiments, an immunogen derivative or epitope derivative of the
disclosure includes
1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-
4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-
15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-
30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10,
5-15, 5-20, 5-30, 5-40,10-15, 15-20, or 20-25 amino acid substitutions
relative to a sequence disclosed
herein (e.g., a wild-type sequence).
In some embodiments, an immunogen derivative or epitope derivative of the
disclosure includes
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino
acid substitutions relative to a
sequence disclosed herein (e.g., a wild-type sequence).
The one or more amino acid substitutions can be at the N-terminus, the C-
terminus, within the
amino acid sequence, or a combination thereof. The amino acid substitutions
can be contiguous, non-
contiguous, or a combination thereof.
In some embodiments, an immunogen derivative or epitope derivative of the
disclosure includes
at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7,
at most 8, at most 9, at most
10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at
most 17, at most 18, at most
19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, at
most 50, at most 60, at most
70, at most 80, at most 90, at most 100, at most 120, at most 140, at most
160, at most 180, or at most
200 amino acid deletions relative to a sequence disclosed herein (e.g., a wild-
type sequence).
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In some embodiments, an immunogen derivative or epitope derivative of the
disclosure includes
1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-
4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-
15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-
30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10,
5-15, 5-20, 5-30, 5-40, 10-15, 15-20, 20-25, 20-30, 30-50, 50-100, or 100-200
amino acid deletions
relative to a wild-type sequence.
In some embodiments, an immunogen derivative or epitope derivative of the
disclosure includes
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino
acid deletions relative to a wild-
type sequence.
The one or more amino acid deletions can be at the N-terminus, the C-terminus,
within the amino
acid sequence, or a combination thereof. The amino acid deletions can be
contiguous, non-contiguous,
or a combination thereof.
In some embodiments, an immunogen derivative or epitope derivative of the
disclosure includes
at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10,
at least 11, at least 12, at least 13, at least 14, at least 15, at least 16,
at least 17, at least 18, at least 19,
at least 20, at least 25, at least 30, at least 35, at least 40, at least 45,
or at least 50 amino acid insertions
relative to a wild-type sequence.
In some embodiments, an immunogen derivative or epitope derivative of the
disclosure includes
at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7,
at most 8, at most 9, at most
10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at
most 17, at most 18, at most
19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or
at most 50 amino acid
insertions relative to a wild-type sequence).
In some embodiments, an immunogen derivative or epitope derivative of the
disclosure includes
1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-
4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-
15, 2-20, 2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-
30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10,
5-15, 5-20, 5-30, 5-40,10-15, 15-20, or 20-25 amino acid insertions relative
to a wild-type sequence.
In some embodiments, an immunogen derivative or epitope derivative of the
disclosure includes
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino
acid insertions relative to a wild-
type sequence.
The one or more amino acid insertions can be at the N-terminus, the C-
terminus, within the
amino acid sequence, or a combination thereof. The amino acid insertions can
be contiguous, non-
contiguous, or a combination thereof.
In some embodiments, the immunogen is expressed by the circular or linear
polyribonucleotide.
In some embodiments, the immunogen is a product of rolling circle
amplification of the circular or linear
polyribonucleotide.
The immunogen may be produced in substantial amounts. As such, the immunogen
may be any
proteinaceous molecule that can be produced. An immunogen can be a polypeptide
that can be secreted
from a cell, or localized to the cytoplasm, nucleus, or membrane compartment
of a cell. In some
embodiments, a polypeptide encoded by a circular or linear polyribonucleotide
of the disclosure includes
a fusion protein including two or more immunogens disclosed herein. In some
embodiments, a
polypeptide encoded by a circular or linear polyribonucleotide of the
disclosure includes an epitope. In
some embodiments, a polypeptide encoded by a circular or linear
polyribonucleotide of the disclosure
includes a fusion protein including two or more epitopes disclosed herein, for
example, an artificial
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peptide sequence including a plurality of predicted epitopes from one or more
microorganisms of the
disclosure.
In some embodiments, an immunogen that can be expressed from the circular or
linear
polyribonucleotide is a membrane protein, for example, including a polypeptide
sequence that is generally
found as a membrane protein, or a polypeptide sequence that is modified to be
a membrane protein. In
some embodiments, exemplary immunogens that can be expressed from the circular
or linear
polyribonucleotide disclosed herein include an intracellular immunogen or
cytosolic immunogen.
In some embodiments, the immunogen has a length of less than about 40,000
amino acids, less
than about 35,000 amino acids, less than about 30,000 amino acids, less than
about 25,000 amino acids,
less than about 20,000 amino acids, less than about 15,000 amino acids, less
than about 10,000 amino
acids, less than about 9,000 amino acids, less than about 8,000 amino acids,
less than about 7,000
amino acids, less than about 6,000 amino acids, less than about 5,000 amino
acids, less than about
4,000 amino acids, less than about 3,000 amino acids, less than about 2,500
amino acids, less than
about 2,000 amino acids, less than about 1,500 amino acids, less than about
1,000 amino acids, less
than about 900 amino acids, less than about 800 amino acids, less than about
700 amino acids, less than
about 600 amino acids, less than about 500 amino acids, less than about 400
amino acids, less than
about 300 amino acids, less than about 250 amino acids, less than about 200
amino acids, less than
about 150 amino acids, less than about 140 amino acids, less than about 130
amino acids, less than
about 120 amino acids, less than about 110 amino acids, less than about 100
amino acids, less than
about 90 amino acids, less than about 80 amino acids, less than about 70 amino
acids, less than about
60 amino acids, less than about 50 amino acids, less than about 40 amino
acids, less than about 30
amino acids, less than about 25 amino acids, less than about 20 amino acids,
less than about 15 amino
acids, less than about 10 amino acids, less than about 5 amino acids, any
amino acid length
therebetween or less may be useful.
In some embodiments, the circular or linear polyribonucleotide includes one or
more immunogen
sequences and is configured for persistent expression in a cell of a subject
in vivo. In some
embodiments, the circular or linear polyribonucleotide is configured such that
expression of the one or
more expression sequences in the cell at a later time point is equal to or
higher than an earlier time point.
In such embodiments, the expression of the one or more immunogen sequences can
be either
maintained at a relatively stable level or can increase over time. The
expression of the immunogen
sequences can be relatively stable for an extended period of time. The
expression of the immunogen
sequences can be relatively stable transiently or for only a limited amount of
time, for example, at most 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 days.
In some embodiments, the circular or linear polyribonucleotide expresses one
or more
immunogens in a subject, e.g., transiently or long term. In certain
embodiments, expression of the
immunogens persists for at least about 1 hr to about 30 days, or at least
about 2 hrs, 6 hrs, 12 hrs, 18
hrs, 24 hrs, 2 days, 3, days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days,
10 days, 11 days, 12 days,
13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21
days, 22 days, 23 days, 24
days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or longer
or any time
therebetween. In certain embodiments, expression of the immunogens persists
for no more than about 30
mins to about 7 days, or no more than about 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs,
6 hrs, 7 hrs, 8 hrs, 9 hrs, 10
hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs,
20 hrs, 21 hrs, 22 hrs, 24 hrs, 36
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hrs, 48 hrs, 60 hrs, 72 hrs, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days,
10 days, 11 days, 12 days, 13
days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days,
22 days, 23 days, 24
days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days, or any
time therebetween.
The immunogen expression includes translating at least a region of the
circular or linear
polyribonucleotide provided herein. For example, a circular or linear
polyribonucleotide can be translated
in a subject to generate polypeptides that include one or more immunogens of
the disclosure, thereby
stimulating production of an adaptive immune response (e.g., antibody response
and/or T cell response)
in the subject. In some embodiments, a circular or linear polyribonucleotide
of the disclosure is translated
to produce one or more immunogens in a human or animal subject, thereby
stimulating production of an
adaptive immune response (e.g., antibody response and/or T cell response) in a
human or animal
subject.
In some embodiments, the methods for immunogen expression includes translation
of at least
10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at
least 90%, or at least 95% of the total length of the circular or linear
polyribonucleotide into polypeptides.
In some embodiments, the methods for immunogen expression includes translation
of the circular or
linear polyribonucleotide into polypeptides of at least 5 amino acids, at
least 10 amino acids, at least 15
amino acids, at least 20 amino acids, at least 50 amino acids, at least 100
amino acids, at least 150
amino acids, at least 200 amino acids, at least 250 amino acids, at least 300
amino acids, at least 400
amino acids, at least 500 amino acids, at least 600 amino acids, at least 700
amino acids, at least 800
amino acids, at least 900 amino acids, or at least 1000 amino acids. In some
embodiments, the methods
for protein expression includes translation of the circular or linear
polyribonucleotide into polypeptides of
about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20
amino acids, about 50 amino
acids, about 100 amino acids, about 150 amino acids, about 200 amino acids,
about 250 amino acids,
about 300 amino acids, about 400 amino acids, about 500 amino acids, about 600
amino acids, about
700 amino acids, about 800 amino acids, about 900 amino acids, or about 1000
amino acids. In some
embodiments, the methods include translation of the circular or linear
polyribonucleotide into continuous
polypeptides as provided herein, discrete polypeptides as provided herein, or
both.
In some embodiments, the methods for immunogen expression include
modification, folding, or
other post-translation modification of the translation product. In some
embodiments, the methods for
immunogen expression include post-translation modification in vivo, e.g., via
cellular machinery.
Signal Sequence
In some embodiments, exemplary immunogens that can be expressed from a
circular or linear
polyribonucleotide disclosed herein include a secreted protein, for example, a
protein (e.g., immunogen)
that naturally includes a signal sequence, or one that does not usually encode
a signal sequence, but is
modified to contain one. In some embodiments, the immunogen(s) encoded for by
the circular or linear
polyribonucleotide includes a secretion signal. For example, the secretion
signal may be the naturally
encoded secretion signal for a secreted protein. In another example, the
secretion signal may be a
modified secretion signal for a secreted protein. In other embodiments, the
immunogen(s) encoded for by
the circular or linear polyribonucleotide do not include a secretion signal.
In some embodiments, a circular or linear polyribonucleotide encodes multiple
copies of the same
immunogen (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or
more) copies of the same
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immunogen. In some embodiments, at least one copy of the immunogen includes a
signal sequence and
at least one copy of the immunogen does not include a signal sequence. In some
embodiments, a
circular or linear polyribonucleotide encodes plurality of immunogens (e.g., a
plurality of different
immunogens or a plurality of immunogens having less than 100% sequence
identity), where at least one
of the plurality of immunogens includes a signal sequence and at least one
copy of the plurality of
immunogens does not include a signal sequence.
In some embodiments, the signal sequence is a wild-type signal sequence that
is present on the
N-terminus of the corresponding wild-type immunogen, e.g., when expressed
endogenously. In some
embodiments, the signal sequence is heterologous to the immunogen, e.g., is
not present when the wild-
type immunogen is expressed endogenously. A polyribonucleotide sequence
encoding an immunogen
may be modified to remove the nucleotide sequence encoding a wild-type signal
sequence and/or add a
sequence encoding a heterologous signal sequence.
The circular or linear polyribonucleotide may further include one or more
adjuvants, each with or
without a signal sequence. In some embodiments, the circular or linear
polyribonucleotide encodes at
least one adjuvant and at least one immunogen. In some embodiments, the at
least one encoded
adjuvant includes a signal sequence and the at least one encoded immunogen
does not include a signal
sequence. In some embodiments, the at least one encoded adjuvant includes a
signal sequence and the
at least one encoded immunogen includes a signal sequence. In some
embodiments, the at least one
encoded adjuvant does not include a signal sequence and the at least one
encoded immunogen includes
.. a signal sequence. In some embodiments, neither the encoded adjuvant nor
the encoded immunogen
includes a signal sequence.
In some embodiments, the signal sequence is a wild-type signal sequence that
is present on the
N-terminus of the corresponding wild-type adjuvant, e.g., when expressed
endogenously. In some
embodiments, the signal sequence is heterologous to the adjuvant, e.g., is not
present when the wild-type
adjuvant is expressed endogenously. A polyribonucleotide sequence encoding an
adjuvant may be
modified to remove the nucleotide sequence encoding a wild-type signal
sequence and/or add a
sequence encoding a heterologous signal sequence.
A polypeptide encoded by a polyribonucleotide (e.g., immunogen or an adjuvant
encoded by a
polyribonucleotide) may include a signal sequence that directs the immunogen
or adjuvant to the
secretory pathway. In some embodiments, the signal sequence may direct the
immunogen or adjuvant to
reside in certain organelles (e.g., the endoplasmic reticulum, Golgi
apparatus, or endosomes). In some
embodiments, the signal sequence directs the immunogen or adjuvant to be
secreted from the cell. For
secreted proteins, the signal sequence may be cleaved after secretion,
resulting in a mature protein. In
other embodiments, the signal sequence may become embedded in the membrane of
the cell or certain
organelles, creating a transmembrane segment that anchors the protein to the
membrane of the cell,
endoplasmic reticulum, or Golgi apparatus. In certain embodiments, the signal
sequence of a
transmembrane protein is a short sequence at the N-terminal of the
polypeptide. In other embodiments,
the first transmembrane domain acts as the first signal sequence, which
targets the protein to the
membrane.
In some embodiments, an adjuvant encoded by a polyribonucleotide includes a
secretion signal
sequence. In some embodiments, an immunogen encoded by a polyribonucleotide
includes either a
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secretion signal sequence, a transmembrane insertion signal sequence, or does
not include a signal
sequence.
Regulatory Element
In some embodiments, a circular or linear polyribonucleotide includes a
regulatory element, e.g.,
a sequence that modifies expression of an expression sequence within the
circular or linear
polyribonucleotide. A regulatory element may include a sequence that is
located adjacent to an
expression sequence that encodes an expression product. A regulatory element
may be operably linked
to the adjacent sequence. A regulatory element may increase an amount of
product expressed as
compared to an amount of the expressed product when no regulatory element is
present. A regulatory
element may be used to increase the expression of one or more immunogen(s)
and/or adjuvant(s)
encoded by a circular or linear polyribonucleotide. Likewise, a regulatory
element may be used to
decrease the expression of one or more immunogen(s) and/or adjuvant(s) encoded
by a circular or linear
polyribonucleotide. In some embodiments, a regulatory element may be used to
increase expression of
an immunogen and/or adjuvant and another regulatory element may be used to
decrease expression of
another immunogen and/or adjuvant on the same circular or linear
polyribonucleotide. In addition, one
regulatory element can increase an amount of products (e.g., an immunogen or
adjuvants) expressed for
multiple expression sequences attached in tandem. Hence, one regulatory
element can enhance the
expression of one or more expression sequences (e.g., immunogens or
adjuvants). Multiple regulatory
.. elements can also be used, for example, to differentially regulate
expression of different expression
sequences. In some embodiments, a regulatory element as provided herein can
include a selective
translation sequence. As used herein, the term "selective translation
sequence" refers to a nucleic acid
sequence that selectively initiates or activates translation of an expression
sequence in the circular or
linear polyribonucleotide, for instance, certain riboswitch aptazymes. A
regulatory element can also
include a selective degradation sequence. As used herein, the term "selective
degradation sequence"
refers to a nucleic acid sequence that initiates degradation of the circular
or linear polyribonucleotide, or
an expression product of the circular or linear polyribonucleotide. In some
embodiments, the regulatory
element is a translation modulator. A translation modulator can modulate
translation of the expression
sequence in the circular or linear polyribonucleotide. A translation modulator
can be a translation
enhancer or suppressor. In some embodiments, a translation initiation sequence
can function as a
regulatory element. Further examples of regulatory elements are described in
paragraphs [0154] ¨ [0161]
of International Patent Publication No. W02019/118919, which is hereby
incorporated by reference in its
entirety.
Nucleotides flanking a codon that initiates translation, such as, but not
limited to, a start codon or
an alternative start codon, are known to affect the translation efficiency,
the length, and/or the structure of
the circular or linear polyribonucleotide. (See e.g., Matsuda and Mauro PLoS
ONE, 20105: 11; the
contents of which are herein incorporated by reference in its entirety).
Masking any of the nucleotides
flanking a codon that initiates translation may be used to alter the position
of translation initiation,
translation efficiency, length and/or structure of the circular or linear
polyribonucleotide.
In one embodiment, a masking agent may be used near the start codon or
alternative start codon
in order to mask or hide the codon to reduce the probability of translation
initiation at the masked start
codon or alternative start codon. In another embodiment, a masking agent may
be used to mask a start
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codon of the circular or linear polyribonucleotide in order to increase the
likelihood that translation will
initiate at an alternative start codon.
Translation Initiation Sequence
In some embodiments, a circular or linear polyribonucleotide encodes an
immunogen and
includes a translation initiation sequence, e.g., a start codon. In some
embodiments, the translation
initiation sequence includes a Kozak or Shine-Dalgarno sequence. In some
embodiments, the translation
initiation sequence includes a Kozak sequence. In some embodiments, the
circular or linear
polyribonucleotide includes the translation initiation sequence, e.g., Kozak
sequence, adjacent to an
expression sequence. In some embodiments, the translation initiation sequence
is a non-coding start
codon. In some embodiments, the translation initiation sequence, e.g., Kozak
sequence, is present on
one or both sides of each expression sequence, leading to separation of the
expression products. In
some embodiments, the circular or linear polyribonucleotide includes at least
one translation initiation
sequence adjacent to an expression sequence. In some embodiments, the
translation initiation sequence
provides conformational flexibility to the circular or linear
polyribonucleotide. In some embodiments, the
translation initiation sequence is within a substantially single stranded
region of the circular or linear
polyribonucleotide. Further examples of translation initiation sequences are
described in paragraphs
[0163] - [0165] of International Patent Publication No. W02019/118919, which
is hereby incorporated by
reference in its entirety.
The circular or linear polyribonucleotide may include more than 1 start codon
such as, but not
limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10,
at least 11, at least 12, at least 13, at least 14, at least 15, at least 16,
at least 17, at least 18, at least 19,
at least 20, at least 25, at least 30, at least 35, at least 40, at least 50,
at least 60 or more than 60 start
codons. Translation may initiate on the first start codon or may initiate
downstream of the first start codon.
In some embodiments, a circular or linear polyribonucleotide may initiate at a
codon which is not
the first start codon, e.g., AUG. Translation of the circular or linear
polyribonucleotide may initiate at an
alternative translation initiation sequence, such as those described in [0164]
of International Patent
Publication No. W02019/118919A1, which is incorporated herein by reference in
its entirety.
In some embodiments, translation is initiated by eukaryotic initiation factor
4A (eIF4A) treatment
with Rocaglates (translation is repressed by blocking 43S scanning, leading to
premature, upstream
translation initiation and reduced protein expression from transcripts bearing
the RocA-elF4A target
sequence, see for example, www.nature.com/articles/nature17978).
IRES
In some embodiments, a circular or linear polyribonucleotide described herein
includes an
internal ribosome entry site (IRES) element. In some embodiments, a circular
or linear polyribonucleotide
described herein includes more than one (e.g., 2, 3, 4, and 5) internal
ribosome entry site (IRES) element.
In some embodiments, the circular or linear polyribonucleotide includes one or
more IRES sequences on
one or both sides of each expression sequence, leading to separation of the
resulting peptide(s) and or
polypeptide(s). In some embodiments, the IRES flanks both sides of at least
one (e.g., 2, 3, 4, 5 or more)
expression sequence. A suitable IRES element to include in a circular or
linear polyribonucleotide can be
an RNA sequence capable of engaging a eukaryotic ribosome. In some
embodiments, the IRES is an
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encephalomyocarditis virus (EMCV) !RES. In some embodiments, the IRES is a
Coxsackievirus (CVB3)
!RES. Further examples of an IRES are described in paragraphs [0166] ¨ [0168]
of International Patent
Publication No. W02019/118919, which is hereby incorporated by reference in
its entirety.
Cleavage Domains
A circular or linear polyribonucleotide of the disclosure can include a
cleavage domain (e.g., a
stagger element or a cleavage sequence).
The term "stagger element" refers to a moiety, such as a nucleotide sequence,
that induces
ribosomal pausing during translation. In some embodiments, the stagger element
is a non-conserved
sequence of amino-acids with a strong alpha-helical propensity followed by the
consensus sequence -
D(V/I)ExNPGP, where x= any amino acid (SEQ ID NO: 7). In some embodiments, the
stagger element
may include a chemical moiety, such as glycerol, a non-nucleic acid linking
moiety, a chemical
modification, a modified nucleic acid, or any combination thereof.
In some embodiments, a circular or linear polyribonucleotide includes at least
one stagger
element adjacent to an expression sequence. In some embodiments, the circular
or linear
polyribonucleotide includes a stagger element adjacent to each expression
sequence. In some
embodiments, the stagger element is present on one or both sides of each
expression sequence, leading
to separation of the expression products, e.g., immunogen(s) and/or
adjuvant(s). In some embodiments,
the stagger element is a portion of the one or more expression sequences. In
some embodiments, the
circular or linear polyribonucleotide includes one or more expression
sequences (e.g., immunogen(s)
and/or adjuvant(s)), and each of the one or more expression sequences is
separated from a succeeding
expression sequence (e.g., immunogen(s) and/or adjuvant(s) by a stagger
element on the circular or
linear polyribonucleotide. In some embodiments, the stagger element prevents
generation of a single
polypeptide (a) from two rounds of translation of a single expression sequence
or (b) from one or more
rounds of translation of two or more expression sequences. In some
embodiments, the stagger element
is a sequence separate from the one or more expression sequences. In some
embodiments, the stagger
element includes a portion of an expression sequence of the one or more
expression sequences.
Examples of stagger elements are described in paragraphs [0172] ¨ [0175] of
International
Patent Publication No. W02019/118919, which is hereby incorporated by
reference in its entirety.
In some embodiments, the plurality of immunogens encoded by a circular or
linear ribonucleotide
may be separated by an IRES between each immunogen. The IRES may be the same
IRES between all
immunogens. The IRES may be different between different immunogens. In other
embodiments, the
plurality of immunogens and/or adjuvants may be separated by a 2A self-
cleaving peptide. Furthermore,
the plurality of immunogens and/or adjuvants encoded by the circular or linear
ribonucleotide may be
separated by both IRES and 2A sequences. For example, an IRES may be between
one immunogens
and/or adjuvant and a second immunogen and/or adjuvant while a 2A peptide may
be between the
second immunogen and/or adjuvant and the third immunogen and/or adjuvant. The
selection of a
particular IRES or 2A self-cleaving peptide may be used to control the
expression level of immunogen
and/or adjuvant under control of the IRES or 2A sequence. For example,
depending on the IRES and or
2A peptide selected, expression on the polypeptide may be higher or lower.
To avoid production of a continuous expression product, e.g., immunogen and/or
adjuvant, while
maintaining rolling circle translation, a stagger element may be included to
induce ribosomal pausing
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during translation. In some embodiments, the stagger element is at 3' end of
at least one of the one or
more expression sequences. The stagger element can be configured to stall a
ribosome during rolling
circle translation of the circular or linear polyribonucleotide. The stagger
element may include, but is not
limited to a 2A-like, or CHYSEL (SEQ ID NO: 8) (cis-acting hydrolase element)
sequence. In some
embodiments, the stagger element encodes a sequence with a C-terminal
consensus sequence that is
XiX2X3EX5NPGP, where X, is absent or G or H, X2 is absent or D or G, X3 is D
or V or I or S or M, and X5
is any amino acid (SEQ ID NO: 9). In some embodiments, this sequence includes
a non-conserved
sequence of amino-acids with a strong alpha-helical propensity followed by the
consensus sequence -
D(V/I)ExNPGP, where x= any amino acid (SEQ ID NO: 7). Some non-limiting
examples of stagger
.. elements includes GDVESNPGP (SEQ ID NO: 10), GDIEENPGP (SEQ ID NO: 11),
VEPNPGP (SEQ ID
NO: 12), IETNPGP (SEQ ID NO: 13), GDIESNPGP (SEQ ID NO: 14), GDVELNPGP (SEQ ID
NO: 15),
GDIETNPGP (SEQ ID NO: 16), GDVENPGP (SEQ ID NO: 17), GDVEENPGP (SEQ ID NO:
18),
GDVEQNPGP (SEQ ID NO: 19), IESNPGP (SEQ ID NO: 20), GDIELNPGP (SEQ ID NO: 21),
HDIETNPGP (SEQ ID NO: 22), HDVETNPGP (SEQ ID NO: 23), HDVEMNPGP (SEQ ID NO:
24),
.. GDMESNPGP (SEQ ID NO: 25), GDVETNPGP (SEQ ID NO: 26), GDIEQNPGP (SEQ ID NO:
27), and
DSEFNPGP (SEQ ID NO: 28).
In some embodiments, a stagger element described herein cleaves an expression
product, such
as between G and P of the consensus sequence described herein. As one non-
limiting example, the
circular or linear polyribonucleotide includes at least one stagger element to
cleave the expression
.. product. In some embodiments, the circular or linear polyribonucleotide
includes a stagger element
adjacent to at least one expression sequence. In some embodiments, the
circular or linear
polyribonucleotide includes a stagger element after each expression sequence.
In some embodiments,
the circular or linear polyribonucleotide includes a stagger element is
present on one or both sides of
each expression sequence, leading to translation of individual peptide(s) and
or polypeptide(s) from each
.. expression sequence.
In some embodiments, a stagger element includes one or more modified
nucleotides or unnatural
nucleotides that induce ribosomal pausing during translation. Unnatural
nucleotides may include peptide
nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as
glycol nucleic acid (GNA)
and threose nucleic acid (TNA). Examples such as these are distinguished from
naturally occurring DNA
or RNA by changes to the backbone of the molecule. Exemplary modifications can
include any
modification to the sugar, the nucleobase, the internucleoside linkage (e.g.
to a linking phosphate / to a
phosphodiester linkage / to the phosphodiester backbone), and any combination
thereof that can induce
ribosomal pausing during translation. Some of the exemplary modifications
provided herein are
described elsewhere herein.
In some embodiments, a stagger element is present in a circular or linear
polyribonucleotide in
other forms. For example, in some exemplary circular or linear
polyribonucleotides, a stagger element
includes a termination element of a first expression sequence in the circular
or linear polyribonucleotide,
and a nucleotide spacer sequence that separates the termination element from a
first translation initiation
sequence of an expression succeeding the first expression sequence. In some
examples, the first stagger
.. element of the first expression sequence is upstream of (5' to) a first
translation initiation sequence of the
expression succeeding the first expression sequence in the circular or linear
polyribonucleotide. In some
cases, the first expression sequence and the expression sequence succeeding
the first expression
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sequence are two separate expression sequences in the circular or linear
polyribonucleotide. The
distance between the first stagger element and the first translation
initiation sequence can enable
continuous translation of the first expression sequence and its succeeding
expression sequence. In
some embodiments, the first stagger element includes a termination element and
separates an
expression product of the first expression sequence from an expression product
of its succeeding
expression sequences, thereby creating discrete expression products. In some
cases, the circular or
linear polyribonucleotide including the first stagger element upstream of the
first translation initiation
sequence of the succeeding sequence in the circular or linear
polyribonucleotide is continuously
translated, while a corresponding circular or linear polyribonucleotide
including a stagger element of a
second expression sequence that is upstream of a second translation initiation
sequence of an
expression sequence succeeding the second expression sequence is not
continuously translated. In
some cases, there is only one expression sequence in the circular or linear
polyribonucleotide, and the
first expression sequence and its succeeding expression sequence are the same
expression sequence.
In some exemplary circular or linear polyribonucleotides, a stagger element
includes a first termination
element of a first expression sequence in the circular or linear
polyribonucleotide, and a nucleotide spacer
sequence that separates the termination element from a downstream translation
initiation sequence. In
some such examples, the first stagger element is upstream of (5' to) a first
translation initiation sequence
of the first expression sequence in the circular or linear polyribonucleotide.
In some cases, the distance
between the first stagger element and the first translation initiation
sequence enables continuous
.. translation of the first expression sequence and any succeeding expression
sequences. In some
embodiments, the first stagger element separates one round expression product
of the first expression
sequence from the next round expression product of the first expression
sequences, thereby creating
discrete expression products. In some cases, the circular or linear
polyribonucleotide including the first
stagger element upstream of the first translation initiation sequence of the
first expression sequence in
the circular or linear polyribonucleotide is continuously translated, while a
corresponding circular or linear
polyribonucleotide including a stagger element upstream of a second
translation initiation sequence of a
second expression sequence in the corresponding circular or linear
polyribonucleotide is not continuously
translated. In some cases, the distance between the second stagger element and
the second translation
initiation sequence is at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x greater
in the corresponding circular or
linear polyribonucleotide than a distance between the first stagger element
and the first translation
initiation in the circular or linear polyribonucleotide. In some cases, the
distance between the first stagger
element and the first translation initiation is at least 2 nt, 3 nt, 4 nt, 5
nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11 nt,
12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30 nt,
35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60
nt, 65 nt, 70 nt, 75 nt, or greater. In some embodiments, the distance between
the second stagger
element and the second translation initiation is at least 2 nt, 3 nt, 4 nt, 5
nt, 6 nt, 7 nt, 8 nt, 9 nt, 10 nt, 11
nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 25 nt, 30
nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt,
60 nt, 65 nt, 70 nt, 75 nt, or greater than the distance between the first
stagger element and the first
translation initiation. In some embodiments, the circular or linear
polyribonucleotide includes more than
one expression sequence.
In some embodiments, a circular or linear polyribonucleotide includes at least
one cleavage
sequence. In some embodiments, the cleavage sequence is adjacent to an
expression sequence. In
some embodiments, the cleavage sequence is between two expression sequences.
In some
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embodiments, cleavage sequence is included in an expression sequence. In some
embodiments, the
circular or linear polyribonucleotide includes between 2 and 10 cleavage
sequences. In some
embodiments, the circular or linear polyribonucleotide includes between 2 and
5 cleavage sequences. In
some embodiments, the multiple cleavage sequences are between multiple
expression sequences; for
example, a circular or linear polyribonucleotide may include three expression
sequences two cleavage
sequences such that there is a cleavage sequence in between each expression
sequence. In some
embodiments, the circular or linear polyribonucleotide includes a cleavage
sequence, such as in an
immolating circRNA or cleavable circRNA or self-cleaving circRNA. In some
embodiments, the circular or
linear polyribonucleotide includes two or more cleavage sequences, leading to
separation of the circular
or linear polyribonucleotide into multiple products, e.g., miRNAs, linear
RNAs, smaller circular or linear
polyribonucleotide, etc.
In some embodiments, a cleavage sequence includes a ribozyme RNA sequence. A
ribozyme
(from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an
RNA molecule that
catalyzes a chemical reaction. Many natural ribozymes catalyze either the
hydrolysis of one of their own
phosphodiester bonds, or the hydrolysis of bonds in other RNA, but they have
also been found to
catalyze the aminotransferase activity of the ribosome. Catalytic RNA can be
"evolved" by in vitro
methods. Similar to riboswitch activity discussed above, ribozymes and their
reaction products can
regulate gene expression. In some embodiments, a catalytic RNA or ribozyme can
be placed within a
larger non-coding RNA such that the ribozyme is present at many copies within
the cell for the purposes
of chemical transformation of a molecule from a bulk volume. In some
embodiments, aptamers and
ribozymes can both be encoded in the same non-coding RNA.
In some embodiments, the cleavage sequence encodes a cleavable polypeptide
linker. For
example, a polyribonucleotide may encode two or more immunogens, e.g., where
the two or more
immunogens are encoded by a single open-reading frame (ORF). For example, two
or more
immunogens may be encoded by a single open-reading frame, the expression of
which is controlled by
an !RES. In some embodiments, the ORF further encodes a polypeptide linker,
e.g., such that the
expression product of the ORF encodes two or more immunogens each separated by
a sequence
encoding a polypeptide linker (e.g., a linker of 5-200, 5 to 100, 5 to 50, 5
to 20, 50 to 100, or 50 to 200
amino acids). The polypeptide linker may include a cleavage site, for example,
a cleavage site
recognized and cleaved by a protease (e.g., an endogenous protease in a
subject following
administration of the polyribonucleotide to that subject). In such
embodiments, a single expression
product including the amino acid sequence of two or more immunogens is cleaved
upon expression, such
that the two or more immunogens are separated following expression. Exemplary
protease cleavage
sites are known to those of skill in the art, for example, amino acid
sequences that act as protease
cleavage sites recognized by a metalloproteinase (e.g., a matrix
metalloproteinase (MMP), such as any
one or more of MMPs 1-28), a disintegrin and metalloproteinase (ADAM, such as
any one or more of
ADAMs 2, 7-12, 15, 17-23, 28-30 and 33), a serine protease, urokinase-type
plasminogen activator,
matriptase, a cysteine protease, an aspartic protease, or a cathepsin
protease. In some embodiments,
the protease is MMP9 or MMP2. In some embodiments, the protease is matriptase.
In some embodiments, a circular or linear polyribonucleotide described herein
is an immolating
circular or linear polyribonucleotide, a cleavable circular or linear
polyribonucleotide, or a self-cleaving
circular or linear polyribonucleotide. A circular or linear polyribonucleotide
can deliver cellular
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components including, for example, RNA, IncRNA, lincRNA, miRNA, tRNA, rRNA,
snoRNA, ncRNA,
siRNA, or shRNA. In some embodiments, a circular or linear polyribonucleotide
includes miRNA
separated by (i) self-cleavable elements; (ii) cleavage recruitment sites;
(iii) degradable linkers; (iv)
chemical linkers; and/or (v) spacer sequences. In some embodiments, circRNA
includes siRNA
separated by (i) self-cleavable elements; (ii) cleavage recruitment sites
(e.g., ADAR); (iii) degradable
linkers (e.g., glycerol); (iv) chemical linkers; and/or (v) spacer sequences.
Non-limiting examples of self-
cleavable elements include hammerhead, splicing element, hairpin, hepatitis
delta virus (HDV), Varkud
Satellite (VS), and glmS ribozymes.
Regulatory Elements and Ratio of Expression Products
In some embodiments, a circular or linear polyribonucleotide includes one or
more regulatory
nucleic acid sequences or includes one or more expression sequences that
encode regulatory nucleic
acid, e.g., a nucleic acid that modifies expression of an endogenous gene
and/or an exogenous gene. In
some embodiments, the expression sequence of a circular or linear
polyribonucleotide as provided herein
can include a sequence that is antisense to a regulatory nucleic acid like a
non-coding RNA, such as, but
not limited to, tRNA, IncRNA, miRNA, rRNA, snRNA, microRNA, siRNA, piRNA,
snoRNA, snRNA,
exRNA, scaRNA, Y RNA, and hnRNA.
Exemplary regulatory nucleic acids are described in paragraphs [0177] - [0194]
of International
Patent Publication No. W02019/118919, which is hereby incorporated by
reference in its entirety.
In some embodiments, the translation efficiency of a circular
polyribonucleotide as provided herein
is greater than a reference, e.g., a linear counterpart, a linear expression
sequence, or a linear
polyribonucleotide for circularization. In some embodiments, a circular
polyribonucleotide as provided
herein has the translation efficiency that is at least about 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%,
200%, 250%,
300%, 350%, 400%, 450%, 500%, 600%, 70%, 800%, 900%, 1000%, 2000%, 5000%,
10000%,
100000%, or more greater than that of a reference. In some embodiments, a
circular polyribonucleotide
has a translation efficiency 10% greater than that of a linear counterpart. In
some embodiments, a
circular polyribonucleotide has a translation efficiency 300% greater than
that of a linear counterpart.
In some embodiments, a circular or linear polyribonucleotide produces
stoichiometric ratios of
.. expression products. Rolling circle translation continuously produces
expression products at substantially
equivalent ratios. In some embodiments, the circular or linear
polyribonucleotide has a stoichiometric
translation efficiency, such that expression products are produced at
substantially equivalent ratios. In
some embodiments, the circular or linear polyribonucleotide has a
stoichiometric translation efficiency of
multiple expression products, e.g., products from 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, or more expression
sequences. In some embodiments, the circular or linear polyribonucleotide
produces substantially
different ratios of expression products. For example, the translation
efficiency of multiple expression
products may have a ratio of 1:10,000; 1:7000, 1:5000, 1:1000, 1:700, 1:500,
1:100, 1:50, 1:10, 1:5, 1:4,
1:3 or 1:2. In some embodiments, the ratio of multiple expression products may
be modified using a
regulatory element.
Translation
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In some embodiments, once translation of a circular polyribonucleotide is
initiated, the ribosome
bound to the circular polyribonucleotide does not disengage from the circular
polyribonucleotide before
finishing at least one round of translation of the circular
polyribonucleotide. In some embodiments, the
circular polyribonucleotide as described herein is competent for rolling
circle translation. In some
embodiments, during rolling circle translation, once translation of the
circular polyribonucleotide is
initiated, the ribosome bound to the circular polyribonucleotide does not
disengage from the circular
polyribonucleotide before finishing at least 2 rounds, at least 3 rounds, at
least 4 rounds, at least 5
rounds, at least 6 rounds, at least 7 rounds, at least 8 rounds, at least 9
rounds, at least 10 rounds, at
least 11 rounds, at least 12 rounds, at least 13 rounds, at least 14 rounds,
at least 15 rounds, at least 20
rounds, at least 30 rounds, at least 40 rounds, at least 50 rounds, at least
60 rounds, at least 70 rounds,
at least 80 rounds, at least 90 rounds, at least 100 rounds, at least 150
rounds, at least 200 rounds, at
least 250 rounds, at least 500 rounds, at least 1000 rounds, at least 1500
rounds, at least 2000 rounds, at
least 5000 rounds, at least 10000 rounds, at least 105 rounds, or at least 106
rounds of translation of the
circular polyribonucleotide.
In some embodiments, the rolling circle translation of a circular
polyribonucleotide leads to
generation of polypeptide product that is translated from more than one round
of translation of the circular
polyribonucleotide ("continuous" expression product). In some embodiments, the
circular
polyribonucleotide includes a stagger element, and rolling circle translation
of the circular
polyribonucleotide leads to generation of polypeptide product that is
generated from a single round of
translation or less than a single round of translation of the circular
polyribonucleotide ("discrete"
expression product). In some embodiments, the circular polyribonucleotide is
configured such that at least
10%, 20%, 30%, 40%, 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% of total polypeptides
(molar/molar) generated
during the rolling circle translation of the circular polyribonucleotide are
discrete polypeptides. In some
embodiments, the circular polyribonucleotide is configured such that at least
99% of the total polypeptides
are discrete polypeptides. In some embodiments, the amount ratio of the
discrete products over the total
polypeptides is tested in an in vitro translation system. In some embodiments,
the in vitro translation
system used for the test of amount ratio includes rabbit reticulocyte lysate.
In some embodiments, the
amount ratio is tested in an in vivo translation system, such as a eukaryotic
cell or a prokaryotic cell, a
cultured cell, or a cell in an organism.
Untranslated Regions
In some embodiments, a circular polyribonucleotide includes untranslated
regions (UTRs). UTRs
of a genomic region including a gene may be transcribed but not translated. In
some embodiments, a
UTR may be included upstream of the translation initiation sequence of an
expression sequence
described herein. In some embodiments, a UTR may be included downstream of an
expression
sequence described herein. In some instances, one UTR for first expression
sequence is the same as or
continuous with or overlapping with another UTR for a second expression
sequence. In some
embodiments, the intron is a human intron. In some embodiments, the intron is
a full-length human
intron, e.g., ZKSCAN1.
Exemplary untranslated regions are described in paragraphs [0197] ¨[201] of
International
Patent Publication No. W02019/118919, which is hereby incorporated by
reference in its entirety.
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In some embodiments, a circular polyribonucleotide includes a poly-A sequence.
Exemplary
poly-A sequences are described in paragraphs [0202] ¨ [0205] of International
Patent Publication No.
W02019/118919, which is hereby incorporated by reference in its entirety. In
some embodiments, a
circular polyribonucleotide lacks a poly-A sequence.
In some embodiments, a circular polyribonucleotide includes a UTR with one or
more stretches of
Adenosines and Uridines embedded within. These AU rich signatures may increase
turnover rates of the
expression product.
Introduction, removal, or modification of UTR AU rich elements (AREs) may be
useful to
modulate the stability, or immunogenicity (e.g., the level of one or more
marker of an immune or
inflammatory response) of the circular polyribonucleotide. When engineering
specific circular
polyribonucleotides, one or more copies of an ARE may be introduced to the
circular polyribonucleotide
and the copies of an ARE may modulate translation and/or production of an
expression product.
Likewise, AREs may be identified and removed or engineered into the circular
polyribonucleotide to
modulate the intracellular stability and thus affect translation and
production of the resultant protein.
It should be understood that any UTR from any gene may be incorporated into
the respective
flanking regions of the circular polyribonucleotide.
In some embodiments, a circular polyribonucleotide lacks a 5'-UTR and is
competent for protein
expression from its one or more expression sequences. In some embodiments, the
circular
polyribonucleotide lacks a 3'-UTR and is competent for protein expression from
its one or more
expression sequences. In some embodiments, the circular polyribonucleotide
lacks a poly-A sequence
and is competent for protein expression from its one or more expression
sequences. In some
embodiments, the circular polyribonucleotide lacks a termination element and
is competent for protein
expression from its one or more expression sequences. In some embodiments, the
circular
polyribonucleotide lacks an internal ribosomal entry site and is competent for
protein expression from its
one or more expression sequences. In some embodiments, the circular
polyribonucleotide lacks a cap
and is competent for protein expression from its one or more expression
sequences. In some
embodiments, the circular polyribonucleotide lacks a 5'-UTR, a 3'-UTR, and an
IRES, and is competent
for protein expression from its one or more expression sequences. In some
embodiments, the circular
polyribonucleotide includes one or more of the following sequences: a sequence
that encodes one or
more miRNAs, a sequence that encodes one or more replication proteins, a
sequence that encodes an
exogenous gene, a sequence that encodes a therapeutic, a regulatory element
(e.g., translation
modulator, e.g., translation enhancer or suppressor), a translation initiation
sequence, one or more
regulatory nucleic acids that targets endogenous genes (e.g., siRNA, IncRNAs,
shRNA), and a sequence
that encodes a therapeutic mRNA or protein.
In some embodiments, a circular polyribonucleotide lacks a 5'-UTR. In some
embodiments, the
circular polyribonucleotide lacks a 3'-UTR. In some embodiments, the circular
polyribonucleotide lacks a
poly-A sequence. In some embodiments, the circular polyribonucleotide lacks a
termination element. In
some embodiments, the circular polyribonucleotide lacks an internal ribosomal
entry site. In some
embodiments, the circular polyribonucleotide lacks degradation susceptibility
by exonucleases. In some
embodiments, the fact that the circular polyribonucleotide lacks degradation
susceptibility can mean that
the circular polyribonucleotide is not degraded by an exonuclease, or only
degraded in the presence of an
exonuclease to a limited extent, e.g., that is comparable to or similar to in
the absence of exonuclease. In
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some embodiments, the circular polyribonucleotide is not degraded by
exonucleases. In some
embodiments, the circular polyribonucleotide has reduced degradation when
exposed to exonuclease. In
some embodiments, the circular polyribonucleotide lacks binding to a cap-
binding protein. In some
embodiments, the circular polyribonucleotide lacks a 5' cap.
Termination Sequence
A circular polyribonucleotide can include one or more expression sequences
(e.g., encoding an
immunogen and/or encoding an adjuvant), and each expression sequence may or
may not have a
termination element. Further examples of termination elements are described in
paragraphs [0169] -
[0170] of International Patent Publication No. W02019/118919, which is hereby
incorporated by
reference in its entirety.
In some embodiments, the circular polyribonucleotide includes a poly-A
sequence. In some
embodiments, the length of a poly-A sequence is greater than 10 nucleotides in
length. In one
embodiment, the poly-A sequence is greater than 15 nucleotides in length
(e.g., at least or greater than
about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140,
160, 180, 200, 250, 300, 350,
400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500,
1,600, 1,700, 1,800, 1,900,
2,000, 2,500, and 3,000 nucleotides). In some embodiments, the poly-A sequence
is designed according
to the descriptions of the poly-A sequence in [0202]-[0204] of International
Patent Publication No.
W02019/118919A1, which is incorporated herein by reference in its entirety.
In some embodiments, a circular polyribonucleotide includes a polyA, lacks a
polyA, or has a
modified polyA to modulate one or more characteristics of the circular
polyribonucleotide. In some
embodiments, the circular polyribonucleotide lacking a polyA or having
modified polyA improves one or
more functional characteristics, e.g., immunogenicity (e.g., the level of one
or more marker of an immune
or inflammatory response), half-life, expression efficiency, etc.
Regulatory Nucleic Acids
In some embodiments, a circular polyribonucleotide includes one or more
expression sequences
that encode regulatory nucleic acid, e.g., that modifies expression of an
endogenous gene and/or an
exogenous gene. In some embodiments, the expression sequence of a circular
polyribonucleotide as
provided herein can include a sequence that is antisense to a regulatory
nucleic acid like a non-coding
RNA, such as, but not limited to, tRNA, IncRNA, miRNA, rRNA, snRNA, microRNA,
siRNA, piRNA,
snoRNA, snRNA, exRNA, scaRNA, Y RNA, and hnRNA.
In one embodiment, the regulatory nucleic acid targets a gene such as a host
gene. The
regulatory nucleic acids may include any of the regulatory nucleic acids
described in [0177] and [0181]-
[0189] of International Patent Publication No. W02019/118919A1, which is
incorporated herein by
reference in its entirety.
In some embodiments, an expression sequence includes one or more of the
features described
herein, e.g., a sequence encoding one or more peptides or proteins, one or
more regulatory element, one
or more regulatory nucleic acids, e.g., one or more non-coding RNAs, other
expression sequences, and
any combination thereof.
In some embodiments, a circular polyribonucleotide includes one or more RNA
binding sites.
microRNAs (or miRNA) are short noncoding RNAs that bind to the 3'UTR of
nucleic acid molecules and
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down-regulate gene expression either by reducing nucleic acid molecule
stability or by inhibiting
translation. The circular polyribonucleotide may include one or more microRNA
target sequences,
microRNA sequences, or microRNA seeds. Such sequences may correspond to any
known microRNA,
such as those taught in US Publication U52005/0261218 and US Publication
U52005/0059005, the
.. contents of which are incorporated herein by reference in their entirety. A
microRNA sequence includes a
"seed" region, i.e., a sequence in the region of positions 2-8 of the mature
microRNA, which sequence
has perfect Watson- Crick complementarity to the miRNA target sequence. A
microRNA seed may
include positions 2-8 or 2-7 of the mature microRNA. In some embodiments, a
microRNA seed may
include 7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA), wherein
the seed-complementary
site in the corresponding miRNA target is flanked by an adenine (A) opposed to
microRNA position 1. In
some embodiments, a microRNA seed may include 6 nucleotides (e.g., nucleotides
2-7 of the mature
microRNA), wherein the seed-complementary site in the corresponding miRNA
target is flanked by an
adenine (A) opposed to microRNA position 1. See for example, Grimson et al;
Mol Cell. 2007 6;27:91-
105; herein incorporated by reference in its entirety.
Protein-Binding
In some embodiments, a circular polyribonucleotide includes one or more
protein binding sites
that enable a protein, e.g., a ribosome, to bind to an internal site in the
RNA sequence. By engineering
protein binding sites, e.g., ribosome binding sites, into the circular
polyribonucleotide, the circular
polyribonucleotide may evade or have reduced detection by the host's immune
system, have modulated
degradation, or modulated translation, by masking the circular
polyribonucleotide from components of the
host's immune system.
In some embodiments, a circular polyribonucleotide includes at least one
immunoprotein binding
site, for example to evade immune responses, e.g., CTL (cytotoxic T
lymphocyte) responses. In some
embodiments, the immunoprotein binding site is a nucleotide sequence that
binds to an immunoprotein
and aids in masking the circular polyribonucleotide as exogenous. In some
embodiments, the
immunoprotein binding site is a nucleotide sequence that binds to an
immunoprotein and aids in hiding
the circular polyribonucleotide as exogenous or foreign.
Traditional mechanisms of ribosome engagement to linear RNA involve ribosome
binding to the
capped 5 end of an RNA. From the 5' end, the ribosome migrates to an
initiation codon, whereupon the
first peptide bond is formed. According to the present disclosure, internal
initiation (i.e., cap-independent)
of translation of the circular polyribonucleotide does not require a free end
or a capped end. Rather, a
ribosome binds to a non-capped internal site, whereby the ribosome begins
polypeptide elongation at an
initiation codon. In some embodiments, the circular polyribonucleotide
includes one or more RNA
sequences including a ribosome binding site, e.g., an initiation codon.
Natural 5'UTRs bear features which play roles in for translation initiation.
They harbor signatures
like Kozak sequences which are commonly known to be involved in the process by
which the ribosome
initiates translation of many genes. Kozak sequences have the consensus
CCR(A/G)CCAUGG (SEQ ID
NO: 29), where R is a purine (adenine or guanine) three bases upstream of the
start codon (AUG), which
is followed by another G. 5 'UTR also have been known to form secondary
structures which are involved
in elongation factor binding.
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In some embodiments, a circular polyribonucleotide encodes a protein binding
sequence that
binds to a protein. In some embodiments, the protein binding sequence targets
or localizes the circular
polyribonucleotide to a specific target. In some embodiments, the protein
binding sequence specifically
binds an arginine-rich region of a protein.
In some embodiments, the protein binding site includes, but is not limited to,
a binding site to the
protein such as ACIN1, AGO, APOBEC3F, APOBEC3G, ATXN2, AUH, BCCIP, CAPRIN1,
CELF2,
CPSF1, CPSF2, CPSF6, CPSF7, CSTF2, CSTF2T, CTCF, DDX21, DDX3, DDX3X, DDX42,
DGCR8,
ElF3A, ElF4A3, ElF4G2, ELAVL1, ELAVL3, FAM120A, FBL, FIP1L1, FKBP4, FMR1, FUS,
FXR1, FXR2,
GNL3, GTF2F1, HNRNPA1, HNRNPA2B1, HNRNPC, HNRNPK, HNRNPL, HNRNPM, HNRNPU,
HNRNPUL1, IGF2BP1, IGF2BP2, IGF2BP3, ILF3, KHDRBS1, LARP7, LIN28A, LIN28B,
m6A, MBNL2,
METTL3, MOV10, MSI1, MSI2, NONO, NONO-, N0P58, NPM1, NUDT21, PCBP2, POLR2A,
PRPF8,
PTBP1, RBFOX2, RBM10, RBM22, RBM27, RBM47, RNPS1, SAFB2, SBDS, SF3A3, SF3B4,
SIRT7,
SLBP, SLTM, SMNDC1, SND1, SRRM4, SRSF1, SRSF3, SRSF7, SRSF9, TAF15, TARDBP,
TIA1,
TNRC6A, TOP3B, TRA2A, TRA2B, U2AF1, U2AF2, UNK, UPF1, WDR33, XRN2, YBX1,
YTHDC1,
YTHDF1, YTHDF2, YWHAG, ZC3H7B, PDK1, AKT1, and any other protein that binds
RNA.
Encryptogen
As described herein, the circular polyribonucleotide includes an encryptogen
to reduce, evade or
avoid the innate immune response of a cell. In one aspect, provided herein are
circular
polyribonucleotide which when delivered to cells, results in a reduced immune
response from the host as
compared to the response triggered by a reference compound, e.g. a linear
polynucleotide corresponding
to the described circular polyribonucleotide or a circular polyribonucleotide
lacking an encryptogen. In
some embodiments, the circular polyribonucleotide has less immunogenicity
(e.g., a lower level of one or
more marker of an immune or inflammatory response) than a counterpart lacking
an encryptogen.
In some embodiments, an encryptogen enhances stability. There is growing body
of evidence
about the regulatory roles played by the UTRs in terms of stability of a
nucleic acid molecule and
translation. The regulatory features of a UTR may be included in the
encryptogen to enhance the stability
of the circular polyribonucleotide.
In some embodiments, 5'- or 3'-UTRs can constitute encryptogens in a circular
polyribonucleotide. For example, removal or modification of UTR AU rich
elements (AREs) may be useful
to modulate the stability or immunogenicity (e.g., the modulate the level of
one or more marker of an
immune or inflammatory response) of the circular polyribonucleotide.
In some embodiments, removal of modification of AU rich elements (AREs) in
expression
sequence, e.g., translatable regions, can be useful to modulate the stability
or immunogenicity (e.g.,
modulate the level of one or more marker of an immune or inflammatory
response) of the circular
polyribonucleotide.
In some embodiments, an encryptogen includes miRNA binding site or binding
site to any other
non-coding RNAs. For example, incorporation of miR-142 sites into the circular
polyribonucleotide
described herein may not only modulate expression in hematopoietic cells, but
also reduce or abolish
immune responses to a protein encoded in the circular polyribonucleotide.
In some embodiments, an encryptogen includes one or more protein binding sites
that enable a
protein, e.g., an immunoprotein, to bind to the RNA sequence. By engineering
protein binding sites into
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the circular polyribonucleotide, the circular polyribonucleotide may evade or
have reduced detection by
the host's immune system, have modulated degradation, or modulated
translation, by masking the
circular polyribonucleotide from components of the host's immune system. In
some embodiments, the
circular polyribonucleotide includes at least one immunoprotein binding site,
for example to evade
immune responses, e.g., CTL responses. In some embodiments, the immunoprotein
binding site is a
nucleotide sequence that binds to an immunoprotein and aids in masking the
circular polyribonucleotide
as exogenous.
In some embodiments, an encryptogen includes one or more modified nucleotides.
Exemplary
modifications can include any modification to the sugar, the nucleobase, the
internucleoside linkage (e.g.
to a linking phosphate / to a phosphodiester linkage / to the phosphodiester
backbone), and any
combination thereof that can prevent or reduce immune response against the
circular polyribonucleotide.
Some of the exemplary modifications are provided herein.
In some embodiments, a circular polyribonucleotide includes one or more
modifications as
described elsewhere herein to reduce an immune response from the host as
compared to the response
triggered by a reference compound, e.g. a circular polyribonucleotide lacking
the modifications. In
particular, the addition of one or more inosine has been shown to discriminate
RNA as endogenous
versus viral. See for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks
dsRNA as "self". Cell
Res. 25, 1283-1284, which is incorporated by reference in its entirety.
In some embodiments, a circular polyribonucleotide includes one or more
expression sequences
for shRNA or an RNA sequence that can be processed into siRNA, and the shRNA
or siRNA targets RIG-
I and reduces expression of RIG-I. RIG-I can sense foreign circular RNA and
leads to degradation of
foreign circular RNA. Therefore, a circular polynucleotide harboring sequences
for RIG-1-targeting
shRNA, siRNA or any other regulatory nucleic acids can reduce immunity, e.g.,
host cell immunity,
against the circular polyribonucleotide.
In some embodiments, a circular polyribonucleotide lacks a sequence, element,
or structure, that
aids the circular polyribonucleotide in reducing, evading, or avoiding an
innate immune response of a cell.
In some such embodiments, the circular polyribonucleotide may lack a polyA
sequence, a 5' end, a 3'
end, phosphate group, hydroxyl group, or any combination thereof.
Nucleotide Spacer Sequences
In some embodiments, a circular polyribonucleotide includes a spacer sequence.
In some
embodiments, the circular polyribonucleotide includes at least one spacer
sequence. In some
embodiments, the circular polyribonucleotide includes 1, 2, 3, 4, 5, 6, 7, or
more spacer sequences.
In some embodiments, a circular polyribonucleotide includes a spacer sequence.
In some
embodiments, elements of a polyribonucleotide may be separated from one
another by a spacer
sequence or linker. Exemplary spacer sequences are described in paragraphs
[0293] ¨ [0302] of
International Patent Publication No.W02019/118919, which is hereby
incorporated by reference in its
entirety.
Non-Nucleic Acid Linkers
A circular polyribonucleotide described herein may include a non-nucleic acid
linker. In some
embodiments, the circular polyribonucleotide has a non-nucleic acid linker
between one or more of the
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sequences or elements described herein. In one embodiment, one or more
sequences or elements
described herein are linked with the linker. The non-nucleic acid linker may
be a chemical bond, e.g., one
or more covalent bonds or non-covalent bonds. In some embodiments, the non-
nucleic acid linker is a
peptide or protein linker. Such a linker may be between 2-30 amino acids, or
longer. The circular
polyribonucleotide described herein may also include a non-nucleic acid
linker. Exemplary non-nucleic
acid linkers are described in paragraphs [0303] ¨ [0307] of International
Patent Publication No.
W02019/118919, which is hereby incorporated by reference in its entirety.
In some embodiments, a circular polyribonucleotide further includes another
nucleic acid
sequence. In some embodiments, the circular polyribonucleotide may include
other sequences that
include DNA, RNA, or artificial nucleic acids. The other sequences may
include, but are not limited to,
genomic DNA, cDNA, or sequences that encode tRNA, mRNA, rRNA, miRNA, gRNA,
siRNA, or other
RNAi molecules. In some embodiments, the circular polyribonucleotide includes
an siRNA to target a
different locus of the same gene expression product as the circular
polyribonucleotide. In some
embodiments, the circular polyribonucleotide includes an siRNA to target a
different gene expression
product than a gene expression product that is present in the circular
polyribonucleotide.
Stability and Half-Life
In some embodiments, a circular polyribonucleotide includes particular
sequence characteristics.
For example, the circular polyribonucleotide may include a particular
nucleotide composition. In some
such embodiments, the circular polyribonucleotide may include one or more
purine (adenine and/or
guanosine) rich regions. In some such embodiments, the circular
polyribonucleotide may include one or
more purine poor regions. In some embodiments, the circular polyribonucleotide
may include one or
more AU rich regions or elements (AREs). In some embodiments, the circular
polyribonucleotide may
include one or more adenine rich regions.
In some embodiments, a circular polyribonucleotide may include one or more
repetitive elements
described elsewhere herein. In some embodiments, the circular
polyribonucleotide includes one or more
modifications described elsewhere herein.
A circular polyribonucleotide may include one or more substitutions,
insertions and/or additions,
deletions, and covalent modifications with respect to reference sequences. For
example, circular
polyribonucleotides with one or more insertions, additions, deletions, and/or
covalent modifications
relative to a parent polyribonucleotide are included within the scope of this
disclosure. Exemplary
modifications are described in paragraphs [0310] ¨ [0325] of International
Patent Publication No.
W02019/118919, which is hereby incorporated by reference in its entirety.
In some embodiments, a circular polyribonucleotide includes a higher order
structure, e.g., a
secondary or tertiary structure. In some embodiments, complementary segments
of the circular
polyribonucleotide fold itself into a double stranded segment, held together
with hydrogen bonds between
pairs, e.g., A-U and C-G. In some embodiments, helices, also known as stems,
are formed intra-
molecularly, having a double-stranded segment connected to an end loop. In
some embodiments, the
circular polyribonucleotide has at least one segment with a quasi-double-
stranded secondary structure.
In some embodiments, one or more sequences of a circular polyribonucleotide
include
substantially single stranded vs double stranded regions. In some embodiments,
the ratio of single
stranded to double stranded may influence the functionality of the circular
polyribonucleotide.
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In some embodiments, one or more sequences of the circular polyribonucleotide
that are
substantially single stranded. In some embodiments, one or more sequences of
the circular
polyribonucleotide that are substantially single stranded may include a
protein- or RNA-binding site. In
some embodiments, the circular polyribonucleotide sequences that are
substantially single stranded may
be conformationally flexible to allow for increased interactions. In some
embodiments, the sequence of
the circular polyribonucleotide is purposefully engineered to include such
secondary structures to bind or
increase protein or nucleic acid binding.
In some embodiments, a circular polyribonucleotide is substantially double
stranded. In some
embodiments, one or more sequences of the circular polyribonucleotide that are
substantially double
stranded may include a conformational recognition site, e.g., a riboswitch or
aptazyme. In some
embodiments, the circular polyribonucleotide sequences that are substantially
double stranded may be
conformationally rigid. In some such instances, the conformationally rigid
sequence may sterically hinder
the circular polyribonucleotide from binding a protein or a nucleic acid. In
some embodiments, the
sequence of the circular polyribonucleotide is purposefully engineered to
include such secondary
structures to avoid or reduce protein or nucleic acid binding.
There are 16 possible base-pairings, however of these, six (AU, GU, GC, UA,
UG, CG) may form
actual base-pairs. The rest are called mismatches and occur at very low
frequencies in helices. In some
embodiments, the structure of the circular polyribonucleotide cannot easily be
disrupted without impact on
its function and lethal consequences, which provide a selection to maintain
the secondary structure. In
some embodiments, the primary structure of the stems (i.e., their nucleotide
sequence) can still vary,
while still maintaining helical regions. The nature of the bases is secondary
to the higher structure, and
substitutions are possible as long as they preserve the secondary structure.
In some embodiments, the
circular polyribonucleotide has a quasi-helical structure. In some
embodiments, the circular
polyribonucleotide has at least one segment with a quasi-helical structure. In
some embodiments, the
circular polyribonucleotide includes at least one of a U-rich or A-rich
sequence or a combination thereof.
In some embodiments, the U-rich and/or A-rich sequences are arranged in a
manner that would produce
a triple quasi-helix structure. In some embodiments, the circular
polyribonucleotide has a double quasi-
helical structure. In some embodiments, the circular polyribonucleotide has
one or more segments (e.g.,
2, 3, 4, 5, 6, or more) having a double quasi-helical structure. In some
embodiments, the circular
polyribonucleotide includes at least one of a C-rich and/or G-rich sequence.
In some embodiments, the
C-rich and/or G-rich sequences are arranged in a manner that would produce
triple quasi-helix structure.
In some embodiments, the circular polyribonucleotide has an intramolecular
triple quasi-helix structure
that aids in stabilization.
In some embodiments, a circular polyribonucleotide has two quasi-helical
structure (e.g.,
separated by a phosphodiester linkage), such that their terminal base pairs
stack, and the quasi-helical
structures become colinear, resulting in a "coaxially stacked" substructure.
In some embodiments, a circular polyribonucleotide includes a tertiary
structure with one or more
motifs, e.g., a pseudoknot, a g-quadruplex, a helix, and coaxial stacking.
Further examples of structure of circular polyribonucleotides as disclosed
herein are described in
paragraphs [0326] ¨ [0333] of International Patent Publication No.
W02019/118919, which is hereby
incorporated by reference in its entirety.
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As a result of its circularization, a circular polyribonucleotide may include
certain characteristics
that distinguish it from linear RNA. For example, the circular
polyribonucleotide is less susceptible to
degradation by exonuclease as compared to linear RNA. As such, a circular
polyribonucleotide can be
more stable than a linear RNA, especially when incubated in the presence of an
exonuclease. The
increased stability of the circular polyribonucleotide compared with linear
RNA can make the circular
polyribonucleotide more useful as a cell transforming reagent to produce
polypeptides (e.g., immunogens
and/or adjuvants). The increased stability of the circular polyribonucleotide
compared with linear RNA
can make the circular polyribonucleotide easier to store for longer than
linear RNA. The stability of the
circular polyribonucleotide treated with exonuclease can be tested using
methods standard in art which
determine whether RNA degradation has occurred (e.g., by gel electrophoresis).
Moreover, unlike linear RNA, a circular polyribonucleotide can be less
susceptible to
dephosphorylation when the circular polyribonucleotide is incubated with
phosphatase, such as calf
intestine phosphatase.
In some embodiments, a circular polyribonucleotide preparation provided herein
has an
increased half-life over a reference, e.g., a linear polyribonucleotide having
the same nucleotide
sequence but is not circularized (e.g., linear counterpart). In some
embodiments, the circular
polyribonucleotide is resistant to degradation, e.g., exonuclease. In some
embodiments, the circular
polyribonucleotide is resistant to self-degradation. In some embodiments, the
circular polyribonucleotide
lacks an enzymatic cleavage site, e.g., a dicer cleavage site. In some
embodiments, the circular
polyribonucleotide has a half-life at least about 5%, at least about 10%, at
least about 20%, at least about
30%, at least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%,
at least about 90%, at least about 100%, at least about 120%, at least about
140%, at least about 150%,
at least about 160%, at least about 180%, at least about 200%, at least about
300%, at least about 400%,
at least about 500%, at least about 600%, at least about 700% at least about
800%, at least about 900%õ
at least about 1000% or at least about 10000%, longer than a reference, e.g.,
a linear counterpart.
In some embodiments, the circular polyribonucleotide persists in a cell during
cell division. In
some embodiments, the circular polyribonucleotide persists in daughter cells
after mitosis. In some
embodiments, the circular polyribonucleotide is replicated within a cell and
is passed to daughter cells. In
some embodiments, the circular polyribonucleotide includes a replication
element that mediates self-
replication of the circular polyribonucleotide. In some embodiments, the
replication element mediates
transcription of the circular polyribonucleotide into a linear
polyribonucleotide that is complementary to the
circular polyribonucleotide (linear complementary). In some embodiments, the
linear complementary
polyribonucleotide can be circularized in vivo in cells into a complementary
circular polyribonucleotide. In
some embodiments, the complementary polyribonucleotide can further self-
replicate into another circular
polyribonucleotide, which has the same or similar nucleotide sequence as the
starting circular
polyribonucleotide. One exemplary self-replication element includes HDV
replication domain (as
described by Beeharry et al, Virol, 2014, 450-451:165-173). In some
embodiments, a cell passes at least
one circular polyribonucleotide to daughter cells with an efficiency of at
least 25%, 50%, 60%, 70%, 80%,
85%, 90%, 95%, or 99%. In some embodiments, cell undergoing meiosis passes the
circular
polyribonucleotide to daughter cells with an efficiency of at least 25%, 50%,
60%, 70%, 80%, 85%, 90%,
95%, or 99%. In some embodiments, a cell undergoing mitosis passes the
circular polyribonucleotide to
daughter cells with an efficiency of at least 25%, 50%, 60%, 70%, 80%, 85%,
90%, 95%, or 99%.
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Further examples of stability and half-life of circular polyribonucleotides as
disclosed herein are
described in paragraphs [0308] ¨ [0309] of International Patent Publication
No. W02019/118919, which is
hereby incorporated by reference in its entirety.
Modifications
A circular polyribonucleotide may include one or more substitutions,
insertions and/or additions,
deletions, and covalent modifications with respect to reference sequences, in
particular, the parent
polyribonucleotide, are included within the scope of this disclosure.
In some embodiments, a circular polyribonucleotide includes one or more post-
transcriptional
modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A
sequence, methylation, acylation,
phosphorylation, methylation of lysine and arginine residues, acetylation, and
nitrosylation of thiol groups
and tyrosine residues, etc.). The one or more post-transcriptional
modifications can be any post-
transcriptional modification, such as any of the more than one hundred
different nucleoside modifications
that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J.
(1999). The RNA Modification
Database: 1999 update. Nucl Acids Res 27: 196-197). In some embodiments, the
first isolated nucleic
acid includes messenger RNA (mRNA). In some embodiments, the mRNA includes at
least one
nucleoside selected from the group such as those described in [0311] of
International Patent Publication
No. W02019/118919A1, which is incorporated herein by reference in its
entirety.
A circular polyribonucleotide may include any useful modification, such as to
the sugar, the
nucleobase, or the internucleoside linkage (e.g., to a linking phosphate / to
a phosphodiester linkage / to
the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may
be replaced or
substituted with optionally substituted amino, optionally substituted thiol,
optionally substituted alkyl (e.g.,
methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments,
modifications (e.g., one or more
modifications) are present in each of the sugar and the internucleoside
linkage. Modifications may be
modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs),
threose nucleic acids
(TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked
nucleic acids (LNAs) or hybrids
thereof). Additional modifications are described herein.
In some embodiments, a circular polyribonucleotide includes at least one
N(6)methyladenosine
(m6A) modification to increase translation efficiency. In some embodiments,
the N(6)methyladenosine
(m6A) modification can reduce immunogenicity (e.g., reduce the level of one or
more marker of an
immune or inflammatory response) of the circular polyribonucleotide.
In some embodiments, a modification may include a chemical or cellular induced
modification.
For example, some non-limiting examples of intracellular RNA modifications are
described by Lewis and
Pan in "RNA modifications and structures cooperate to guide RNA-protein
interactions" from Nat Reviews
Mol Cell Biol, 2017, 18:202-210.
In some embodiments, chemical modifications to the ribonucleotides of a
circular
polyribonucleotide may enhance immune evasion. The circular polyribonucleotide
may be synthesized
and/or modified by methods well established in the art, such as those
described in "Current protocols in
nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons,
Inc., New York, NY, USA, which
is hereby incorporated herein by reference. Modifications include, for
example, end modifications, e.g., 5'
end modifications (phosphorylation (mono-, di- and tri-), conjugation,
inverted linkages, etc.), 3 end
modifications (conjugation, DNA nucleotides, inverted linkages, etc.), base
modifications (e.g.,
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replacement with stabilizing bases, destabilizing bases, or bases that base
pair with an expanded
repertoire of partners), removal of bases (abasic nucleotides), or conjugated
bases. The modified
ribonucleotide bases may also include 5-methylcytidine and pseudouridine. In
some embodiments, base
modifications may modulate expression, immune response, stability, subcellular
localization, to name a
.. few functional effects, of the circular polyribonucleotide. In some
embodiments, the modification includes
a bi-orthogonal nucleotide, e.g., an unnatural base. See for example, Kimoto
et al, Chem Commun
(Camb), 2017, 53:12309, DOI: 10.1039/c7cc06661a, which is hereby incorporated
by reference.
In some embodiments, sugar modifications (e.g., at the 2 position or 4'
position) or replacement
of the sugar one or more ribonucleotides of the circular polyribonucleotide
may, as well as backbone
.. modifications, include modification or replacement of the phosphodiester
linkages. Specific examples of
circular polyribonucleotide include, but are not limited to, circular
polyribonucleotide including modified
backbones or no natural internucleoside linkages such as internucleoside
modifications, including
modification or replacement of the phosphodiester linkages. Circular
polyribonucleotides having modified
backbones include, among others, those that do not have a phosphorus atom in
the backbone. For the
purposes of this application, and as sometimes referenced in the art, modified
RNAs that do not have a
phosphorus atom in their internucleoside backbone can also be considered to be
oligonucleosides. In
particular embodiments, the circular polyribonucleotide will include
ribonucleotides with a phosphorus
atom in its internucleoside backbone.
Modified circular polyribonucleotide backbones may include, for example,
phosphorothioates,
.. chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and
other alkyl phosphonates such as 3'-alkylene phosphonates and chiral
phosphonates, phosphinates,
phosphoramidates such as 3'-amino phosphoramidate and
aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters,
and boranophosphates
having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having
inverted polarity wherein the
adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-
2'. Various salts, mixed salts and
free acid forms are also included. In some embodiments, the circular
polyribonucleotide may be
negatively or positively charged.
The modified nucleotides, which may be incorporated into the circular
polyribonucleotide, can be
modified on the internucleoside linkage (e.g., phosphate backbone). Herein, in
the context of the
polynucleotide backbone, the phrases "phosphate" and "phosphodiester" are used
interchangeably.
Backbone phosphate groups can be modified by replacing one or more of the
oxygen atoms with a
different substituent. Further, the modified nucleosides and nucleotides can
include the wholesale
replacement of an unmodified phosphate moiety with another internucleoside
linkage as described
herein. Examples of modified phosphate groups include, but are not limited to,
phosphorothioate,
phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen
phosphonates,
phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and
phosphotriesters.
Phosphorodithioates have both non-linking oxygens replaced by sulfur. The
phosphate linker can also be
modified by the replacement of a linking oxygen with nitrogen (bridged
phosphoramidates), sulfur
(bridged phosphorothioates), and carbon (bridged methylenephosphonates).
The a-thio substituted phosphate moiety is provided to confer stability to RNA
and DNA polymers
through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA
and RNA have
increased nuclease resistance and subsequently a longer half-life in a
cellular environment.
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Phosphorothioate linked to the circular polyribonucleotide is expected to
reduce the innate immune
response through weaker binding/activation of cellular innate immune
molecules.
In specific embodiments, a modified nucleoside includes an alpha-thio-
nucleoside (e.g., 5.-0-(l-
thiophosphate)-adenosine, 5.-0-(l-thiophosphate)-cytidine (a- thio-cytidine),
5.-0-(l-thiophosphate)-
guanosine, 5.-0-(l-thiophosphate)-uridine, or 5'-0- (1 -thiophosphate)-
pseudouridine).
Other internucleoside linkages that may be employed according to the present
disclosure,
including internucleoside linkages which do not contain a phosphorous atom,
are described herein.
In some embodiments, a circular polyribonucleotide may include one or more
cytotoxic
nucleosides. For example, cytotoxic nucleosides may be incorporated into
circular polyribonucleotide,
such as bifunctional modification. Cytotoxic nucleoside may include, but are
not limited to, adenosine
arabinoside, 5-azacytidine, 4'-thio- aracytidine, cyclopentenylcytosine,
cladribine, clofarabine, cytarabine,
cytosine arabinoside, I-(2-C-cyano-2-deoxy-beta-D-arabino- pentofuranosyl)-
cytosine, decitabine, 5-
fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur
and uracil, tegafur ((RS)-5-
fluoro-I-(tetrahydrofuran-2- yOpyrimidine-2,4(1H,3H)-dione), troxacitabine,
tezacitabine, 2'- deoxy-2'-
methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include
fludarabine phosphate,
N4-behenoyl-l-beta-D- arabinofuranosylcytosine, N4-octadecyl- 1 -beta-D-
arabinofuranosylcytosine, N4-
palmitoyl-I-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-
4055 (cytarabine 5'-
elaidic acid ester).
A circular polyribonucleotide may or may not be uniformly modified along the
entire length of the
molecule. For example, one or more or all types of nucleotide (e.g., naturally-
occurring nucleotides,
purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or
may not be uniformly modified
in the circular polyribonucleotide, or in a given predetermined sequence
region thereof. In some
embodiments, the circular polyribonucleotide includes a pseudouridine. In some
embodiments, the
circular polyribonucleotide includes an inosine, which may aid in the immune
system characterizing the
circular polyribonucleotide as endogenous versus viral RNAs. The incorporation
of inosine may also
mediate improved RNA stability/reduced degradation. See for example, Yu, Z. et
al. (2015) RNA editing
by ADAR1 marks dsRNA as "self". Cell Res. 25, 1283-1284, which is incorporated
by reference in its
entirety.
In some embodiments, all nucleotides in a circular polyribonucleotide (or in a
given sequence
region thereof) are modified. In some embodiments, the modification may
include an m6A, which may
augment expression; an inosine, which may attenuate an immune response;
pseudouridine, which may
increase RNA stability, or translational readthrough (stagger element), an
m5C, which may increase
stability; and a 2,2,7-trimethylguanosine, which aids subcellular
translocation (e.g., nuclear localization).
Different sugar modifications, nucleotide modifications, and/or
internucleoside linkages (e.g.,
backbone structures) may exist at various positions in a circular
polyribonucleotide. One of ordinary skill
in the art will appreciate that the nucleotide analogs or other
modification(s) may be located at any
position(s) of the circular polyribonucleotide, such that the function of the
circular polyribonucleotide is not
substantially decreased. A modification may also be a non-coding region
modification. The circular
polyribonucleotide may include from about 1% to about 100% modified
nucleotides (either in relation to
overall nucleotide content, or in relation to one or more types of nucleotide,
i.e. any one or more of A, G,
U or C) or any intervening percentage (e.g., from 1% to 20%>, from 1% to 25%,
from 1% to 50%, from
1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%,
from 10% to 20%, from
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10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to
80%, from 10% to
90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from
20% to 60%, from
20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to
100%, from 50% to
60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from
50% to 100%, from
70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to
90%, from 80% to
95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to
100%).
Structure
In some embodiments, a circular polyribonucleotide includes a higher order
structure, e.g., a
secondary or tertiary structure. In some embodiments, complementary segments
of the circular
polyribonucleotide fold itself into a double stranded segment, held together
with hydrogen bonds between
pairs, e.g., A-U and C-G. In some embodiments, helices, also known as stems,
are formed intra-
molecularly, having a double-stranded segment connected to an end loop. In
some embodiments, the
circular polyribonucleotide has at least one segment with a quasi-double-
stranded secondary structure.
In some embodiments, a segment having a quasi-double-stranded secondary
structure has at least 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more paired nucleotides. In
some embodiments, the circular
polyribonucleotide has one or more segments (e.g., 2, 3, 4, 5, 6, or more)
having a quasi-double-stranded
secondary structure. In some embodiments, the segments are separated by 3, 4,
5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, or more nucleotides.
In some embodiments, one or more sequences of a circular polyribonucleotide
include
substantially single stranded vs double stranded regions. In some embodiments,
the ratio of single
stranded to double stranded may influence the functionality of the circular
polyribonucleotide.
In some embodiments, one or more sequences of a circular polyribonucleotide
are substantially
single stranded. In some embodiments, one or more sequences of the circular
polyribonucleotide that
are substantially single stranded may include a protein- or RNA-binding site.
In some embodiments, the
circular polyribonucleotide sequences that are substantially single stranded
may be conformationally
flexible to allow for increased interactions. In some embodiments, the
sequence of the circular
polyribonucleotide is purposefully engineered to include such secondary
structures to bind or increase
protein or nucleic acid binding.
In some embodiments, a circular polyribonucleotide has at least one binding
site, e.g., at least
one protein binding site, at least one miRNA binding site, at least one IncRNA
binding site, at least one
tRNA binding site, at least one rRNA binding site, at least one snRNA binding
site, at least one siRNA
binding site, at least one piRNA binding site, at least one snoRNA binding
site, at least one snRNA
binding site, at least one exRNA binding site, at least one scaRNA binding
site, at least one Y RNA
binding site, at least one hnRNA binding site, and/or at least one tRNA motif.
In some embodiments, a circular polyribonucleotide is configured to include a
higher order
structure, such as those described in International Patent Publication No.
W02019/118919A1, which is
incorporated herein by reference in its entirety.
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Production Methods
In some embodiments, a circular polyribonucleotide includes a deoxyribonucleic
acid sequence
that is non-naturally occurring and can be produced using recombinant
technology (e.g., derived in vitro
using a DNA plasmid), chemical synthesis, or a combination thereof.
It is within the scope of the disclosure that a DNA molecule used to produce
an RNA circle can
include a DNA sequence of a naturally-occurring original nucleic acid
sequence, a modified version
thereof, or a DNA sequence encoding a synthetic polypeptide not normally found
in nature (e.g., chimeric
molecules or fusion proteins, such as fusion proteins including multiple
immunogens). DNA and RNA
molecules can be modified using a variety of techniques including, but not
limited to, classic mutagenesis
techniques and recombinant techniques, such as site-directed mutagenesis,
chemical treatment of a
nucleic acid molecule to induce mutations, restriction enzyme cleavage of a
nucleic acid fragment,
ligation of nucleic acid fragments, polymerase chain reaction (PCR)
amplification and/or mutagenesis of
selected regions of a nucleic acid sequence, synthesis of oligonucleotide
mixtures and ligation of mixture
groups to "build" a mixture of nucleic acid molecules and combinations
thereof.
A circular polyribonucleotide may be prepared according to any available
technique including, but
not limited to chemical synthesis and enzymatic synthesis. In some
embodiments, a linear primary
construct or linear mRNA may be cyclized, or concatemerized to create a
circular polyribonucleotide
described herein. The mechanism of cyclization or concatemerization may occur
through methods such
as, but not limited to, chemical, enzymatic, splint ligation), or ribozyme
catalyzed methods. The newly
formed 5 '-/3 '-linkage may be an intramolecular linkage or an intermolecular
linkage.
Methods of making circular polyribonucleotides described herein are described
in, for example,
Khudyakov & Fields, Artificial DNA: Methods and Applications, CRC Press
(2002); in Zhao, Synthetic
Biology: Tools and Applications, (First Edition), Academic Press (2013); and
Egli & Herdewijn, Chemistry
and Biology of Artificial Nucleic Acids, (First Edition), Wiley-VCH (2012).
Various methods of synthesizing circular polyribonucleotides are also
described in the art (see,
e.g., US Patent No. U5621 0931, US Patent No. U55773244, US Patent No.
U55766903, US Patent No.
U55712128, US Patent No. U55426180, US Publication No. U520100137407,
International Publication
No. W01992001813 and International Publication No. W02010/084371; the contents
of each of which
are herein incorporated by reference in their entireties).
Circularization
In some embodiments, a linear polyribonucleotide for circularization may be
cyclized, or
concatemerized. In some embodiments, the linear polyribonucleotide for
circularization may be cyclized
in vitro prior to formulation and/or delivery. In some embodiments, the linear
polyribonucleotide for
circularization may be cyclized within a cell.
Extracellular Circularization
In some embodiments, a linear polyribonucleotide for circularization is
cyclized, or
concatemerized using a chemical method to form a circular polyribonucleotide.
In some chemical
methods, the 5'-end and the 3'-end of the nucleic acid (e.g., a linear
polyribonucleotide for circularization)
includes chemically reactive groups that, when close together, may form a new
covalent linkage between
the 5'-end and the 3'-end of the molecule. The 5'-end may contain an NHS-ester
reactive group and the
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3'-end may contain a 3'-amino-terminated nucleotide such that in an organic
solvent the 3'-amino-
terminated nucleotide on the 3'-end of a linear RNA molecule will undergo a
nucleophilic attack on the 5'-
NHS-ester moiety forming a new 5.43.-amide bond.
In some embodiments, a DNA or RNA ligase is used to enzymatically link a 5'-
phosphorylated
nucleic acid molecule (e.g., a linear polyribonucleotide for circularization)
to the 3'-hydroxyl group of a
nucleic acid (e.g., a linear nucleic acid) forming a new phosphorodiester
linkage. In an example reaction,
a linear polyribonucleotide for circularization is incubated at 37 C for 1
hour with 1-10 units of T4 RNA
ligase (New England Biolabs, Ipswich, MA) according to the manufacturer's
protocol. The ligation
reaction may occur in the presence of a linear nucleic acid capable of base-
pairing with both the 5'- and
3'- region in juxtaposition to assist the enzymatic ligation reaction. In some
embodiments, the ligation is
splint ligation. For example, a splint ligase, like SplintRe ligase, can be
used for splint ligation. For splint
ligation, a single stranded polynucleotide (splint), like a single stranded
RNA, can be designed to
hybridize with both termini of a linear polyribonucleotide, so that the two
termini can be juxtaposed upon
hybridization with the single-stranded splint. Splint ligase can thus catalyze
the ligation of the juxtaposed
.. two termini of the linear polyribonucleotide, generating a circular
polyribonucleotide.
In some embodiments, a DNA or RNA ligase is used in the synthesis of the
circular
polynucleotides. In some embodiments, either the 5'-or 3'-end of the linear
polyribonucleotide for
circularization can encode a ligase ribozyme sequence such that during in
vitro transcription, the resultant
linear polyribonucleotide for circularization includes an active ribozyme
sequence capable of ligating the
5'-end of the linear polyribonucleotide for circularization to the 3'-end of
the linear polyribonucleotide for
circularization. The ligase ribozyme may be derived from the Group I Intron,
Hepatitis Delta Virus, Hairpin
ribozyme or may be selected by SELEX (systematic evolution of ligands by
exponential enrichment). The
ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and
37 C.
In some embodiments, a linear polyribonucleotide for circularization is
cyclized or
concatermerized by using at least one non-nucleic acid moiety. In one aspect,
the at least one non-
nucleic acid moiety may react with regions or features near the 5 terminus
and/or near the 3' terminus of
the linear polyribonucleotide for circularization in order to cyclize or
concatermerize the linear
polyribonucleotide for circularization. In another aspect, the at least one
non-nucleic acid moiety may be
located in or linked to or near the 5' terminus and/or the 3' terminus of the
linear polyribonucleotide for
circularization. The non-nucleic acid moieties contemplated may be homologous
or heterologous. As a
non-limiting example, the non-nucleic acid moiety may be a linkage such as a
hydrophobic linkage, ionic
linkage, a biodegradable linkage, and/or a cleavable linkage. As another non-
limiting example, the non-
nucleic acid moiety is a ligation moiety. As yet another non-limiting example,
the non-nucleic acid moiety
may be an oligonucleotide or a peptide moiety, such as an aptamer or a non-
nucleic acid linker as
.. described herein.
In some embodiments, a linear polyribonucleotide for circularization is
cyclized or
concatermerized due to a non-nucleic acid moiety that causes an attraction
between atoms, molecular
surfaces at, near or linked to the 5' and 3' ends of the linear
polyribonucleotide for circularization. As a
non-limiting example, one or more linear polyribonucleotides for
circularization may be cyclized or
.. concatemerized by intermolecular forces or intramolecular forces. Non-
limiting examples of
intermolecular forces include dipole-dipole forces, dipole-induced dipole
forces, induced dipole-induced
dipole forces, Van der Waals forces, and London dispersion forces. Non-
limiting examples of
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intramolecular forces include covalent bonds, metallic bonds, ionic bonds,
resonant bonds, agnostic
bonds, dipolar bonds, conjugation, hyperconjugation and antibonding.
In some embodiments, a linear polyribonucleotide for circularization may
include a ribozyme RNA
sequence near the 5 terminus and near the 3' terminus. The ribozyme RNA
sequence may covalently
.. link to a peptide when the sequence is exposed to the remainder of the
ribozyme. In one aspect, the
peptides covalently linked to the ribozyme RNA sequence near the 5' terminus
and the 3 'terminus may
associate with each other causing a linear polyribonucleotide for
circularization to cyclize or
concatemerize. In another aspect, the peptides covalently linked to the
ribozyme RNA near the 5'
terminus and the 3' terminus may cause the linear primary construct or linear
mRNA to cyclize or
concatemerize after being subjected to ligated using various methods known in
the art such as, but not
limited to, protein ligation. Non-limiting examples of ribozymes for use in
the linear primary constructs or
linear RNA of the present disclosure or a non-exhaustive listing of methods to
incorporate and/or
covalently link peptides are described in US patent application No.
US20030082768, the contents of
which is here in incorporated by reference in its entirety.
In some embodiments, a linear polyribonucleotide for circularization may
include a 5' triphosphate
of the nucleic acid converted into a 5' monophosphate, e.g., by contacting the
5' triphosphate with RNA 5'
pyrophosphohydrolase (RppH) or an ATP diphosphohydrolase (apyrase).
Alternately, converting the 5'
triphosphate of the linear polyribonucleotide for circularization into a 5'
monophosphate may occur by a
two-step reaction including: (a) contacting the 5' nucleotide of the linear
polyribonucleotide for
circularization with a phosphatase (e.g., Antarctic Phosphatase, Shrimp
Alkaline Phosphatase, or Calf
Intestinal Phosphatase) to remove all three phosphates; and (b) contacting the
5' nucleotide after step (a)
with a kinase (e.g., Polynucleotide Kinase) that adds a single phosphate.
In some embodiments, circularization efficiency of the circularization methods
provided herein is
at least about 10%, at least about 15%, at least about 20%, at least about
25%, at least about 30%, at
.. least about 35%, at least about 40%, at least about 45%, at least about
50%, at least about 60%, at least
about 70%, at least about 80%, at least about 90%, at least about 95%, or
100%. In some embodiments,
the circularization efficiency of the circularization methods provided herein
is at least about 40%. In some
embodiments, the circularization method provided has a circularization
efficiency of between about 10%
and about 100%; for example, the circularization efficiency may be about 15%,
about 20%, about 25%,
about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about
70%, about 75%, about 80%, about 85%, about 90%, about 95%, and about 99%. In
some embodiments,
the circularization efficiency is between about 20% and about 80%. In some
embodiments, the
circularization efficiency is between about 30% and about 60%. In some
embodiments the circularization
efficiency is about 40%.
Splicing Element
In some embodiment, a circular polyribonucleotide includes at least one
splicing element.
Exemplary splicing elements are described in paragraphs [0270] - [0275] of
International Patent
Publication No. W02019/118919, which is hereby incorporated by reference in
its entirety.
In some embodiments, a circular polyribonucleotide includes at least one
splicing element. In a
circular polyribonucleotide as provided herein, a splicing element can be a
complete splicing element that
can mediate splicing of the circular polyribonucleotide. Alternatively, the
splicing element can also be a
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residual splicing element from a completed splicing event. For instance, in
some cases, a splicing
element of a linear polyribonucleotide can mediate a splicing event that
results in circularization of the
linear polyribonucleotide, thereby the resultant circular polyribonucleotide
includes a residual splicing
element from such splicing-mediated circularization event. In some cases, the
residual splicing element
is not able to mediate any splicing. In other cases, the residual splicing
element can still mediate splicing
under certain circumstances. In some embodiments, the splicing element is
adjacent to at least one
expression sequence. In some embodiments, the circular polyribonucleotide
includes a splicing element
adjacent each expression sequence. In some embodiments, the splicing element
is on one or both sides
of each expression sequence, leading to separation of the expression products,
e.g., peptide(s) and or
polypeptide(s).
In some embodiments, a circular polyribonucleotide includes an internal
splicing element that
when replicated the spliced ends are joined together. Some examples may
include miniature introns
(<100 nt) with splice site sequences and short inverted repeats (30-40 nt)
such as AluSq2, AluJr, and
AluSz, inverted sequences in flanking introns, Alu elements in flanking
introns, and motifs found in
(suptable4 enriched motifs) cis-sequence elements proximal to backsplice
events such as sequences in
the 200 bp preceding (upstream of) or following (downstream from) a backsplice
site with flanking exons.
In some embodiments, the circular polyribonucleotide includes at least one
repetitive nucleotide
sequence described elsewhere herein as an internal splicing element. In such
embodiments, the
repetitive nucleotide sequence may include repeated sequences from the Alu
family of introns. In some
embodiments, a splicing-related ribosome binding protein can regulate circular
polyribonucleotide
biogenesis (e.g. the Muscleblind and Quaking (QKI) splicing factors).
In some embodiments, a circular polyribonucleotide may include canonical
splice sites that flank
head-to-tail junctions of the circular polyribonucleotide.
In some embodiments, a circular polyribonucleotide may include a bulge-helix-
bulge motif,
including a 4-base pair stem flanked by two 3-nucleotide bulges. Cleavage
occurs at a site in the bulge
region, generating characteristic fragments with terminal 5'-hydroxyl group
and 2', 3'-cyclic phosphate.
Circularization proceeds by nucleophilic attack of the 5'-OH group onto the
2', 3'-cyclic phosphate of the
same molecule forming a 3', 5'-phosphodiester bridge.
In some embodiments, a circular polyribonucleotide may include a multimeric
repeating RNA
sequence that harbors a HPR element. The HPR includes a 2',3'-cyclic phosphate
and a 5'-OH terminus.
The HPR element self-processes the 5'- and 3'-ends of the linear
polyribonucleotide for circularization,
thereby ligating the ends together.
In some embodiments, a circular polyribonucleotide may include a self-splicing
element. For
example, the circular polyribonucleotide may include an intron from the
cyanobacteria Anabaena.
In some embodiments, a circular polyribonucleotide may include a sequence that
mediates self-
ligation. In one embodiment, the circular polyribonucleotide may include a HDV
sequence (e.g., HDV
replication domain conserved sequence,
GGCUCAUCUCGACAAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUG
CUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC (SEQ ID NO: 5) or
GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGC
CGCCCGAGCC (SEQ ID NO: 6) to self-ligate. In one embodiment, the circular
polyribonucleotide may
include loop E sequence (e.g., in PSTVd) to self-ligate. In another
embodiment, the circular
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polyribonucleotide may include a self-circularizing intron, e.g., a 5' and 3'
slice junction, or a self-
circularizing catalytic intron such as a Group I, Group II or Group III
Introns. Non-limiting examples of
group I intron self-splicing sequences may include self-splicing permuted
intron-exon sequences derived
from T4 bacteriophage gene td, and the intervening sequence (IVS) rRNA of
Tetrahymena.
Other Circularization Methods
In some embodiments, linear polyribonucleotides for circularization may
include complementary
sequences, including either repetitive or nonrepetitive nucleic acid sequences
within individual introns or
across flanking introns. Repetitive nucleic acid sequence are sequences that
occur within a segment of
the circular polyribonucleotide. In some embodiments, the circular
polyribonucleotide includes a
repetitive nucleic acid sequence. In some embodiments, the repetitive
nucleotide sequence includes poly
CA or poly UG sequences. In some embodiments, the circular polyribonucleotide
includes at least one
repetitive nucleic acid sequence that hybridizes to a complementary repetitive
nucleic acid sequence in
another segment of the circular polyribonucleotide, with the hybridized
segment forming an internal
double strand. In some embodiments, the circular polyribonucleotide includes
between 1 and 10 (e.g., 2,
3, 4, 5, 6, 7, 8, 9, and 10) repetitive nucleic acid sequences that hybridize
to a complementary repetitive
nucleic acid sequence in another segment of the circular polyribonucleotide,
with the hybridized segment
forming an internal double strand. In some embodiments, the circular
polyribonucleotide includes 2
repetitive nucleic acid sequences that hybridize to a complementary repetitive
nucleic acid sequence in
another segment of the circular polyribonucleotide, with the hybridized
segment forming an internal
double strand. In some embodiments, repetitive nucleic acid sequences and
complementary repetitive
nucleic acid sequences from two separate circular polyribonucleotides
hybridize to generate a single
circularized polyribonucleotide, with the hybridized segments forming internal
double strands. In some
embodiments, the complementary sequences are found at the 5' and 3' ends of
the linear
polyribonucleotides for circularization. In some embodiments, the
complementary sequences include
about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more paired
nucleotides.
In some embodiments, chemical methods of circularization may be used to
generate the circular
polyribonucleotide. Such methods may include, but are not limited to click
chemistry (e.g., alkyne and
azide-based methods, or clickable bases), olefin metathesis, phosphoramidate
ligation, hemiaminal-imine
crosslinking, base modification, and any combination thereof.
In some embodiments, enzymatic methods of circularization may be used to
generate the circular
polyribonucleotide. In some embodiments, a ligation enzyme, e.g., DNA or RNA
ligase, may be used to
generate a template of the circular polyribonuclease or complement, a
complementary strand of the
circular polyribonuclease, or the circular polyribonuclease.
Circularization of the circular polyribonucleotide may be accomplished by
methods known in the
art, for example, those described in "RNA circularization strategies in vivo
and in vitro" by Petkovic and
Muller from Nucleic Acids Res, 2015, 43(4): 2454-2465, and "In vitro
circularization of RNA" by Muller and
Appel, from RNA Biol, 2017, 14(8):1018-1027.
The circular polyribonucleotide may encode a sequence and/or motif useful for
replication.
Exemplary replication elements are described in paragraphs [0280] - [0286] of
International Patent
Publication No. W02019/118919, which is hereby incorporated by reference in
its entirety.
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Purification of Circular Polyribonucleotides
In some embodiments, the circular polyribonucleotide is purified, e.g., free
ribonucleic acids,
linear or nicked RNA, DNA, proteins, etc. are removed. In some embodiments,
the circular
polyribonucleotides may be purified by any known method commonly used in the
art. Examples of
nonlimiting purification methods include, column chromatography, gel excision,
size exclusion, etc.
Detection of Linear and Circular RNA
The presence of linear RNA in pharmaceutical circular RNA preparations can
have unexpected
and sometimes undesirable effects. Thus, circular RNAs may be enriched,
separated, and/or purified
relative to linear RNA; methods (e.g., methods of manufacturing circular RNA
preparations) whereby
linear RNAs can be monitored, evaluated and/or controlled; and methods of
using such pharmaceutical
compositions and preparations, e.g., to deliver an effector, such as a
therapeutic effector or scaffold (e.g.,
an aptamer sequence), to a cell, tissue or subject. In some embodiments, a
circular RNA preparation has
no more than a threshold level of linear RNA, e.g., a circular RNA preparation
is enriched over linear RNA
or purified to reduce linear RNA.
Generally, detection and quantitation of an element in a pharmaceutical
preparation includes the
use of a reference standard that is either the component of interest (e.g.,
circular RNA, linear RNA,
fragment, impurity, etc.) or is a similar material (e.g., using a linear RNA
structure of the same sequence
as a circular RNA structure as a standard for circular RNA), or includes the
use of an internal standard or
signal from a test sample. In some embodiments, the standard is used to
establish the response from a
detector for a known or relative amount of material (response factor). In some
embodiments, the
response factor is determined from a standard at one or multiple
concentrations (e.g., using linear
regression analysis). In some embodiments, the response factor is then used to
determine the amount of
the material of interest from the signal due to that component. In some
embodiments, the response factor
is a value of one or is assumed to have a value of one.
In some embodiments, detection and quantification of linear versus circular
RNA in the
pharmaceutical composition is determined using a comparison to a linear
version of the circular
polyribonucleotides. In some embodiments, the mass of total ribonucleotide in
the pharmaceutical
composition is determined using a standard curve generated using a linear
version of the circular
polyribonucleotide and assuming a response factor of one. In some embodiments,
a w/w percentage of
circular polyribonucleotide in the pharmaceutical preparation is determined by
a comparison of a standard
curve generated by band intensities of multiple known amounts of a linear
version of the circular
polyribonucleotide to a band intensity of the circular polyribonucleotide in
the pharmaceutical preparation.
In some embodiments, the bands are produced during gel-base electrophoresis,
and the band intensities
are measured by a gel imager (e.g., an E-gel Imager). In some embodiments, a
circular
polyribonucleotide preparation includes less than a threshold amount (e.g.,
where the threshold amount is
a reference criterion, e.g., a pharmaceutical release specification for the
circular polyribonucleotide
preparation) of linear polyribonucleotide molecules when evaluated as
described herein.
In some embodiments, detection and quantification of nicked versus total RNA
in the
pharmaceutical composition is determined by sequencing after gel extraction of
the preparation including
the circular RNA. In some embodiments, detection and quantification of nicked
versus linear RNA in the
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pharmaceutical composition is determined by sequencing after gel extraction of
the preparation including
the circular RNA. In some embodiments, a circular polyribonucleotide
preparation includes less than a
threshold amount (e.g., where the threshold amount is a reference criterion,
e.g., a pharmaceutical
release specification for the circular polyribonucleotide preparation) of
nicked RNA, linear RNA, or
combined linear and nicked RNA when evaluated as described herein. For
example, the reference
criterion for the amount of linear polyribonucleotide molecules present in the
preparation is no more than
30%, 20%, 15%, 10%, 1%, 0.5%, or 0.1% linear polyribonucleotide molecules, or
any percentage
therebetween, relative to total ribonucleotide molecules in the preparation.
In some embodiments, the
reference criterion for the amount of nicked polyribonucleotide molecules
present in the preparation is no
more than 30%, 20%, 15%, 10%, 1%, 0.5%, or 0.1%, or any percentage
therebetween, nicked
polyribonucleotide molecules relative to total ribonucleotide molecules in the
preparation. In some
embodiments, the reference criterion for the amount of linear and nicked
polyribonucleotide molecules
present in the preparation is no more than 40%, 30%, 20%, 15%, 10%, 1%, 0.5%,
or 0.1%, or any
percentage therebetween, combined linear polyribonucleotide and nicked
polyribonucleotide molecules
relative to total ribonucleotide molecules in the preparation.
In some embodiments, the standard is run under the same conditions as the
sample. For
example, the standard is run with the same type of gel, same buffer, and same
exposure as the sample.
In further embodiments, the standard is run in parallel with the sample. In
some embodiments, a
quantification of an element is repeated (e.g., twice or in triplicate) in a
plurality of samples from the
subject preparation to obtain a mean result. In some embodiments, quantitation
of a linear RNA is
measured using parallel capillary electrophoresis (e.g., using a Fragment
Analyzer or analytical HPLC
with UV detection).
Methods of Purification
Circular polyribonucleotides may be separated, enriched, or purified from
unwanted substances
(such as unwanted (e.g., linear) RNA, enzymes, DNA). In some embodiments, the
unwanted substances
are present in, or originating from, a process of making and/or manufacturing
the circular
polyribonucleotides. Circular polyribonucleotides described herein may be
enriched and/or purified prior
to formulation in a pharmaceutical preparation, pharmaceutical composition,
pharmaceutical drug
substance, or pharmaceutical drug product. Circular polyribonucleotides
described herein may be
enriched and/or purified during or after formulation in a pharmaceutical
preparation, pharmaceutical
composition, pharmaceutical drug substance, or pharmaceutical drug product.
In some embodiments, a circular polyribonucleotide may be purified during or
after production to
remove undesirable elements, e.g., linear RNA, or nicked RNA, as well as
recognized impurities, e.g.,
free ribonucleic acids (e.g., monoribonucleic acids, diribonucleic acids, or
triribonucleic acids), DNA (e.g.,
cell DNA, such as host cell DNA), cell or process-related protein impurities
(e.g., cell or process-related
impurities), etc. In some embodiments, an impurity is a process-related
impurity. In some embodiments,
the process-related impurity is a protein (e.g., a cell protein), a nucleic
acid (e.g., a cell nucleic acid), a
buffer or buffer reagent, an enzyme, a media/reagent component (e.g., media,
media additive, transition
metal, or vitamin), a preparatory or analytical gel component (e.g.,
acrylamide debris), DNA, or a
chromatographic material. A buffer reagent can be MgCl2, DTT, ATP, SDS, Na,
glycogen, Tris-HCL, or
Et0H. A buffer reagent can include, but is not limited to, acetate, Tris,
bicarbonate, phosphate, citric acid,
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lactate, or TEA. An enzyme can be a ligase. A ligase can be T4 RNA ligase 2.
In some embodiments,
an impurity is a buffer reagent, a media/reagent component, a salt, a ligase,
a nuclease, an RNase
inhibitor, RNase R, linear polyribonucleotide molecules, deoxyribonucleotide
molecules, acrylamide
debris, or mononucleotide molecules.
In some embodiments, circular polyribonucleotides may be enriched or purified
by any known
method commonly used in the art. Examples of non-limiting purification methods
include column
chromatography, gel excision, size exclusion, etc.
In some embodiments, a circular polyribonucleotide is purified by gel. For
example, a circular
RNA may be resolved on a denaturing PAGE and bands corresponding to the
circular RNAs may be
excised and the circular RNA may be eluted from the band using known methods.
The eluted circular
RNA may then be analyzed.
In some embodiments, a circular polyribonucleotide is purified by
chromatography, e.g.,
hydrophobic interaction chromatography (H IC), mixed-mode chromatography,
liquid chromatography,
e.g., reverse-phase ion-pair chromatography (IP-RP), ion-exchange
chromatography (1E), affinity
chromatography (AC), and size-exclusion chromatography (SEC), and any
combinations thereof.
In some embodiments, a circular polyribonucleotide is purified by utilizing a
structural feature of
the circular polyribonucleotide to separate it from a linear RNA or an
impurity. In some embodiments, the
circular polyribonucleotide is purified by utilizing a structural feature
(e.g., a lack of free ends). For
example, circular RNA is enriched from a preparation including a mixed pool of
circular RNA and linear
RNA counterpart containing the same nucleotide sequences using polyadenylation
of the linear RNA
counterpart or fragments thereof. The 3' end of the linear RNA counterpart or
fragments thereof can be
polyadenylated using poly(A) polymerase, resulting in the addition of a 3'
polyadenine tail. In some
embodiments, the 3' polyadenine tail enables a pulldown of the linear RNA and
fragments thereof using a
column, such as an affinity column, to enrich for the circular RNA. Poly(A)
polymerase can also
incorporate modified adenines such as the biotinylated N6-ATP analog. This
addition biotinylated N6-
ATP analog into the 3' polyadenine tail of enables a pulldown of the linear
RNA and fragments thereof in
a system such as a biotin-streptavidin binding system. In contrast,
circularized RNA does not have a 3'
end, and therefore is not polyadenylated by the poly(A) polymerase, does not
have a polyadenylated tail
for conjugation, and is not captured in the pulldown. Therefore, the circular
RNA is enriched in the
preparation after the pulldown.
In some embodiments, a circular polyribonucleotide is purified by utilizing a
structural feature of
the linear RNA (e.g., presence of free ends). For example, circular RNA is
enriched from a preparation
including a mixed pool of circular RNA and linear RNA counterpart containing
the same nucleotide
sequences using polyadenylation of the linear RNA counterpart. Exonucleases
can be added to the
mixed pool to hydrolyze the linear RNA. In some embodiments, an exonuclease
can be 3' exonuclease
or a 5' exonuclease. In some embodiments, a 3' exonuclease and a 5'
exonuclease can be used.
In an embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
polyribonucleotide preparation) is at least 25% (w/w), 30% (w/w), 40% (w/w),
50% (w/w), 60% (w/w), 70%
(w/w), 80% (w/w), 85% (w/w), 90% (w/w), 91% (w/w), 92% (w/w), 93% (w/w), 94%
(w/w), 95% (w/w), 96%
(w/w), 97% (w/w), 98% (w/w), 99% (w/w), or 100% (w/w) pure on a mass basis.
Purity may be measured
by any one of a number of analytical techniques known to one skilled in the
art, such as, but not limited
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to, the use of separation technologies such as chromatography (using a column,
using a paper, using a
gel, using HPLC, using UHPLC, etc., or by IC, by SEC, by reverse phase, by
anion exchange, by mixed
mode, etc.) or electrophoresis (UREA PAGE, chip-based, polyacrylamide gel,
RNA, capillary, c-IEF, etc.)
with or without pre- or post-separation derivatization methodologies using
detection techniques based on
.. mass spectrometry, UV-visible, fluorescence, light scattering, refractive
index, or that use silver or dye
stains or radioactive decay for detection. Alternatively, purity may be
determined without the use of a
separation technology by mass spectrometry, by microscopy, by circular
dichroism (CD) spectroscopy, by
UV or UV-vis spectrophotometry, by fluorometry (e.g., Qubit), by RNase H
analysis, by surface plasmon
resonance (SPR), or by methods that utilize silver or dye stains or
radioactive decay for detection.
In some embodiments, purity can be measured by biological test methodologies
(e.g., cell-based
or receptor-based tests). In some embodiments, at least 25% (w/w), 30% (w/w),
40% (w/w), 50% (w/w),
60% (w/w), 70% (w/w), 80% (w/w), 85% (w/w), 90% (w/w), 91% (w/w), 92% (w/w),
93% (w/w), 94% (w/w),
95% (w/w), 96% (w/w), 97% (w/w), 98% (w/w), 99% (w/w) or 100% (w/w) of the
total of mass
ribonucleotide in the a preparation described herein is contained in circular
polyribonucleotide molecules.
.. The percent may be measured by any one of a number of analytical techniques
known to one skilled in
the art such as, but not limited to, the use of a separation technology such
as chromatography (using a
column, using a paper, using a gel, using HPLC, using UHPLC, etc., or by IC,
by SEC, by reverse phase,
by anion exchange, by mixed mode, etc.) or electrophoresis (UREA PAGE, chip-
based, polyacrylamide
gel, RNA, capillary, c-IEF, etc.) with or without pre- or post-separation
derivatization methodologies using
detection techniques based on mass spectrometry, UV-visible, fluorescence,
light scattering, refractive
index, or that use silver or dye stains or radioactive decay for detection.
Alternatively, purity may be
determined without the use of separation technologies by mass spectrometry, by
microscopy, by circular
dichroism (CD) spectroscopy, by UV or UV-vis spectrophotometry, by fluorometry
(e.g., Qubit), by RNase
H analysis, by surface plasmon resonance (SPR), or by methods that utilize
silver or dye stains or
radioactive decay for detection.
In an embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
polyribonucleotide preparation) has a circular polyribonucleotide
concentration of at least 0.1 ng/mL, 0.5
ng/mL, 1 ng/mL, 5 ng/mL, 10 ng/mL, 50 ng/mL, 0.1 g/mL, 0.5 g/mL,1 g/mL, 2
g/mL, 5 g/mL, 10
.. g/mL, 20 g/mL, 30 g/mL, 40 g/mL, 50 g/mL, 60 g/mL, 70 g/mL, 80
g/mL, 100 g/mL, 200
g/mL, 300 g/mL, 500 g/mL, 1000 g/mL, 5000 g/mL, 10,000 g/mL, 100,000
g/mL, 200 mg/mL,
300 mg/mL, 400 mg/mL, 500 mg/mL, 600 mg/mL, 650 mg/mL, 700 mg/mL, or 750
mg/mL. In an
embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide pharmaceutical
preparation or composition or an intermediate in the production of the
circular polyribonucleotide
.. preparation) is substantially free of mononucleotide or has a
mononucleotide content of no more than 1
pg/ml, 10 pg/ml, 0.1 ng/ml, 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25
ng/ml, 30 ng/ml, 35 ng/ml,
ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 200 ng/ml,
300 ng/ml, 400 ng/ml,
500 ng/ml, 1000 g/mL, 5000 g/mL, 10,000 g/mL, or 100,000 g/mL. In an
embodiment, a circular
polyribonucleotide preparation (e.g., a circular polyribonucleotide
pharmaceutical preparation or
40 .. composition or an intermediate in the production of the circular
polyribonucleotide preparation) has a
mononucleotide content from the limit of detection up to 1 pg/ml, 10 pg/ml,
0.1 ng/ml, 1 ng/ml, 5 ng/ml, 10
ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 50 ng/ml,
60 ng/ml, 70 ng/ml, 80 ng/ml,
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90 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 1000 g/mL,
5000 g/mL, 10,000
g/mL, or 100,000 g/mL.
In an embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
polyribonucleotide preparation) has mononucleotide content no more than 0.1%
(w/w), 0.2% (w/w), 0.3%
(w/w), 0.4% (w/w), 0.5% (w/w), 0.6% (w/w), 0.7% (w/w), 0.8% (w/w), 0.9% (w/w),
1% (w/w), 2% (w/w), 3%
(w/w), 4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w),
15% (w/w), 20% (w/w),
25% (w/w), 30% (w/w), or any percentage therebetween of total nucleotides on a
mass basis, wherein
total nucleotide content is the total mass of deoxyribonucleotide molecules
and ribonucleotide molecules.
In an embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
polyribonucleotide preparation) has a linear RNA content, e.g., linear RNA
counterpart or RNA fragments,
of no more than 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30
ng/ml, 35 ng/ml, 40 ng/ml,
50 ng/ml, 6 Ong/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 200 ng/ml, 300
ng/ml, 400 ng/ml, 500ng/ml,
600 ng/ml, 1 g/ ml, 10 g/ml, 50 g/ml, 100 g/ml, 200 g/ml, 300 g/ml, 400
g/ml, 500 g/ml, 600
g/ml, 700 g/ml, 800 g/ml, 900 g/ml, 1 mg/ml, 1.5 mg/ml, 2mg/ml, 5 mg/mL, 10
mg/mL, 50 mg/mL,
100 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/mL, 500 mg/mL, 600 mg/mL, 650 mg/mL,
700 mg/mL, or
750 mg/mL. In an embodiment, a circular polyribonucleotide preparation (e.g.,
a circular
polyribonucleotide pharmaceutical preparation or composition or an
intermediate in the production of the
.. circular polyribonucleotide preparation) has a linear RNA content, e.g.,
linear RNA counterpart or RNA
fragments, from the limit of detection of up to 1 ng/ml, 5 ng/ml, 10 ng/ml, 15
ng/ml, 20 ng/ml, 25 ng/ml, 30
ng/ml, 35 ng/ml, 40 ng/ml, 50 ng/ml, 6 Ong/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml,
100 ng/ml, 200 ng/ml, 300
ng/ml, 400 ng/ml, 500ng/ml, 600 ng/ml, 1 g/ ml, 10 g/ml, 50 g/ml, 100
g/ml, 200 g/ml, 300 g/ml, 400
g/ml, 500 g/ml, 600 g/ml, 700 g/ml, 800 g/ml, 900 g/ml, 1 mg/ml, 1.5
mg/ml, 2mg/ml, 5 mg/ml, 10
.. mg/ml, 50 mg/ml, 100 mg/ml, 200 mg/ml, 300 mg/ml, 400 mg/ml, 500 mg/ml, 600
mg/ml, 650 mg/ml, 700
mg/ml, or 750 mg/ml.
In an embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
polyribonucleotide preparation) has a nicked RNA content of no more than 10%
(w/w), 9.9% (w/w), 9.8%
(w/w), 9.7% (w/w), 9.6% (w/w), 9.5% (w/w), 9.4% (w/w), 9.3% (w/w), 9.2% (w/w),
9.1% (w/w), 9% (w/w),
8% (w/w), 7% (w/w), 6% (w/w), 5% (w/w), 4% (w/w), 3% (w/w), 2% (w/w), 1%
(w/w), 0.5% (w/w), or 0.1%
(w/w), or percentage therebetween. In an embodiment, a circular
polyribonucleotide preparation (e.g., a
circular polyribonucleotide pharmaceutical preparation or composition or an
intermediate in the production
of the circular polyribonucleotide preparation) has a nicked RNA content that
as low as zero or is
substantially free of nicked RNA.
In an embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
polyribonucleotide preparation) has a combined linear RNA and nicked RNA
content of no more than
70% (w/w), 60% (w/w), 50% (w/w), 40% (w/w), 30% (w/w), 25% (w/w), 20% (w/w),
15% (w/w), 10% (w/w),
9% (w/w), 8% (w/w), 7% (w/w), 6% (w/w), 5% (w/w), 4% (w/w), 3% (w/w), 2%
(w/w), 1% (w/w), 0.5%
(w/w), or 0.1% (w/w), or percentage therebetween. In an embodiment, a circular
polyribonucleotide
preparation (e.g., a circular polyribonucleotide pharmaceutical preparation or
composition or an
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intermediate in the production of the circular polyribonucleotide preparation)
has a combined nicked RNA
and linear RNA content that is as low as zero or is substantially free of
nicked and linear RNA.
In some embodiments, a circular polyribonucleotide preparation (e.g., a
circular
polyribonucleotide pharmaceutical preparation or composition or an
intermediate in the production of the
circular polyribonucleotide preparation) has a linear RNA content, e.g.,
linear RNA counterpart or RNA
fragments, of no more than the detection limit of analytical methodologies,
such as methods utilizing
mass spectrometry, UV spectroscopic or fluorescence detectors, light
scattering techniques, surface
plasmon resonance (SPR) with or without the use of methods of separation
including HPLC, by HPLC,
chip or gel based electrophoresis with or without using either pre or post
separation derivatization
methodologies, methods of detection that use silver or dye stains or
radioactive decay, or microscopy,
visual methods or a spectrophotometer.
In an embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
polyribonucleotide preparation) has no more than 0.1% (w/w), 1% (w/w), 2%
(w/w), 3% (w/w), 4% (w/w),
5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 15% (w/w), 20%
(w/w), 25% (w/w), 30%
(w/w), 35% (w/w), 40% (w/w), 45% (w/w), 50% (w/w) of linear RNA, e.g., as
measured by the methods in
Example 2.
In some embodiments, the linear polyribonucleotide molecules of a circular
polyribonucleotide
preparation include the linear counterpart or a fragment thereof of the
circular polyribonucleotide
molecule. In some embodiments, the linear polyribonucleotide molecules of the
circular
polyribonucleotide preparation include the linear counterpart (e.g., a pre-
circularized version). In some
embodiments, the linear polyribonucleotide molecules of the circular
polyribonucleotide preparation
include a non-counterpart or fragment thereof to the circular
polyribonucleotide. In some embodiments,
the linear polyribonucleotide molecules of the circular polyribonucleotide
preparation include a non-
counterpart to the circular polyribonucleotide. In some embodiments, the
linear polyribonucleotide
molecules include a combination of the counterpart of the circular
polyribonucleotide and a non-
counterpart or fragment thereof of the circular polyribonucleotide. In some
embodiments, the linear
polyribonucleotide molecules include a combination of the counterpart of the
circular polyribonucleotide
and a non-counterpart of the circular polyribonucleotide. In some embodiments,
a linear
polyribonucleotide molecule fragment is a fragment that is at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 20, 30, 40,
50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000,
6000, 7000, 8000, 9000,
10000, 11000, 12000, or more nucleotides in length, or any nucleotide number
therebetween.
In some embodiments, a circular polyribonucleotide preparation (e.g., a
circular
polyribonucleotide pharmaceutical preparation or composition or an
intermediate in the production of the
circular polyribonucleotide preparation) has an A260/A280 absorbance ratio
from about 1.6 to about 2.3,
e.g., as measured by spectrophotometer. In some embodiments, the A260/A280
absorbance ratio is
about 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or any
number therebetween. In some
embodiments, a circular polyribonucleotide (e.g., a circular
polyribonucleotide pharmaceutical preparation
or composition or an intermediate in the production of the circular
polyribonucleotide) has an A260/A280
absorbance ratio greater than about 1.8, e.g., as measured by
spectrophotometer. In some
embodiments, the A260/A280 absorbance ratio is about 1.6, 1.7, 1.8, 1.9, 2.0,
2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, or greater.
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In some embodiments, a circular polyribonucleotide preparation (e.g., a
circular
polyribonucleotide pharmaceutical preparation or composition or an
intermediate in the production of the
circular polyribonucleotide preparation) is substantially free of an impurity.
In various embodiments, the
level of at least one impurity in a composition including the circular
polyribonucleotide is reduced by at
least 30% (w/w), at least 40% (w/w), at least 50% (w/w), at least 60% (w/w),
at least 70% (w/w), at least
80% (w/w), at least 90% (w/w), or at least 95% (w/w) as compared to that of
the composition prior to
purification or treatment to remove the impurity. In some embodiments, the
level of at least one process-
related impurity is reduced by at least 30% (w/w), at least 40% (w/w), at
least 50% (w/w), at least 60%
(w/w), at least 70% (w/w), at least 80% (w/w), at least 90% (w/w), or at least
95% (w/w) as compared to
that of the composition prior to purification or treatment to remove the
impurity. In some embodiments, the
level of at least one product-related substance is reduced by at least 30%
(w/w), at least 40% (w/w), at
least 50% (w/w), at least 60% (w/w), at least 70% (w/w), at least 80% (w/w),
at least 90% (w/w), or at
least 95% (w/w) as compared to that of the a composition prior to purification
or treatment to remove the
impurity. In some embodiments, a circular polyribonucleotide preparation
(e.g., a circular
polyribonucleotide pharmaceutical preparation or composition or an
intermediate in the production of the
circular polyribonucleotide preparation) is further substantially free of a
process-related impurity. In some
embodiments, the process-related impurity includes a protein (e.g., a cell
protein, such as a host cell
protein), a deoxyribonucleic acid (e.g., a cell deoxyribonucleic acid, such as
a host cell deoxyribonucleic
acid), monodeoxyribonucleotide or dideoxyribonucleotide molecules, an enzyme
(e.g., a nuclease, such
as an endonuclease or exonuclease, or ligase), a reagent component, a gel
component, or a
chromatographic material. In some embodiments, the impurity is selected from:
a buffer reagent, a ligase,
a nuclease, RNase inhibitor, RNase R, deoxyribonucleotide molecules,
acrylamide gel debris, and
monodeoxyribonucleotide molecules. In some embodiments, the pharmaceutical
preparation includes
protein (e.g., cell protein, such as a host cell protein) contamination of
less than 0.1 ng, 1 ng, 5 ng, 10 ng,
15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng,
100 ng, 200 ng, 300 ng, 400
ng, or 500 ng of protein contamination per milligram (mg) of the circular
polyribonucleotide molecules.
In an embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
polyribonucleotide preparation) is substantially free of DNA content e.g.,
template DNA or cell DNA (e.g.,
host cell DNA)õ has a DNA content, as low as zero, or has a DNA content of no
more than 1 pg/ml, 10
pg/ml, 0.1 ng/ml, 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30
ng/ml, 35 ng/ml, 40 ng/ml,
50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 200 ng/ml, 300
ng/ml, 400 ng/ml, 500 ng/ml,
1000 g/mL, 5000 g/mL, 10,000 g/mL, or 100,000 g/mL.
In an embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
polyribonucleotide preparation) is substantially free of DNA content, has a
DNA content as low as zero, or
has DNA content no more than 0.001% (w/w), 0.01% (w/w), 0.1% (w/w), 1% (w/w),
2% (w/w), 3% (w/w),
4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 15%
(w/w), 20% (w/w), 25%
(w/w), 30% (w/w), 35% (w/w), 40% (w/w), 45% (w/w), 50% (w/w) of total
nucleotides on a mass basis,
wherein total nucleotide molecules is the total mass of deoxyribonucleotide
content and ribonucleotide
molecules. In an embodiment, a circular polyribonucleotide preparation (e.g.,
a circular polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
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polyribonucleotide preparation) is substantially free of DNA content, has DNA
content as low as zero, or
has DNA content no more than 0.001% (w/w), 0.01% (w/w), 0.1% (w/w), 1% (w/w),
2% (w/w), 3% (w/w),
4% (w/w), 5% (w/w), 6% (w/w), 7% (w/w), 8% (w/w), 9% (w/w), 10% (w/w), 15%
(w/w), 20% (w/w), 25%
(w/w), 30% (w/w), 35% (w/w), 40% (w/w), 45% (w/w), 50% (w/w) of total
nucleotides on a mass basis as
measured after a total DNA digestion by enzymes that digest nucleosides by
quantitative liquid
chromatography-mass spectrometry (LC-MS), in which the content of DNA is back
calculated from a
standard curve of each base (i.e., A, C, G, T) as measured by LC-MS.
In an embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
polyribonucleotide preparation) has a protein (e.g., cell protein (CP), e.g.,
enzyme, a production-related
protein, e.g., carrier protein) contamination of no more than 0.1 ng/ml, 1
ng/ml, 5 ng/ml, 10 ng/ml, 15
ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35 ng/ml, 40 ng/ml, 50 ng/ml, 60 ng/ml,
70 ng/ml, 80 ng/ml, 90 ng/ml,
100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, or 500 ng/ml. In an embodiment, a
circular
polyribonucleotide (e.g., a circular polyribonucleotide pharmaceutical
preparation or composition or an
intermediate in the production of the circular polyribonucleotide) has a
protein (e.g., production-related
protein such as a cell protein (CP), e.g., enzyme) contamination from the
limit of detection of up to 0.1
ng/ml, 1 ng/ml, 5 ng/ml, 10 ng/ml, 15 ng/ml, 20 ng/ml, 25 ng/ml, 30 ng/ml, 35
ng/ml, 40 ng/ml, 50 ng/ml,
60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, 200 ng/ml, 300 ng/ml, 400
ng/ml, or 500 ng/ml.
In an embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
polyribonucleotide preparation) has a protein (e.g., production-related
protein such as a cell protein (CP),
e.g., enzyme) contamination of less than 0.1 ng, 1 ng, 5 ng, 10 ng, 15 ng, 20
ng, 25 ng, 30 ng, 35 ng, 40
ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng, 300 ng, 400 ng, or 500
ng per milligram (mg) of the
circular polyribonucleotide. In an embodiment, a circular polyribonucleotide
(e.g., a circular
polyribonucleotide pharmaceutical preparation or composition or an
intermediate in the production of the
circular polyribonucleotide) has a protein (e.g., production-related protein
such as a cell protein (CP),
e.g., enzyme) contamination from the level of detection up to 0.1 ng, 1 ng, 5
ng, 10 ng, 15 ng, 20 ng, 25
ng, 30 ng, 35 ng, 40 ng, 50 ng, 60 ng, 70 ng, 80 ng, 90 ng, 100 ng, 200 ng,
300 ng, 400 ng, or 500 ng per
milligram (mg) of the circular polyribonucleotide.
In an embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
polyribonucleotide preparation) has low levels or is substantially absent of
endotoxins, e.g., as measured
by the Limulus amebocyte lysate (LAL) test. In some embodiments, the
pharmaceutical preparation or
compositions or an intermediate in the production of the circular
polyribonucleotides includes less than 20
EU/kg (weight), 10 EU/kg, 5 EU/kg, 1 EU/kg, or lacks endotoxin as measured by
the Limulus amebocyte
lysate test. In an embodiment, a circular polyribonucleotide composition has
low levels or absence of a
nuclease or a ligase.
In some embodiments, a circular polyribonucleotide preparation (e.g., a
circular
polyribonucleotide pharmaceutical preparation or composition or an
intermediate in the production of the
circular polyribonucleotide preparation) includes no greater than about 50%
(w/w), 45% (w/w), 40% (w/w),
35% (w/w), 30% (w/w), 25% (w/w), 20% (w/w), 19% (w/w), 18% (w/w), 17% (w/w),
16% (w/w), 15% (w/w),
14% (w/w), 13% (w/w), 12% (w/w), 11% (w/w), 10% (w/w), 9% (w/w), 8% (w/w), 7%
(w/w), 6% (w/w), 5%
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(w/w), 4% (w/w), 3% (w/w), 2% (w/w), 1% (w/w) of at least one enzyme, e.g.,
polymerase, e.g., RNA
polymerase.
In an embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
polyribonucleotide preparation) is sterile or substantially free of
microorganisms, e.g., the composition or
preparation supports the growth of fewer than 100 viable microorganisms as
tested under aseptic
conditions, the composition or preparation meets the standard of USP <71>,
and/or the composition or
preparation meets the standard of USP <85>. In some embodiments, the
pharmaceutical preparation
includes a bioburden of less than 100 CFU/100 ml, 50 CFU/100 ml, 40 CFU/100
ml, 30 CFU/100 ml, 200
CFU/100 ml, 10 CFU/100 ml, or 10 CFU/100 ml before sterilization.
In some embodiments, a circular polyribonucleotide preparation can be further
purified using
known techniques in the art for removing impurities, such as column
chromatography or pH/vial
inactivation.
In some embodiments, a circular polyribonucleotide preparation produces a
reduced level of one
more markers of an immune or inflammatory response after administration to a
subject when the circular
polyribonucleotide preparation has undergone a purification step (or a
plurality of purification steps)
compared to prior to the purification step(s). In some embodiments, the one or
more markers of an
immune or inflammatory response is a cytokine or immune response related gene.
In some
embodiments, the one or more markers of an immune or inflammatory response is
expression of a gene,
such as RIG-I, MDA5, PKR, IFN-beta, OAS, and OASL.
In an embodiment, a circular polyribonucleotide preparation (e.g., a circular
polyribonucleotide
pharmaceutical preparation or composition or an intermediate in the production
of the circular
polyribonucleotide preparation) expresses an expression product, e.g.,
protein, e.g., in-vitro translation
activity.
Delivery
A circular or linear polyribonucleotide described herein may be included in
pharmaceutical
compositions with a carrier or without a carrier.
Pharmaceutical compositions described herein may be formulated for example
including a
carrier, such as a pharmaceutical carrier and/or a polymeric carrier, e.g., a
liposome, and delivered by
known methods to a subject in need thereof (e.g., a human or non-human
agricultural or domestic animal,
e.g., cattle, dog, cat, horse, poultry). Such methods include, but not limited
to, transfection (e.g., lipid-
mediated, cationic polymers, calcium phosphate, dendrimers); electroporation
or other methods of
membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus,
retrovirus, adenovirus, AAV),
microinjection, microprojectile bombardment ("gene gun"), fugene, direct sonic
loading, cell squeezing,
optical transfection, protoplast fusion, impalefection, magnetofection,
exosome-mediated transfer, lipid
nanoparticle-mediated transfer, and any combination thereof. Methods of
delivery are also described,
e.g., in Gori et al., Delivery and Specificity of CRISPR/Cas9 Genome Editing
Technologies for Human
Gene Therapy. Human Gene Therapy. July 2015, 26(7): 443-451.
doi:10.1089/hum.2015.074; and Zuris
et al. Cationic lipid-mediated delivery of proteins enables efficient protein-
based genome editing in vitro
and in vivo. Nat Biotechnol. 2014 Oct 30;33(1):73-80.
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In some embodiments, circular or linear polyribonucleotides may be delivered
in a "naked"
delivery formulation. A naked delivery formulation delivers a circular
polyribonucleotide to a cell without
the aid of a carrier and without covalent modification of the circular or
linear polyribonucleotide or partial
or complete encapsulation of the circular or linear polyribonucleotide.
A naked delivery formulation is a formulation that is free from a carrier and
wherein the circular or
linear polyribonucleotide is without a covalent modification that binds a
moiety that aids in delivery to a
cell and the circular or linear polyribonucleotide is not partially or
completely encapsulated. In some
embodiments, an circular or linear polyribonucleotide without covalent
modification that binds to a moiety
that aids in delivery to a cell may be a polyribonucleotide that is not
covalently bound to a moiety, such as
a protein, small molecule, a particle, a polymer, or a biopolymer that aids in
delivery to a cell. In some
embodiments, circular or linear polyribonucleotides may be delivered in a
delivery formulation with
protamine or a protamine salt (e.g., protamine sulfate).
A polyribonucleotide without covalent modification that binds to a moiety that
aids in delivery to a
cell may not contain a modified phosphate group. For example, a
polyribonucleotide without covalent
modification that binds to a moiety that aids in delivery to a cell may not
contain phosphorothioate,
phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen
phosphonates,
phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, or
phosphotriesters.
In some embodiments, a naked delivery formulation may be free of any or all
of: transfection
reagents, cationic carriers, carbohydrate carriers, nanoparticle carriers, or
protein carriers. For example,
a naked delivery formulation may be free from phtoglycogen octenyl succinate,
phytoglycogen beta-
dextrin, anhydride-modified phytoglycogen beta-dextrin, lipofectamine,
polyethylenimine,
poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine,
aminoglycoside-polyamine, dideoxy-
diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl
methacrylate, poly(lysine),
poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-
Dioleoy1-3-
Trimethylammonium-Propane(DOTAP), N-[ 1 -(2,3-dioleoyloxy)propyI]-N,N,N-
trimethylammonium
chloride (DOTMA), l-[2-(oleoyloxy)ethy1]-2-oley1-3-(2-
hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-
dioleyloxy-N- [2(sperminecarboxamido)ethyq-N,N-dimethyl-l-propanaminium
trifluoroacetate (DOSPA),
3B-[N¨ (N\N'-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride (DC-
Cholesterol HC1),
diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N- dimethylammonium
bromide (DDAB),
N-(l,2-dimyristyloxyprop-3-y1)-N,N-dimethyl-N- hydroxyethyl ammonium bromide
(DMRIE), N,N-dioleyl-
N,N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low-density
lipoprotein (LDL),
high- density lipoprotein (HDL), or globulin.
A naked delivery formulation may include a non-carrier excipient. In some
embodiments, a non-
carrier excipient may include an inactive ingredient that does not exhibit an
active cell-penetrating effect.
In some embodiments, a non-carrier excipient may include a buffer, for example
PBS. In some
embodiments, a non-carrier excipient may be a solvent, a non-aqueous solvent,
a diluent, a suspension
aid, a surface-active agent, an isotonic agent, a thickening agent, an
emulsifying agent, a preservative, a
polymer, a peptide, a protein, a cell, a hyaluronidase, a dispersing agent, a
granulating agent, a
disintegrating agent, a binding agent, a buffering agent, a lubricating agent,
or an oil.
In some embodiments, a naked delivery formulation may include a diluent, such
as a parenterally
acceptable diluent. A diluent (e.g., a parenterally acceptable diluent) may be
a liquid diluent or a solid
diluent. In some embodiments, a diluent (e.g., a parenterally acceptable
diluent) may be an RNA
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solubilizing agent, a buffer, or an isotonic agent. Examples of an RNA
solubilizing agent include water,
ethanol, methanol, acetone, formamide, and 2-propanol. Examples of a buffer
include 2-(N-
morpholino)ethanesulfonic acid (M ES), Bis-Tris, 2-[(2-amino-2-oxoethyl)-
(carboxymethyl)amino]acetic
acid (ADA), N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-N,N'-
bis(2-ethanesulfonic
acid) (PIPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-
yl]amino]ethanesulfonic acid (TES), 3-(N-
morpholino)propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES),
Tris, Tricine, Gly-Gly, Bicine, or phosphate. Examples of an isotonic agent
include glycerin, mannitol,
polyethylene glycol, propylene glycol, trehalose, or sucrose.
In some embodiments, the pharmaceutical preparation as disclosed herein, the
pharmaceutical
composition as disclosed herein, the pharmaceutical drug substance of as
disclosed, or the
pharmaceutical drug product as disclosed herein is in parenteral nucleic acid
delivery system. The
parental nucleic acid delivery system may include the pharmaceutical
preparation as disclosed herein, the
pharmaceutical composition as disclosed herein, the pharmaceutical drug
substance of as disclosed, or
the pharmaceutical drug product as disclosed herein, and a parenterally
acceptable diluent. In some
embodiments, the pharmaceutical preparation as disclosed herein, the
pharmaceutical composition as
disclosed herein, the pharmaceutical drug substance of as disclosed, or the
pharmaceutical drug product
as disclosed herein in the parenteral nucleic acid delivery system is free of
any carrier.
The disclosure is further directed to a host or host cell including the
circular or linear
polyribonucleotide described herein. In some embodiments, the host or host
cell is a vertebrate, mammal
(e.g., human), or other organism or cell.
In some embodiments, the circular polyribonucleotide has a decreased, or fails
to produce a,
undesired response by the host's immune system as compared to the response
triggered by a reference
compound, e.g., a linear polynucleotide corresponding to the described
circular polyribonucleotide or a
circular polyribonucleotide lacking an encryptogen. In embodiments, the
circular polyribonucleotide is
non-immunogenic in the host. Some immune responses include, but are not
limited to, humoral immune
responses (e.g. production of immunogen-specific antibodies) and cell-mediated
immune responses (e.g.,
lymphocyte proliferation).
In some embodiments, a host or a host cell is contacted with (e.g., delivered
to or administered
to) the circular polyribonucleotide or linear. In some embodiments, the host
is a mammal, such as a
human. The amount of the circular polyribonucleotide or linear, expression
product, or both in the host
can be measured at any time after administration. In certain embodiments, a
time course of host growth
in a culture is determined. If the growth is increased or reduced in the
presence of the circular
polyribonucleotide or linear, the circular polyribonucleotide or expression
product or both is identified as
being effective in increasing or reducing the growth of the host.
A method of delivering a circular or linear polyribonucleotide molecule as
described herein to a
cell, tissue, or subject, includes administering the pharmaceutical
composition, pharmaceutical drug
substance or pharmaceutical drug product as described herein to the cell,
tissue, or subject.
In some embodiments, the cell is a eukaryotic cell. In some embodiments, the
cell is a
mammalian cell. In some embodiments, the cell is an ungulate cell. In some
embodiments, the cell is an
animal cell. In some embodiments, the cell is an immune cell. In some
embodiments, the tissue is a
connective tissue, a muscle tissue, a nervous tissue, or an epithelial tissue.
In some embodiments, the
tissue is an organ (e.g., liver, lung, spleen, kidney, etc.).
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In some embodiments, the method of delivering is an in vivo method. For
example, a method of
delivery of a circular polyribonucleotide as described herein includes
parenterally administering to a
subject in need thereof, the pharmaceutical composition, pharmaceutical drug
substance or
pharmaceutical drug product as described herein to the subject in need
thereof. As another example, a
method of delivering a circular polyribonucleotide to a cell or tissue of a
subject, includes administering
parenterally to the cell or tissue the pharmaceutical composition,
pharmaceutical drug substance or
pharmaceutical drug product as described herein. In some embodiments, the
circular polyribonucleotide
is in an amount effective to elicit a biological response in the subject. In
some embodiments, the circular
polyribonucleotide is an amount effective to have a biological effect on the
cell or tissue in the subject. In
some embodiments, the pharmaceutical composition, pharmaceutical drug
substance or pharmaceutical
drug product as described herein includes a carrier. In some embodiments the
pharmaceutical
composition, pharmaceutical drug substance or pharmaceutical drug product as
described herein
includes a diluent and is free of any carrier.
In some embodiments the pharmaceutical composition, the pharmaceutical drug
substance, or
the pharmaceutical drug product is administered parenterally. In some
embodiments the pharmaceutical
composition, the pharmaceutical drug substance, or the pharmaceutical drug
product is administered
intravenously, intraarterially, intraperitoneally, intradermally,
intracranially, intrathecally, intralymphaticly,
subcutaneously, or intramuscularly. In some embodiments, parenteral
administration is intravenously,
intramuscularly, ophthalmically, subcutaneously, intradermally or topically.
In some embodiments, the pharmaceutical composition, pharmaceutical drug
substance or
pharmaceutical drug product as described herein is administered
intramuscularly. In some embodiments,
the pharmaceutical composition, pharmaceutical drug substance or
pharmaceutical drug product as
described herein is administered subcutaneously. In some embodiments, the
pharmaceutical
composition, pharmaceutical drug substance or pharmaceutical drug product as
described herein is
administered topically. In some embodiments, the pharmaceutical composition,
the pharmaceutical drug
substance, or the pharmaceutical drug product is administered intratracheally.
In some embodiments the pharmaceutical composition, pharmaceutical drug
substance or
pharmaceutical drug product is administered by injection. The administration
can be systemic
administration or local administration. In some embodiments, any of the
methods of delivery as described
herein are performed with a carrier. In some embodiments, any methods of
delivery as described herein
are performed without the aid of a carrier or cell penetrating agent.
In some embodiments, the circular polyribonucleotide or a product translated
from the circular
polyribonucleotide is detected in the cell, tissue, or subject at least 1 day,
at least 2 days, at least 3 days,
at least 4 days, or at least 5 days after the administering step. In some
embodiments, the presence of the
circular polyribonucleotide or a product translated from the circular
polyribonucleotide is evaluated in the
cell, tissue, or subject before the administering step. In some embodiments,
the presence of the circular
polyribonucleotide or a product translated from the circular
polyribonucleotide is evaluated in the cell,
tissue, or subject after the administering step.
Formulations
In some embodiments, a pharmaceutical formulation disclosed herein can
include: (i) a
compound (e.g., circular polyribonucleotide) disclosed herein; (ii) a buffer;
(iii) a non-ionic detergent; (iv) a
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tonicity agent; and/or (v) a stabilizer. In some embodiments, a pharmaceutical
formulation disclosed
herein can include: (i) a compound (e.g., linear polyribonucleotide) disclosed
herein; (ii) a buffer; (iii) a
non-ionic detergent; (iv) a tonicity agent; and/or (v) a stabilizer. In some
embodiments, the
pharmaceutical formulation disclosed herein is a stable liquid pharmaceutical
formulation. In some
embodiments, the pharmaceutical formulation disclosed herein includes
protamine or a protamine salt
(e.g., protamine sulfate).
The disclosure provides immunogenic compositions including a circular
polyribonucleotide
described herein. The disclosure provides immunogenic compositions including a
linear
polyribonucleotide described herein. Immunogenic compositions of the
disclosure may include a diluent
or a carrier, adjuvant, or any combination thereof. Immunogenic compositions
of the disclosure may also
include one or more immunoregulatory agents, e.g., one or more adjuvants. The
adjuvants may include a
TH1 adjuvant and/or a TH2 adjuvant, further discussed below. In some
embodiments, the immunogenic
composition includes a diluent free of any carrier and is used for naked
delivery of the circular
polyribonucleotide to a subject. In some embodiments, the immunogenic
composition includes a diluent
free of any carrier and is used for naked delivery of the linear
polyribonucleotide to a subject.
Immunogenic compositions of the disclosure are used to raise an immune
response in a subject.
The immune response is preferably protective and preferably involves an
antibody response (usually
including IgG) and/or a cell-mediated immune response. For example, a subject
is immunized with an
immunogenic composition including a circular polyribonucleotide of the
disclosure to induce an immune
response. In another example, a subject is immunized with an immunogenic
composition including a
linear polyribonucleotide including an immunogen to stimulate production of
antibodies that bind to the
immunogen. By raising an immune response in the subject by these uses and
methods, the subject can
be protected against various diseases and/or infections e.g. against bacterial
and/or viral diseases as
discussed above. In certain embodiments, the immunogenic compositions are
vaccine compositions.
Vaccines according to the disclosure may either be prophylactic (i.e. to
prevent infection) or therapeutic
(i.e. to treat infection) but will typically be prophylactic. In some
embodiments, the subject is a mammal.
In some embodiments, the subject is an animal, preferably a mammal, e.g., a
human. In one
embodiment, the subject is a human. In other embodiments the subject is a non-
human mammal, e.g.,
selected from a cow (e.g., dairy and beef cattle), a sheep, a goat, a pig, a
horse, a dog, or a cat. In other
embodiments the subject is a bird, e.g., a hen or rooster, turkey, parrot. In
some embodiments, the
animal is not a mouse or a rabbit or a cow. In a particular embodiment, where
the immunogenic
composition is for prophylactic use, the human is a child (e.g. a toddler or
infant) or a teenager. In
another embodiment, where the immunogenic composition is for therapeutic use,
the human is a
teenager or an adult. An immunogenic composition intended for children may
also be administered to
adults e.g. to assess safety, dosage, immunogenicity, etc.
Immunogenic composition prepared according to the disclosure may be used to
treat both
children and adults. A human subject may be less than 1 year old, less than 5
years old, 1-5 years old, 5-
15 years old, 15-55 years old, or at least 55 years old. In a particular
embodiment, a human subject for
receiving the immunogenic compositions are the elderly (e.g., 50 years old, 60
years old, and 65
years), the young (e.g., years old), hospitalized patients, healthcare
workers, armed service and
military personnel, pregnant women, the chronically ill, or immunodeficient
patients. The immunogenic
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compositions are not suitable solely for these groups, however, and may be
used more generally in a
population.
In some embodiments, the subject is further immunized with an adjuvant. In
some embodiments
the subject is further immunized with a vaccine.
Immunization
In some embodiments, methods of the disclosure include immunizing a subject
with an
immunogenic composition including a circular polyribonucleotide as disclosed
herein. In some
embodiments, an immunogen is expressed from the circular polyribonucleotide.
In some embodiments,
immunization induces an immune response in a subject against the immunogen
expressed from the
circular polyribonucleotide. In some embodiments, immunization induces an
immune response in a
subject (e.g., induces the production of antibodies that bind to the immunogen
expressed from the circular
polyribonucleotide). In some embodiments, an immunogenic composition includes
the circular
polyribonucleotide and a diluent, carrier, first adjuvant or a combination
thereof in a single composition.
In some embodiments, the subject is further immunized with a second adjuvant.
In some embodiments,
the subject is further immunized with a vaccine.
In some embodiments, methods of the disclosure include immunizing a subject
with an
immunogenic composition including a linear polyribonucleotide as disclosed
herein. In some
embodiments, an immunogen is expressed from the linear polyribonucleotide. In
some embodiments,
immunization induces an immune response in a subject against the immunogen
expressed from the
linear polyribonucleotide. In some embodiments, immunization induces the
production of antibodies that
bind to the immunogen expressed from the linear polyribonucleotide. In some
embodiments,
immunization induces a cell-mediated immune response. In some embodiments, an
immunogenic
composition includes the linear polyribonucleotide and a diluent, carrier,
first adjuvant or a combination
thereof in a single composition. In some embodiments, the subject is further
immunized with a second
adjuvant. In some embodiments, the subject is further immunized with a
vaccine.
The subject is immunized with one or more immunogenic composition(s) including
any number of
circular polyribonucleotides. The subject is immunized with, for example, one
or more immunogenic
composition(s) including at least 1 circular polyribonucleotide. A non-human
animal having a non-
humanized immune system is immunized with, for example, one or more
immunogenic composition(s)
including at least 2, at least 3, at least 4, at least 5, at least 6, at least
7, at least 8, at least 9, at least 10,
at least 11, at least 12, at least 13, at least 14, at least 15, at least 20
different circular
polyribonucleotides, or more different circular polyribonucleotides. In some
embodiments, a subject is
immunized with one or more immunogenic composition(s) including at most 1
circular polyribonucleotide.
In some embodiments, a non-human animal having a humanized immune system is
immunized with one
or more immunogenic composition(s) including at most 2, at most 3, at most 4,
at most 5, at most 6, at
most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13,
at most 14, at most 15, at
most 20 different circular polyribonucleotides, or less than 21 different
circular polyribonucleotides. In
some embodiments, a subject is immunized with one or more immunogenic
composition(s) including
about 1 circular polyribonucleotide. In some embodiments, a non-human animal
having a humanized
immune system is immunized with one or more immunogenic composition(s)
including about 2, about 3,
about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11,
about 12, about 13, about 14,
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about 15, or about 20 different circular polyribonucleotides. In some
embodiments, a subject is
immunized with one or more immunogenic composition(s) including about 1-20, 1-
15, 1-10, 1-9, 1-8, 1-7,
1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3,
3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-
6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 4-4, 4-3, 5-20, 5-15,
5-10, 5-9, 5-8, 5-7, 5-6, 5-10, 10-
15, or 15-20 different circular polyribonucleotides. Different circular
polyribonucleotides have different
sequences from each other. For example, they can include or encode different
immunogens, overlapping
immunogens, similar immunogens, or the same immunogens (for example, with the
same or different
regulatory elements, initiation sequences, promoters, termination elements, or
other elements of the
disclosure). In cases where a subject is immunized with one or more
immunogenic composition(s)
including two or more different circular polyribonucleotides, the two or more
different circular
polyribonucleotides can be in the same or different immunogenic compositions
and immunized at the
same time or at different times. The immunogenic compositions including two or
more different circular
polyribonucleotides can be administered to the same anatomical location or
different anatomical
locations.
The subject can be immunized with one or more immunogenic composition(s)
including any
number of linear polyribonucleotides. The subject is immunized with, for
example, one or more
immunogenic composition(s) including at least 1 linear polyribonucleotide. A
non-human animal having a
non-humanized immune system is immunized with, for example, one or more
immunogenic
composition(s) including at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, at least
9, at least 10, at least 11, at least 12, at least 13, at least 14, at least
15, at least 20 different linear
polyribonucleotides, or more different linear polyribonucleotides. In some
embodiments, a subject is
immunized with one or more immunogenic composition(s) including at most 1
linear polyribonucleotide.
In some embodiments, a non-human animal having a humanized immune system is
immunized with one
or more immunogenic composition(s) including at most 2, at most 3, at most 4,
at most 5, at most 6, at
most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13,
at most 14, at most 15, at
most 20 different linear polyribonucleotides, or less than 21 different linear
polyribonucleotides. In some
embodiments, a subject is immunized with one or more immunogenic
composition(s) including about 1
linear polyribonucleotide. In some embodiments, a non-human animal having a
humanized immune
system is immunized with one or more immunogenic composition(s) including
about 2, about 3, about 4,
about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12,
about 13, about 14, about 15,
or about 20 different linear polyribonucleotides. In some embodiments, a
subject is immunized with one or
more immunogenic composition(s) including about 1-20, 1-15, 1-10, 1-9, 1-8, 1-
7, 1-6, 1-5, 1-4, 1-3, 1-2,
2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-
8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15,
4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 4-4, 4-3, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6,
5-10, 10-15, or 15-20 different
linear polyribonucleotides. Different linear polyribonucleotides can have
different sequences from each
other. For example, they can include or encode different immunogens,
overlapping immunogens, similar
immunogens, or the same immunogens (for example, with the same or different
regulatory elements,
initiation sequences, promoters, termination elements, or other elements of
the disclosure). In cases
where a subject is immunized with one or more immunogenic composition(s)
including two or more
different linear polyribonucleotides, the two or more different linear
polyribonucleotides can be in the
same or different immunogenic compositions and immunized at the same time or
at different times. The
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immunogenic compositions including two or more different linear
polyribonucleotides can be administered
to the same anatomical location or different anatomical locations.
The two or more different linear polyribonucleotides can include or encode
immunogens from the
same source, different source, or different combinations of sources disclosed
herein. The two or more
different linear polyribonucleotides can include or encode immunogens from the
same virus or from
different viruses, for example, different isolates.
In some embodiments, the subject is immunized with one or more immunogenic
composition(s)
including any number of circular polyribonucleotides and one or more
immunogenic composition(s)
including any number of linear polyribonucleotides as disclosed herein. In
some embodiments, an
immunogenic composition disclosed herein includes one or more circular
polyribonucleotides and one or
more linear polyribonucleotides as disclosed herein.
In some embodiments, an immunogenic composition includes a circular
polyribonucleotide and a
diluent, a carrier, a first adjuvant, or a combination thereof. In a
particular embodiment, an immunogenic
composition includes a circular polyribonucleotide described herein and a
carrier or a diluent free of any
carrier. In some embodiments, an immunogenic composition including a circular
polyribonucleotide with
a diluent free of any carrier is used for naked delivery of the circular
polyribonucleotide to a subject. In
another particular embodiment, an immunogenic composition includes a circular
polyribonucleotide
described herein and a first adjuvant.
In certain embodiments, a subject is further administered a second adjuvant.
An adjuvant
enhances the innate immune response, which in turn, enhances the adaptive
immune response in a
subject. An adjuvant can be any adjuvant as discussed below. In certain
embodiments, an adjuvant is
formulated with the circular polyribonucleotide as a part of an immunogenic
composition. In certain
embodiments, an adjuvant is not part of an immunogenic composition including
the circular
polyribonucleotide. In certain embodiments, an adjuvant is administered
separately from an
immunogenic composition including the circular polyribonucleotide. In this
aspect, the adjuvant is co-
administered (e.g., administered simultaneously) or administered at a
different time than an immunogenic
composition including the circular polyribonucleotide to the subject. For
example, the adjuvant is
administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45
minutes, 60 minutes, 90
minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9
hours, 10 hours, 12 hours, 14
hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or
hour therebetween, after an
immunogenic composition including the circular polyribonucleotide. In some
embodiments, the adjuvant is
administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45
minutes, 60 minutes, 90
minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9
hours, 10 hours, 12 hours, 14
hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or
hour therebetween, before
an immunogenic composition including the circular polyribonucleotide. For
example, the adjuvant is
administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or
84 days, or any day
therebetween, after an immunogenic composition including the circular
polyribonucleotide. In some
embodiments, the adjuvant is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35,
42, 49, 56, 63, 70, 77, or 84
days, or any day therebetween, before an immunogenic composition including the
circular
.. polyribonucleotide. The adjuvant is administered to the same anatomical
location or different anatomical
location as the immunogenic composition including the circular
polyribonucleotide.
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In some embodiments, an immunogenic composition includes a linear
polyribonucleotide and a
diluent, a carrier, a first adjuvant, or a combination thereof. In a
particular embodiment, an immunogenic
composition includes a linear polyribonucleotide described herein and a
carrier or a diluent free of any
carrier. In some embodiments, an immunogenic composition including a linear
polyribonucleotide with a
diluent free of any carrier is used for naked delivery of the linear
polyribonucleotide to a subject. In
another particular embodiment, an immunogenic composition includes a linear
polyribonucleotide
described herein and a first adjuvant.
In certain embodiments, a subject is further administered a second adjuvant.
An adjuvant
enhances the innate immune response, which in turn, enhances the adaptive
immune response in a
subject. An adjuvant can be any adjuvant as discussed below. In certain
embodiments, an adjuvant is
formulated with the linear polyribonucleotide as a part of an immunogenic
composition. In certain
embodiments, an adjuvant is not part of an immunogenic composition including
the linear
polyribonucleotide. In certain embodiments, an adjuvant is administered
separately from an
immunogenic composition including the linear polyribonucleotide. In this
aspect, the adjuvant is co-
administered (e.g., administered simultaneously) or administered at a
different time than an immunogenic
composition including the linear polyribonucleotide to the subject. For
example, the adjuvant is
administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45
minutes, 60 minutes, 90
minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9
hours, 10 hours, 12 hours, 14
hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or
hour therebetween, after an
immunogenic composition including the linear polyribonucleotide. In some
embodiments, the adjuvant is
administered 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45
minutes, 60 minutes, 90
minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9
hours, 10 hours, 12 hours, 14
hours, 16 hours, 18 hours, 20 hours, 22 hours, or 24 hours, or any minute or
hour therebetween, before
an immunogenic composition including the linear polyribonucleotide. For
example, the adjuvant is
administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or
84 days, or any day
therebetween, after an immunogenic composition including the linear
polyribonucleotide. In some
embodiments, the adjuvant is administered 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35,
42, 49, 56, 63, 70, 77, or 84
days, or any day therebetween, before an immunogenic composition including the
linear
polyribonucleotide. The adjuvant is administered to the same anatomical
location or different anatomical
location as the immunogenic composition including the linear
polyribonucleotide.
In some embodiments, a subject is further immunized with a second agent, e.g.,
a vaccine (as
described below) that is not a circular polyribonucleotide. The vaccine is co-
administered (e.g.,
administered simultaneously) or administered at a different time than an
immunogenic composition
including the circular polyribonucleotide to the subject. For example, the
vaccine is administered 1
minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes,
90 minutes, 2 hours, 3
hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12
hours, 14 hours, 16 hours, 18
hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween,
after an immunogenic
composition including the circular polyribonucleotide. In some embodiments,
the vaccine is administered
1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60
minutes, 90 minutes, 2 hours, 3
hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12
hours, 14 hours, 16 hours, 18
hours, 20 hours, 22 hours, or 24 hours, or any minute or hour therebetween,
before an immunogenic
composition including the circular polyribonucleotide. For example, the
vaccine is administered 1, 2, 3, 4,
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5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day
therebetween, after an immunogenic
composition including the circular polyribonucleotide. In some embodiments,
the vaccine is administered
1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or
any day therebetween, before an
immunogenic composition including the circular polyribonucleotide.
In some embodiments, a subject is further immunized with a second agent, e.g.,
a vaccine (as
described below) that is not a linear polyribonucleotide. The vaccine is co-
administered (e.g.,
administered simultaneously) or administered at a different time than an
immunogenic composition
including the linear polyribonucleotide to the subject. For example, the
vaccine is administered 1 minute,
5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90
minutes, 2 hours, 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14
hours, 16 hours, 18 hours, 20
hours, 22 hours, or 24 hours, or any minute or hour therebetween, after an
immunogenic composition
including the linear polyribonucleotide. In some embodiments, the vaccine is
administered 1 minute, 5
minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90
minutes, 2 hours, 3 hours, 4
hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14
hours, 16 hours, 18 hours, 20
hours, 22 hours, or 24 hours, or any minute or hour therebetween, before an
immunogenic composition
including the linear polyribonucleotide. For example, the vaccine is
administered 1, 2, 3, 4, 5, 6, 7, 14, 21,
28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day therebetween, after an
immunogenic composition
including the linear polyribonucleotide. In some embodiments, the vaccine is
administered 1, 2, 3, 4, 5, 6,
7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, or 84 days, or any day
therebetween, before an immunogenic
composition including the linear polyribonucleotide.
A subject can be immunized with an immunogenic composition, adjuvant, vaccine
(e.g., protein
subunit vaccine), or a combination thereof any suitable number of times to
achieve a desired response.
For example, a prime-boost immunization strategy can be utilized to elicit
systemic and/or mucosal
immunity. A subject can be immunized with an immunogenic composition,
adjuvant, vaccine (e.g.,
.. protein subunit vaccine), or a combination thereof, of the disclosure, for
example, at least 1, at least 2, at
least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least
9, at least 10, or at least 15 times, or
more.
In some embodiments, a subject can be immunized with an immunogenic
composition, adjuvant,
vaccine (e.g., protein subunit vaccine), or a combination thereof, of the
disclosure at most 2, at most 3, at
.. most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10,
at most 15, or at most 20 times,
or less.
In some embodiments, a subject can be immunized with an immunogenic
composition, adjuvant,
vaccine (e.g., protein subunit vaccine), or a combination thereof, of the
disclosure about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, or 20 times.
In some embodiments, a subject can be immunized with an immunogenic
composition, adjuvant,
vaccine (e.g., protein subunit vaccine), or a combination thereof, of the
disclosure once. In some
embodiments, a subject can be immunized with an immunogenic composition,
adjuvant, vaccine (e.g.,
protein subunit vaccine), or a combination thereof, of the disclosure twice.
In some embodiments, a
subject can be immunized with an immunogenic composition, adjuvant, vaccine
(e.g., protein subunit
vaccine), or a combination thereof, of the disclosure three times. In some
embodiments, a subject can be
immunized with an immunogenic composition, adjuvant, vaccine (e.g., protein
subunit vaccine), or a
combination thereof, of the disclosure four times. In some embodiments, a
subject can be immunized
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with an immunogenic composition, adjuvant, vaccine (e.g., protein subunit
vaccine), or a combination
thereof, of the disclosure five times. In some embodiments, a subject can be
immunized with an
immunogenic composition, adjuvant, vaccine (e.g., protein subunit vaccine), or
a combination thereof, of
the disclosure seven times.
Suitable time intervals can be selected for spacing two or more immunizations.
The time
intervals can apply to multiple immunizations with the same immunogenic
composition, adjuvant, or
vaccine (e.g., protein subunit vaccine), or combination thereof, for example,
the same immunogenic
composition, adjuvant, or vaccine (e.g., protein subunit vaccine), or
combination thereof, can be
administered in the same amount or a different amount, via the same
immunization route or a different
immunization route. The time intervals can apply to multiple immunizations
with a different immunogenic
composition, adjuvant, or vaccine (e.g., protein subunit vaccine), or
combination thereof, for example, a
different immunogenic composition, adjuvant, or vaccine (e.g., protein subunit
vaccine), or combination
thereof, can be administered in the same amount or a different amount, via the
same immunization route
or a different immunization route. The time intervals can apply to
immunizations with different agents, for
example, a first immunogenic composition including a first circular
polyribonucleotide and a second
immunogenic composition including a second circular polyribonucleotide. The
time intervals can apply to
immunizations with different agents, for example, a first immunogenic
composition including a first circular
polyribonucleotide and a second immunogenic composition including a protein
immunogen (e.g., a
protein subunit). The time intervals can apply to a first immunogenic
composition including a first linear
polyribonucleotide and a second immunogenic composition including a second
linear polyribonucleotide.
For regimens including three or more immunizations, the time intervals between
immunizations can be
the same or different. In some examples, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 14, 16, 17 ,18, 20, 22,
24, 26, 28, 30, 32, 34, 36, 40, 48, or 72 hours elapse between two
immunizations. In some embodiments,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 17, 18, 20, 21, 24, 28,
or 30 days elapse between two
immunizations. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, or 8 weeks
elapse between two
immunizations. In some embodiments, about 1, 2, 3, 4, 5, 6, 7, or 8 months
elapse between two
immunizations.
In some embodiments, at least 1, at least 2, at least 3, at least 4, at least
5, at least 6, at least 7,
at least 8, at least 9, at least 10, at least 15, at least 20, at least 24, at
least 36, or at least 72 hours, or
more elapse between two immunizations. In some embodiments, at most 1, at most
2, at most 3, at most
4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most
15, at most 20, at most 24, at
most 36, or at most 72 hours, or less elapse between two immunizations.
In some embodiments, at least 1, at least 2, at least 3, at least 4, at least
5, at least 6, at least 7,
at least 8, at least 9, at least 10, at least 15, at least 20, at least 21, at
least 22, at least 23, at least 24, at
least 25, at least 26 at least 27, at least 28, at least 29, or at least 30
days, or more, elapse between two
immunizations. In some embodiments, at most 2, at most 3, at most 4, at most
5, at most 6, at most 7, at
most 8, at most 9, at most 10, at most 15, at most 20, at most 21, at most 22,
at most 23, at most 24, at
most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most
32, at most 34, or at most 36
days, or less elapse between two immunizations.
In some embodiments, at least 1, at least 2, at least 3, at least 4, at least
5, at least 6, at least 7,
or at least 8 weeks, or more elapse between two immunizations. In some
embodiments, at most 2, at
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most 3, at most 4, at most 5, at most 6, at most 7, at most 8 weeks, or less
elapse between two
immunizations.
In some embodiments, at least 1, at least 2, at least 3, at least 4, at least
5, at least 6, at least 7,
or at least 8 months, or more elapse between two immunizations. In some
embodiments, at most 2, at
most 3, at most 4, at most 5, at most 6, at most 7, at most 8 months, at most
9 months, at most 10
months, at most 11 months, or at most 12 months or less elapse between two
immunizations.
In some embodiments, the method includes pre-administering to the subject an
agent to improve
immunogenic responses to a circular polyribonucleotide including a sequence
encoding an immunogen.
In some embodiments, the agent is the immunogen as disclosed herein (e.g., a
protein immunogen). For
example, the method includes administering the protein immunogen from 1 to 7
days prior to
administration of the circular polyribonucleotide including the sequence
encoding the protein immunogen.
In some embodiments, the protein immunogen is administered 1, 2, 3, 4, 5, 6,
or 7 days prior to
administration of the circular polyribonucleotide including the sequence
encoding the protein immunogen.
For example, the method includes administering the protein immunogen from 1 to
7 days prior to
administration of the linear polyribonucleotide including the sequence
encoding the protein immunogen.
In some embodiments, the protein immunogen is administered 1, 2, 3, 4, 5, 6,
or 7 days prior to
administration of the linear polyribonucleotide including the sequence
encoding the protein immunogen.
The protein immunogen may be administered as a protein preparation, encoded in
a plasmid (pDNA),
presented in a virus-like particle (VLP), formulated in a lipid nanoparticle,
or the like.
In some embodiments, the method includes administering to the subject an agent
to improve
immunogenic responses to a circular polyribonucleotide including a sequence
encoding an immunogen
after the subject has been administered the circular polyribonucleotide
including a sequence encoding an
immunogen. In some embodiments, the agent is the immunogen as disclosed herein
(e.g., a protein
immunogen). In some embodiments, the circular polyribonucleotide includes a
sequence encoding a
protein immunogen. For example, the method includes administering the protein
immunogen within 1
year (e.g., within 11 months, 10 months, 9 months, 8 months, 7 months, 6
months, 5 months, 4 months, 3
months, 2 months, and 1 month) of administering the circular
polyribonucleotide including a sequence
encoding the immunogen to the subject. In some embodiments, the method
includes administering any
one of the circular polyribonucleotides described herein or any one of the
immunogenic compositions
described herein and a protein subunit to the subject.
In some embodiments, the protein immunogen has the same amino acid sequence as
the
immunogen encoded by circular polyribonucleotide. For example, the polypeptide
immunogen may
correspond to (e.g., shares 90%, 95%, 96%, 97%, 98%, or 100%) amino acid
sequence identity with a
polypeptide immunogen encoded by a sequence of the circular
polyribonucleotide. In some
embodiments, the protein immunogen has a different amino acid sequence from
the amino acid
sequence of the immunogen encoded by the circular polyribonucleotide. For
example, the polypeptide
immunogen may share less than 90% (e.g., 80%, 70%, 30%, 20%, or 10%) amino
acid sequence identity
with the polypeptide immunogen encoded by a sequence of the circular
polyribonucleotide.
A subject can be immunized with an immunogenic composition, an adjuvant, or a
vaccine (e.g.,
protein subunit vaccine), or a combination thereof, at any suitable number
anatomical sites. The same
immunogenic composition, an adjuvant, a vaccine (e.g., protein subunit
vaccine), or a combination
thereof can be administered to multiple anatomical sites, different
immunogenic compositions including
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the same or different circular polyribonucleotides, adjuvants, vaccines (e.g.,
protein subunit vaccine) or a
combination thereof can be administered to different anatomical sites,
different immunogenic
compositions including the same or different circular polyribonucleotides,
adjuvants, vaccines (e.g.,
protein subunit vaccines) or a combination thereof can be administered to the
same anatomical site, or
any combination thereof. For example, an immunogenic composition including a
circular
polyribonucleotide can be administered in to two different anatomical sites,
and/or an immunogenic
composition including a circular polyribonucleotide can be administered to one
anatomical site, and an
adjuvant can be administered to a different anatomical site. The same
immunogenic composition, an
adjuvant, a vaccine (e.g., protein subunit vaccine), or a combination thereof
can be administered to
multiple anatomical sites, different immunogenic compositions including the
same or different linear
polyribonucleotides, adjuvants, vaccines (e.g., protein subunit vaccine) or a
combination thereof can be
administered to different anatomical sites, different immunogenic compositions
including the same or
different linear polyribonucleotides, adjuvants, vaccines (e.g., protein
subunit vaccines) or a combination
thereof can be administered to the same anatomical site, or any combination
thereof. For example, an
immunogenic composition including a linear polyribonucleotide can be
administered in to two different
anatomical sites, and/or an immunogenic composition including a linear
polyribonucleotide can be
administered to one anatomical site, and an adjuvant can be administered to a
different anatomical site.
Immunization at any two or more anatomical routes can be via the same route of
immunization
(e.g., intramuscular) or by two or more routes of immunization. In some
embodiments, an immunogenic
composition including a circular polyribonucleotide, an adjuvant, or a vaccine
(e.g., protein subunit
vaccine), or a combination thereof, of the disclosure is immunized to at least
1, at least 2, at least 3, at
least 4, at least 5, or at least 6 anatomical sites of a subject. In some
embodiments, an immunogenic
composition including a circular polyribonucleotide, an adjuvant, or a vaccine
(e.g., protein subunit
vaccine), or a combination thereof, of the disclosure is immunized to at most
2, at most 3, at most 4, at
most 5, at most 6, at most 7, at most 8, at most 9, or at most 10 anatomical
sites of the subject, or less. In
some embodiments, an immunogenic composition including a circular
polyribonucleotide or an adjuvant
of the disclosure is immunized to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 anatomical
sites of a subject. In some
embodiments, an immunogenic composition including a linear polyribonucleotide,
an adjuvant, or a
vaccine (e.g., protein subunit vaccine), or a combination thereof, of the
disclosure is immunized to at least
1, at least 2, at least 3, at least 4, at least 5, or at least 6 anatomical
sites of a subject. In some
embodiments, an immunogenic composition including a linear polyribonucleotide,
an adjuvant, or a
vaccine (e.g., protein subunit vaccine), or a combination thereof, of the
disclosure is immunized to at most
2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most
9, or at most 10 anatomical
sites of the subject, or less. In some embodiments, an immunogenic composition
including a linear
polyribonucleotide or an adjuvant of the disclosure is immunized to 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 anatomical
sites of a subject.
Immunization can be by any suitable route. Non-limiting examples of
immunization routes
include intravenous, intramuscular, intraarterial, intrathecal, intracapsular,
intraorbital, intracardiac,
intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular,
subarachnoid, intraspinal, epidural, intrasternal, intracerebral, intraocular,
intralesional,
intracerebroventricular, intracisternal, or intraparenchymal, e.g., injection
and infusion. In some cases,
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immunization can be via inhalation. Two or more immunizations can be done by
the same route or by
different routes.
Any suitable amount of a circular polyribonucleotide can be administered to a
subject of the
disclosure. For example, a subject can be immunized with at least about 1 ng,
at least about 10 ng, at
least about 100 ng, at least about 1 pg, at least about 10 pg, at least about,
at least about 100 pg, at least
about 1 mg, at least about 10 mg, at least about 100 mg, or at least about 1 g
of a circular
polyribonucleotide. In some embodiments, a subject can be immunized with at
most about 1 ng, at most
about 10 ng, at most about 100 ng, at most about 1 pg, at most about 10 pg, at
most about, at most about
100 pg, at most about 1 mg, at most about 10 mg, at most about 100 mg, or at
most about 1 g of a
circular polyribonucleotide. In some embodiments, a subject can be immunized
with about 1 ng, about 10
ng, about 100 ng, about 1 pg, about 10 pg, about, about 100 pg, about 1 mg,
about 10 mg, about 100 mg,
or about 1 g of a circular polyribonucleotide.
In some embodiments, the method further includes evaluating the subject for
antibody response
to the immunogen. In some embodiments, the evaluating is before and/or after
administration of the
circular polyribonucleotide including a sequence encoding an immunogen. In
some embodiments, the
evaluating is before and/or after administration of the linear
polyribonucleotide including a sequence
encoding an immunogen.
In some embodiments, the circular polyribonucleotide, immunogenic composition,
pharmaceutical
preparation, or pharmaceutical composition described herein is administered to
a subject between birth
and 15 months according to the dosing schedule provided in Table 1 or is
administered to a subject
between 18 months and 18 years according to the dosing schedule of Table 2.
Dosing may be performed
according to dosing scheduled known in the art, for example, as described by
the Centers of Disease
Control and Prevention (CDC) or the National Institutes of Health (NIH).
Tables 1 and 2 provide an
abbreviated summary of the dosing schedules for vaccination for certain
disorders indicated on the CDC
website as of August 29, 2020.
Table 1. Dosing birth to 15 months
Indication Birth 1 ma 2 mos 4 mos 6
mos 9 mos 12 mos 15 mos
(Vaccine)
Hepatitis B 1st 2nd dose 3rd dose
(HepB) dose
Measles, 15t dose
mumps, rubella
(MMR); varicella
(VAR)
Hepatitis A 2 dose series
(HepA)
Rotavirus 1St dose 2nd dose 3rd dose*
(RV1 or RV5)
Diphtheria, 1St dose 2nd dose 3rd dose
4th dose
tetanus,
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acellular
pertussis (DTap)
Haemophilus 1St dose 2nd dose 3rd dose* 3rd or 4th
dose
Influenzae B
(Hib)
Pneumococcal 1St dose 2nd dose 3rd dose 4th dose
conjugate
(PCV13)
Inactivated polio 15t dose 2nd dose 3rd dose
(IPV)
Influenza (IIV)
Annual vaccination 1 or 2 doses
*optional
Table 2. Dosing 18 months to 18 years
Vaccine 18 19-23 2-3 4-6 4-6 7-10 11-12 13-15 16 17-18
mos yrs yrs yrs yrs yrs yrs yrs yrs
yrs
Hep B 3rd
(HepB) dose
Diphtheria, 4th 5th
tetanus, dose dose
acellular
pertussis
(DTap)
Inactivated 3rd 4th
polio (IPV) dose dose
Influenza (IIV) Annual
vaccination 1 or 2 doses Annual vaccination 1 dose only
Influenza Annual vaccine 1 or 2 Annual
vaccination 1 dose only
(LAIV) doses
Hepatitis A 2 dose series
(HepA)
Meningococcal 15t 2nd
(MenACWY-D; dose dose
MenACWY-
CRM)
Measles, 2nd
mumps, dose
rubella (MMR);
varicella (VAR)
Diphtheria, TDap
tetanus,
acellular
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pertussis
(DTap)
Topical Delivery
In some aspects, the present disclosure provides compositions and
pharmaceutical compositions
for topically delivering a polyribonucleotide that produces an immune response
in the subject. The
compositions and pharmaceutical compositions can comprise an alcohol (e.g.,
ethanol) for delivering the
polyribonucleotide to a cell. The compositions and pharmaceutical compositions
can comprise a cell-
penetrating agent. The cell-penetrating agent is configured to enhance
delivery of the polyribonucleotide
into a cell. The polyribonucleotide can be present in either linear or
circular form.
In some embodiments, the compositions or pharmaceutical compositions described
herein are
directly administered to a surface area (e.g., a topical surface area). In
some embodiments, the
compositions or pharmaceutical compositions described herein are applied to a
surface area of a subject
after application of a sterilizing agent. In some embodiments, the
compositions or pharmaceutical
compositions described herein are applied to a surface area of a subject after
application of an alcohol.
The compositions disclosed herein can include a mixture including a
polyribonucleotide and an
alcohol, such as ethanol. The methods disclosed herein can include delivering
a polyribonucleotide in a
composition comprising a mixture of the polyribonucleotide and an alcohol,
such as ethanol. In some
aspects, the present disclosure provides a kit comprising a polyribonucleotide
and an alcohol (e.g.,
ethanol) for delivering the polyribonucleotide into a cell. In some
embodiments, the kit comprises a
sterilizing agent. In a particular embodiment, the kit comprises a
polyribonucleotide and an alcohol wipe
(e.g., ethanol wipe, isopropyl wipe).
The compositions disclosed herein can include a mixture including a
polyribonucleotide and a
cell-penetrating agent. The methods disclosed herein can include delivering a
polyribonucleotide in a
composition comprising a mixture of the polyribonucleotide and a cell-
penetrating agent. In some
aspects, the present disclosure provides a kit comprising a polyribonucleotide
and a cell-penetrating
agent for delivery of the polyribonucleotide into a cell. In some embodiments,
the kit comprises a
sterilizing agent.
The compositions disclosed herein can be a composition free of any carrier
comprising a
polyribonucleotide and a diluent. This composition can be used in a method of
delivery to an epithelial
cell.
In some embodiments, the compositions, therapeutic compositions, or
pharmaceutical
compositions described herein are directly administered to a surface area
(e.g., a topical surface area).
In some embodiments, the compositions, therapeutic compositions, or
pharmaceutical compositions
described herein are applied to a surface area of a subject after application
of a sterilizing agent.
The compositions, methods, and kits provided herein can offer a simple and
effective solution in
which to deliver polyribonucleotides into cells. A polyribonucleotide can be
delivered into a cell more
efficiently in the presence of the cell-penetrating agent than in the absence
of the cell-penetrating agent.
In some cases, the cell-penetrating agent described herein can increase the
efficiency of delivery of the
polynucleotide by at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 100%, 120%,
150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%,
5000%, 8000%,
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10,000%, 20,000%, or 50,000% as compared to the efficiency of delivery of the
polynucleotide in the
absence of the cell-penetrating agent.
As used herein, the term "topical delivery" means the delivery of a substance
to skin or an
epithelial layer accessible though non-invasive means. Topical delivery
includes topical delivery to
intestinal and other gastrointestinal (GI) epithelia or the vaginal
epithelium. Topical delivery of a
pharmaceutical composition can have a local pharmacodynamic effect on the
subject, e.g., the topically
delivered pharmaceutical composition has a pharmacodynamic effect at or
proximate to the particular part
of the body (e.g. skin) where the pharmaceutical composition is delivered. In
some other embodiments,
topical delivery of a pharmaceutical composition as discussed herein is used
only to refer to the delivery
mode (locally to, e.g., a specific surface area), whereas the pharmaceutical
composition can have either a
local or systemic pharmacodynamic effect. For instance, the pharmaceutical
composition can either stay
local at or proximate to the administration site, or can enter a circulation
system (e.g., blood or lymphoid
system) of the subject body, through which the pharmaceutical composition can
be transported to remote
parts of the body that are typically not reachable by the pharmaceutical
composition via routes other than
the circulation systems.
In some embodiments, a composition or pharmaceutical composition described
herein is
formulated for direct application on a skin area (e.g., percutaneous or dermal
administration). In some
embodiments, a composition or pharmaceutical composition described herein is
formulated for
administration via the nasal passages of a subject. In some embodiments, a
composition or
pharmaceutical composition described herein is formulated for oral
administration. In some
embodiments, a composition or pharmaceutical composition described herein is
formulated for
ophthalmic administration. In some embodiments, a composition or
pharmaceutical composition
described herein is formulated for administration as a suppository, for
vaginal administration, or for
mucosal administration.
A composition or pharmaceutical composition described herein can be applied to
a surface after
application of a sterilizing agent to that surface (e.g., pre-treatment of the
surface with a sterilizing agent
for delivery of the polyribonucleotide). The surface can be a surface area of
a subject. The surface area
of the subject comprises cells, such as epithelial cells.
A sterilizing agent can be any agent that is bactericidal, bacteriostatic,
and/or actively kills
microorganisms, inactivates microorganism, or prevents microorganisms from
growing. In some
embodiments, the sterilizing agent is an alcohol, iodine, or hydrogen
peroxide. The sterilizing agent can
be UV light or laser light. In some embodiments, the sterilizing agent is heat
delivered electrically or
through other means, such as by vapor or contact.
The sterilizing agent can be applied to the surface area of the subject by
various non-invasive
methods. For example, a sterilizing agent can be applied by a wipe or swab
comprising the sterilizing
agent. In some embodiments, the sterilizing agent is applied as a spray.
Various devices can be used to
apply the sterilizing agent. For example, a device that produces UV light or
laser light can be used. In
other embodiments, a device that produces heat can be used.
A composition or pharmaceutical composition described herein can be applied to
a surface after
application of an alcohol, such as ethanol (e.g., pre-treatment of the surface
with an alcohol for delivery of
the polyribonucleotide). The surface can be a surface area of a subject. The
surface area of the subject
comprises cells, such as epithelial cells. Alcohol used to pre-treat a surface
prior to the administration
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(e.g., application) of a polyribonucleotide to that surface may act as a
sterilizing agent and/or enhance the
cell penetration of the polyribonucleotide. In some, alcohol is applied to the
surface in the form of a wipe
or a swap. In some embodiments, the alcohol in permitted to try on the surface
prior to administration
(e.g., application) of a polyribonucleotide.
The composition or pharmaceutical composition described herein can be a liquid
preparation
such as a suspension, syrup, or elixir. In some cases, aqueous solutions are
packaged for use as is, or
lyophilized, and the lyophilized preparation being combined with a sterile
solution prior to administration.
The composition can be delivered as a solution or as a suspension. In general,
formulations such as a
gel (e.g., DMSO gel), jellies, creams, lotions (e.g., Johnson & Johnson
lotion), suppositories and
ointments can provide an area with more extended exposure to one or more
active agents, while
formulations in solution, e.g., sprays, can provide more immediate, short-term
exposure.
The composition or pharmaceutical composition applied to the surface area
after pre-treatment
can be free of any carrier and comprise the polyribonucleotide and a diluent.
The polyribonucleotide can
be a linear polyribonucleotide or a circular polyribonucleotide.
In some cases, the compositions provided herein are suitable for in vivo use
for eliciting an
immune response against an immunogen of interest when the composition is
administered to a subject.
The polyribonucleotide can be present in either linear or circular form.
In a particular embodiment, the compositions and pharmaceutical compositions
as described
herein, e.g., formulated for topical delivery, can be used to induce an immune
response against a viral
immunogen and/or for the treatment or prevention of a viral infection, for
example, a viral infection
selected from influenza (e.g., seasonal influenza, pandemic influenza,
influenza A, influenza Hi Ni
subtype), small pox, chikungunya virus (e.g. chikungunya virus), a coronavirus
(e.g., SARS-CoV-2),
dengue, zika, yellow fever virus, a coxsackievirus (e.g. hand, foot, and mouth
disease), HIV, AIDS,
ebolavirus (e.g., Ebola Hemorrhagic Fever), Marburg virus (e.g., Marburg
Hemorrhagic Fever), viral
Bronchiolitis, Respiratory Syncytial Virus (RSV), a hepatitis virus (e.g.
Hepatitis B), a herpes virus (e.g.,
genital herpes), a rotavirus infection, a measles infection, a mumps
infection, and a rubella infection.
In another embodiment, the compositions and pharmaceutical compositions as
described herein,
e.g., formulated for topical delivery, can be used to induce an immune
response against a bacterial
immunogen and/or for the treatment or prevention of a bacterial infection, for
example, a bacterial
infection selected from Q fever, smallpox, pertussis (whooping cough),
Haemophilus influenzae,
streptococcus (e.g., streptococcal pneumonia), an E. coli infection, bacterial
diarrhea, Francisella
tularensis bacteria (e.g., tularemia), diphtheria, and tetanus.
In another embodiment, the compositions and pharmaceutical compositions as
described herein,
e.g., formulated for topical delivery, can be used to induce an immune
response against a parasite
immunogen and/or for the treatment or prevention of a parasitic infection, for
example, malaria.
In another embodiment, the compositions and pharmaceutical compositions as
described herein,
e.g., formulated for topical delivery, can be used to induce an immune
response against a toxin and/or for
the treatment or prevention of toxicity associated with exposure to a toxin,
for example, a toxin selected
from botulin toxin and anthrax.
In another embodiment, the compositions and pharmaceutical compositions as
described herein,
e.g., formulated for topical delivery, can be used to induce an immune
response against an allergen
and/or for the treatment or prevention of symptoms associated with exposure to
an allergen, for example,
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an allergen selected from a peanut allergen, a food allergen, a birch pollen
allergen, a grass pollen
allergen, a cat allergen, a cedar pollen allergen, a dust mite allergen and a
ragweed allergen.
In another embodiment, the compositions and pharmaceutical compositions as
described herein
may be formulated for transdermal delivery. In some embodiments, transdermal
delivery may be used to
induce an immune response against and/or for the treatment or prevention of
any of the following: Q
fever, smallpox, a peanut allergen, botulin toxin, viral Bronchiolitis,
Respiratory Syncytial Virus (RSV),
anthrax, tetanus, and a dust mite allergen.
In another embodiment, the compositions and pharmaceutical compositions as
described herein
may be formulated for cutaneous delivery. In some embodiments, cutaneous
delivery may be used to
induce an immune response against and/or for the treatment or prevention of
any of the following:
influenza (e.g., seasonal influenza, pandemic influenza, influenza A,
influenza Hi Ni subtype), small pox,
chikungunya virus (e.g. chikungunya virus), a coronavirus (e.g., SARS-CoV-2),
dengue, zika, yellow fever
virus, a coxsackievirus (e.g. hand, foot, and mouth disease), HIV, AIDS,
ebolavirus (e.g., Ebola
Hemorrhagic Fever), Marburg virus (e.g., Marburg Hemorrhagic Fever),
Respiratory Syncytial Virus
(RSV), a hepatitis virus (e.g. Hepatitis B), a herpes virus (e.g., genital
herpes), a rotavirus infection, a
measles infection, a mumps infection, a rubella infection, pertussis (whooping
cough), streptococcus
(e.g., streptococcal pneumonia), an E. coli infection, bacterial diarrhea,
Francisella tularensis bacteria
(e.g., tularemia), diphtheria, tetanus, malaria, anthrax, a food allergen, a
birch pollen allergen, a grass
pollen allergen, a cat allergen, a cedar pollen allergen, and a dust mite
allergen a ragweed allergen.
Alcohol
Alcohol as described herein can be used for the delivery of a
polyribonucleotide into a cell. An
alcohol can be in a mixture with a polyribonucleotide as described herein for
delivery of the
polyribonucleotide into a cell. The mixture can comprise the alcohol in at
least about 0.3% v/v alcohol to
about 75% v/v. The alcohol can be ethanol. In some embodiments, the mixture is
applied to a surface
area of a subject. In some embodiments, the mixture is a pharmaceutical
composition.
An alcohol can be any alcohol that comprises one or more hydroxyl function
groups. In some cases, the
alcohol is, but is not limited to, a monohydric alcohol, a polyhydric alcohol,
an unsaturated aliphatic
alcohol, or an alicyclic alcohol. The alcohol can include one or more of
methanol, ethanol, isopropanol,
phenoxyethanol, triethanolamine, phenethyl alcohol, butanol, pentanol, cetyl
alcohol, ethylene glycol,
propylene glycol, denatured alcohol, benzyl alcohol, specially denatured
alcohol, glycol, stearyl alcohol,
cetearyl alcohol, menthol, polyethylene glycols (PEG)-400, ethoxylated fatty
acids, or
hydroxyethylcellulose. In certain embodiments, the alcohol is ethanol.
In other cases, the compositions, pharmaceutical compositions, and methods
provided herein
only include an alcohol and do not have or use any other agent to enhance the
delivery of the
polyribonucleotide into a cell. In some cases, the alcohol is ethanol and the
composition, pharmaceutical
composition, and methods do not have or use any other agent to enhance
delivery of polyribonucleotide
into a cell. In some cases, the alcohol is a cell-penetrating agent. In some
cases, the alcohol is not a
cell-penetrating agent.
The composition disclosed herein can include a mixture comprising an alcohol
and a
polyribonucleotide. In some cases, the polyribonucleotide is present in a pre-
mixed mixture with the
alcohol. In some cases, the polyribonucleotides is provided separately from
the alcohol prior to contact to
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a cell. In these instances, the polyribonucleotide is contacted with the
alcohol when being applied to a
cell and becomes mixed together for delivery of the polyribonucleotide into
the cell. Without being bound
to a certain theory, the concentration of the alcohol in the mixture can
contribute to the efficiency of
delivery. Therefore, in some cases, the alcohol is provided at a predetermined
concentration in the
mixture. In some other cases, when the alcohol and the polyribonucleotide are
separate initially but
mixed together when being applied for delivery, the alcohol is provided at a
sufficient amount relative to
the polyribonucleotide that would ensure it reach a minimum predetermined
concentration in the mixture.
In some cases, the alcohol constitutes at least about 0.01%, at least about
0.02%, at least about
0.03%, at least about 0.04%, at least about 0.05%, at least about 0.06%, at
least about 0.07%, at least
about 0.08%, at least about 0.09%, at least about 0.1%, at least about 0.2%,
at least about 0.3%, at least
about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at
least about 0.9%, at least
about 1%, at least about 2%, at least about 3%, at least about 4%, at least
about 5%, at least about 6%,
at least about 7%, at least about 8%, at least about 9%, at least about 10%,
at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about
80%, at least about 90%, at least about 95%, or at least about 98% volume per
volume (v/v) of the
mixture. In some cases, the alcohol constitutes at most about 0.01%, at most
about 0.02%, at most about
0.03%, at most about 0.04%, at most about 0.05%, at most about 0.06%, at most
about 0.07%, at most
about 0.08%, at most about 0.09%, at most about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,
0.6%, 0.7%, 0.9%, 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%
v/v of the
mixture. In some cases, the alcohol constitutes about 10%, about 20%, about
30%, about 40%, about
50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or
about 100% v/v of the
mixture.
In some cases, the alcohol constitutes at least about 0.01%, at least about
0.02%, at least about
0.03%, at least about 0.04%, at least about 0.05%, at least about 0.06%, at
least about 0.07%, at least
about 0.08%, at least about 0.09%, at least about 0.1%, at least about 0.2%,
at least about 0.3%, at least
about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at
least about 0.9%, at least
about 1%, at least about 2%, at least about 3%, at least about 4%, at least
about 5%, at least about 6%,
at least about 7%, at least about 8%, at least about 9%, at least about 10%,
at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about
80%, at least about 90%, at least about 95%, or at least about 98% weight per
weight (w/w) of the
mixture. In some cases, the alcohol constitutes at most about 0.01%, at most
about 0.02%, at most about
0.03%, at most about 0.04%, at most about 0.05%, at most about 0.06%, at most
about 0.07%, at most
about 0.08%, at most about 0.09%, at most about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,
0.6%, 0.7%, 0.9%, 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%
w/w of the
mixture. In some cases, the cell-penetrating agent constitutes about 10%,
about 20%, about 30%, about
40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or
about 98% w/w of the
mixture. In some cases, the alcohol constitutes about 10% v/v of the mixture.
In some embodiments, the alcohol constitutes at least about 0.5% v/v to about
75% v/v, at least
about 1% v/v to about 75% v/v, at least about 5% v/v to about 75% v/v, at
least about 10% v/v to about
75% v/v, at least about 15% v/v to about 75% v/v, at least about 20% v/v to
about 75% v/v, at least about
30% v/v to about 75% v/v, at least about 40% v/v to about 75% v/v, at least
about 50% v/v to about 75%
v/v, at least about 60% v/v to about 75% v/v, or at least about 70% v/v to
about 75% v/v of the mixture, or
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any percentage v/v therebetween. In some embodiments, the alcohol constitutes
at least about 0.3% v/v
to about 70% v/v, at least about 0.3% v/v to about 60% v/v, at least about
0.3% v/v to about 50% v/v, at
least about 0.3% v/v to about 40% v/v, at least about 30% v/v to about 20%
v/v, at least about 0.3% v/v to
about 15% v/v, at least about 0.3% v/v to about 10% v/v, at least about 0.3%
v/v to about 5% v/v, at least
about 0.3% v/v to about 1% v/v, or at least about 0.3% v/v to about 0.5% v/v
of the mixture, or any
percentage v/v therebetween.
In some cases, the mixture described herein is a liquid solution. For
instance, the alcohol is a
liquid substance itself. In these cases, the polyribonucleotide can also be
dissolved in the liquid solution.
In some cases, ethanol constitutes at least about 0.1%, at least about 0.2%,
at least about 0.3%, at least
about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at
least about 0.9%, at least
about 1%, at least about 2%, at least about 3%, at least about 4%, at least
about 5%, at least about 6%,
at least about 7%, at least about 8%, at least about 9%, at least about 10%,
at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about 60%, at
least about 70%, at least about
80%, at least about 90%, at least about 95%, or at least about 98% volume per
volume (v/v) of the
mixture. In some cases, ethanol constitutes at most about 0.1%, 0.2%, 0.3%,
0.4%, 0.5%, 0.6%, 0.7%,
0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, or 90% v/v
of the mixture. In some cases, ethanol constitutes about 10%, about 20%, about
30%, about 40%, about
50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or
about 100% v/v of the
mixture. In some cases, ethanol constitutes about 10% v/v of the mixture.
In some embodiments, the ethanol constitutes at least about 0.5% v/v to about
75% v/v, at least
about 1% v/v to about 75% v/v, at least about 5% v/v to about 75% v/v, at
least about 10% v/v to about
75% v/v, at least about 15% v/v to about 75% v/v, at least about 20% v/v to
about 75% v/v, at least about
30% v/v to about 75% v/v, at least about 40% v/v to about 75% v/v, at least
about 50% v/v to about 75%
v/v, at least about 60% v/v to about 75% v/v, or at least about 70% v/v to
about 75% v/v of the mixture, or
any percentage v/v therebetween. In some embodiments, the ethanol constitutes
at least about 0.3% v/v
to about 70% v/v, at least about 0.3% v/v to about 60% v/v, at least about
0.3% v/v to about 50% v/v, at
least about 0.3% v/v to about 40% v/v, at least about 30% v/v to about 20%
v/v, at least about 0.3% v/v to
about 15% v/v, at least about 0.3% v/v to about 10% v/v, at least about 0.3%
v/v to about 5% v/v, at least
about 0.3% v/v to about 1% v/v, or at least about 0.3% v/v to about 0.5% v/v
of the mixture, or any
percentage v/v therebetween.
Cell-Penetrating Agents
The cell-penetrating agent described herein can include any substance that
enhances delivery of
a polyribonucleotide into a cell. The cell-penetrating agent can include an
organic compound or an
inorganic molecule. In some cases, the cell-penetrating agent is an organic
compound having one or
more functional groups such as, but not limited to, alkane, alkene, and arene;
halogen-substituted alkane,
alkenes, and arenes; alcohols, phenols (derivatives of benzene), ethers,
aldehydes, ketones, and
carboxylic acids; amines and nitriles; and organosulfurs (e.g., dimethyl
sulfoxide). In some embodiments,
the cell-penetrating agent is soluble in polar solvents. In some embodiments,
the cell-penetrating agent is
insoluble in polar solvents. The polyribonucleotide can be present in either
linear or circular form.
The cell-penetrating agent can include organic compounds such as alcohols
having one or more
hydroxyl function groups. In some cases, the cell-penetrating agent includes
an alcohol such as, but not
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limited to, monohydric alcohols, polyhydric alcohols, unsaturated aliphatic
alcohols, and alicyclic alcohols.
The cell-penetrating agent can include one or more of methanol, ethanol,
isopropanol, phenoxyethanol,
triethanolamine, phenethyl alcohol, butanol, pentanol, cetyl alcohol, ethylene
glycol, propylene glycol,
denatured alcohol, benzyl alcohol, specially denatured alcohol, glycol,
stearyl alcohol, cetearyl alcohol,
menthol, polyethylene glycols (PEG)-400, ethoxylated fatty acids, or
hydroxyethylcellulose. In certain
embodiments, the cell-penetrating agent comprises ethanol.
In other cases, the compositions and methods provided herein only include an
alcohol as the cell-
penetrating agent, and do not have or use any other agent to enhance the
delivery of the
polyribonucleotide into a cell. In some cases, the cell-penetrating agent
comprises ethanol and any other
alcohol that can enhance delivery of polyribonucleotide into a cell. In some
cases, the cell-penetrating
agent comprises ethanol and any other organic or inorganic molecules that can
enhance delivery of
polyribonucleotide into a cell. In some cases, the cell-penetrating agent
comprises ethanol and liposome
or nanoparticles such as those described in International Publication Nos.
W02013/006825,
W02016/036735, W02018/112282A1, and W02012/031043A1, each of which is
incorporated herein by
reference in its entirety. In some cases, the cell-penetrating agent comprises
ethanol and cell-penetrating
peptides or proteins such as those described in Bechara et al, Cell-
penetrating peptides: 20 years later,
where do we stand? FEBS Letters 587(12):1693-1702 (2013); Langel, Cell-
Penetrating Peptides:
Processes and Applications (CRC Press, Boca Raton FL, 2002); El-Andaloussi et
al., Curr. Pharm. Des.
11(28):3597-611 (2003); Deshayes et al, Cell. Mol. Life Sci. 62(16):1839-49
(2005), US Patent
Publication Nos. US20130129726, US20130137644 and US20130164219, each of which
is herein
incorporated by reference in its entirety). In some cases, the ratio of
ethanol versus other cell-penetrating
agent is about 1:0.001, 1:0.002, 1: 005, 1:008, 1:0.01, 1:0.02, 1:0.05,
1:0.08, 1: 0.1, 1: 0.2, 1: 0.3, 1:0.4,
1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.2, 1: 1.5, 1: 1.8, 1: 2, 1:2.5,
1:3, 1:3.5, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,
1:10, 1:15, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:120,
1:150, 1:200, 1:250, 1:500, or
1:1000. In some cases, the ratio of ethanol versus other cell-penetrating
agent is at least about 1:0.001,
1:0.002, 1: 005, 1:008, 1:0.01, 1:0.02, 1:0.05, 1:0.08, 1: 0.1, 1: 0.2, 1:
0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8,
1:0.9, 1:1, 1:1.2, 1: 1.5, 1: 1.8, 1: 2, 1:2.5, 1:3, 1:3.5, 1:4, 1:5, 1:6,
1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:30,
1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:120, 1:150, 1:200, 1:250, or
1:500.
The composition disclosed herein can include a mixture of a cell-penetrating
agent and a
polyribonucleotide. In some cases, the polyribonucleotide is present in a pre-
mixed mixture with the cell-
penetrating agent. In some cases, the polyribonucleotides is provided
separately from the cell-
penetrating agent prior to contact to a cell. In these instances, the
polyribonucleotide is contacted with
the cell-penetrating agent when being applied to a cell and becomes mixed
together for delivery of the
polyribonucleotide into the cell. Without being bound to a certain theory, the
concentration of the cell-
penetrating agent in the mixture can contribute to the efficiency of delivery.
Therefore, in some cases, the
cell-penetrating agent is provided at a predetermined concentration in the
mixture. In some other cases,
when the cell-penetrating agent and the polyribonucleotide are separate
initially but mixed together when
being applied for delivery, the cell-penetrating agent is provided at a
sufficient amount relative to the
polyribonucleotide that would ensure it reach a minimum predetermined
concentration in the mixture.
In some cases, the cell-penetrating agent constitutes at least about 0.01%, at
least about 0.02%,
at least about 0.03%, at least about 0.04%, at least about 0.05%, at least
about 0.06%, at least about
0.07%, at least about 0.08%, at least about 0.09%, at least about 0.1%, at
least about 0.2%, at least
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about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at
least about 0.7%, at least
about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least
about 4%, at least about 5%,
at least about 6%, at least about 7%, at least about 8%, at least about 9%, at
least about 10%, at least
about 20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about
70%, at least about 80%, at least about 90%, at least about 95%, or at least
about 98% volume per
volume (v/v) of the mixture. In some cases, the cell-penetrating agent
constitutes at most about 0.01%, at
most about 0.02%, at most about 0.03%, at most about 0.04%, at most about
0.05%, at most about
0.06%, at most about 0.07%, at most about 0.08%, at most about 0.09%, at most
about 0.1%, 0.2%,
0.3 /0, 0.4 /0, 0.5 /0, 0.6 /0, 0.70/0, 0.90/0, 10/0, 20/0, 3O/0, 4 /0, 5 /0,
6O/0, 70/0, 80/0, 90/0, 10 /0, 20 /0, 30 /0, 40 /0,
50%, 60%, 70%, 80%, or 90% v/v of the mixture. In some cases, the cell-
penetrating agent constitutes
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,
about 80%, about
90%, about 95%, about 98%, or about 100% v/v of the mixture.
In some cases, the cell-penetrating agent constitutes at least about 0.01%, at
least about 0.02%,
at least about 0.03%, at least about 0.04%, at least about 0.05%, at least
about 0.06%, at least about
0.07%, at least about 0.08%, at least about 0.09%, at least about 0.1%, at
least about 0.2%, at least
about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at
least about 0.7%, at least
about 0.9%, at least about 1%, at least about 2%, at least about 3%, at least
about 4%, at least about 5%,
at least about 6%, at least about 7%, at least about 8%, at least about 9%, at
least about 10%, at least
about 20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about
70%, at least about 80%, at least about 90%, at least about 95%, or at least
about 98% weight per weight
(w/w) of the mixture. In some cases, the cell-penetrating agent constitutes at
most about 0.01%, at most
about 0.02%, at most about 0.03%, at most about 0.04%, at most about 0.05%, at
most about 0.06%, at
most about 0.07%, at most about 0.08%, at most about 0.09%, at most about
0.1%, 0.2%, 0.3%, 0.4%,
0.5 /0, 0.6 /0, 0.70/0, 0.90/0, 10/0, 20/0, 3O/0, 4 /0, 5 /0, 6 /0, 70/0,
80/0, 90/0, 10 /0, 20 /0, 30 /0, 40 /0, 50 /0, 60 /0,
70%, 80%, or 90% w/w of the mixture. In some cases, the cell-penetrating agent
constitutes about 10%,
about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,
about 90%, about
95%, or about 98% w/w of the mixture. In some cases, the cell-penetrating
agent constitutes about 10%
v/v of the mixture.
In some cases, the mixture described herein is a liquid solution. For
instance, the cell-
penetrating agent is a liquid substance itself. Alternatively, the cell-
penetrating agent is a solid, liquid, or
gas substance and dissolved in a liquid carrier, e.g., water. In these cases,
the polyribonucleotide can
also be dissolved in the liquid solution.
In some cases, ethanol constitutes at least about 0.1%, at least about 0.2%,
at least about 0.3%,
at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about
0.7%, at least about 0.9%, at
least about 1%, at least about 2%, at least about 3%, at least about 4%, at
least about 5%, at least about
6%, at least about 7%, at least about 8%, at least about 9%, at least about
10%, at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least about 60%,
at least about 70%, at least
about 80%, at least about 90%, at least about 95%, or at least about 98%
volume per volume (v/v) of the
mixture. In some cases, ethanol constitutes at most about 0.1%, 0.2%, 0.3%,
0.4%, 0.5%, 0.6%, 0.7%,
0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, or 90% v/v
of the mixture. In some cases, ethanol constitutes about 10%, about 20%, about
30%, about 40%, about
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50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or
about 100% v/v of the
mixture. In some cases, ethanol constitutes about 10% v/v of the mixture.
Preservatives
A composition or pharmaceutical composition provided herein can comprise
material for a single
administration, or can comprise material for multiple administrations (e.g., a
"multidose" kit). The
polyribonucleotide can be present in either linear or circular form. The
composition or pharmaceutical
composition can include one or more preservatives such as thiomersal or 2-
phenoxyethanol.
Preservatives can be used to prevent microbial contamination during use.
Suitable preservatives include:
benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl
paraben, phenylethyl alcohol,
edetate disodium, sorbic acid, Onamer M, or other agents known to those
skilled in the art. In ophthalmic
products, e.g., such preservatives can be employed at a level of from 0.004%
to 0.02%. In the
compositions described herein the preservative, e.g., benzalkonium chloride,
can be employed at a level
of from 0.001% to less than 0.01%, e.g., from 0.001% to 0.008%, preferably
about 0.005% by weight.
Polyribonucleotides can be susceptible to RNase that can be abundant in
ambient environment.
Compositions provided herein can include reagents that inhibit RNase activity,
thereby preserving the
polyribonucleotide from degradation. In some cases, the composition or
pharmaceutical composition
includes any RNase inhibitor known to one skilled in the art. Alternatively or
additionally, the
polyribonucleotide, and cell-penetrating agent and/or pharmaceutically
acceptable diluents or carriers,
vehicles, excipients, or other reagents in the composition provided herein can
be prepared in RNase-free
environment. The composition can be formulated in RNase-free environment.
In some cases, a composition provided herein can be sterile. The composition
can be formulated
as a sterile solution or suspension, in suitable vehicles, known in the art.
The composition can be
sterilized by conventional, known sterilization techniques, e.g., the
composition can be sterile filtered.
Salts
In some cases, a composition or pharmaceutical composition provided herein
comprises one or
more salts. For controlling the tonicity, a physiological salt such as sodium
salt can be included a
composition provided herein. Other salts can comprise potassium chloride,
potassium dihydrogen
phosphate, disodium phosphate, and/or magnesium chloride, or the like. In some
cases, the composition
is formulated with one or more pharmaceutically acceptable salts. The one or
more pharmaceutically
acceptable salts can comprise those of the inorganic ions, such as, for
example, sodium, potassium,
calcium, magnesium ions, and the like. Such salts can comprise salts with
inorganic or organic acids,
such as hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid,
sulfuric acid, methanesulfonic
acid, p-toluenesulfonic acid, acetic acid, fumaric acid, succinic acid, lactic
acid, mandelic acid, malic acid,
citric acid, tartaric acid, or maleic acid. The polyribonucleotide can be
present in either linear or circular
form.
Buffers/pH
A composition or pharmaceutical composition provided herein can comprise one
or more buffers,
such as a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer
(e.g., with an aluminum
hydroxide adjuvant); or a citrate buffer. Buffers, in some cases, are included
in the 5-20 mM range.
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A composition or pharmaceutical composition provided herein can have a pH
between about 5.0
and about 8.5, between about 6.0 and about 8.0, between about 6.5 and about
7.5, or between about 7.0
and about 7.8. The composition or pharmaceutical composition can have a pH of
about 7. The
polyribonucleotide can be present in either linear or circular form.
Detergents/surfactants
A composition or pharmaceutical composition provided herein can comprise one
or more
detergents and/or surfactants, depending on the intended administration route,
e.g., polyoxyethylene
sorbitan esters surfactants (commonly referred to as "Tweens"), e.g.,
polysorbate 20 and polysorbate 80;
copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide
(BO), sold under the
DOWFAXTM tradename, such as linear EO/PO block copolymers; octoxynols, which
can vary in the
number of repeating ethoxy (oxy-1,2-ethanediy1) groups, e.g., octoxyno1-9
(Triton X-100, or t-
octylphenoxypolyethoxyethanol); (octylphenoxy)polyethoxyethanol (IGEPAL CA-
630/NP-40);
phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates,
such as the TergitolTm NP
series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and
oleyl alcohols (known as Brij
surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and
sorbitan esters (commonly known
as "SPANs"), such as sorbitan trioleate (Span 85) and sorbitan monolaurate, an
octoxynol (such as
octoxyno1-9 (Triton X-100) or t-octylphenoxypolyethoxyethanol), a cetyl
trimethyl ammonium bromide
("CTAB"), or sodium deoxycholate. The one or more detergents and/or
surfactants can be present only at
trace amounts. In some cases, the composition can include less than 1 mg/ml of
each of octoxynol-10
and polysorbate 80. Non-ionic surfactants can be used herein. Surfactants can
be classified by their
"HLB" (hydrophile/lipophile balance). In some cases, surfactants have a HLB of
at least 10, at least 15,
and/or at least 16. The polyribonucleotide can be present in either linear or
circular form.
Diluents
In some embodiments, an immunogenic composition of the disclosure includes a
circular
polyribonucleotide and a diluent. In some embodiments, an immunogenic
composition of the disclosure
includes a linear polyribonucleotide and a diluent.
A diluent can be a non-carrier excipient. A non-carrier excipient serves as a
vehicle or medium
for a composition, such as a circular polyribonucleotide as described herein.
A non-carrier excipient
serves as a vehicle or medium for a composition, such as a linear
polyribonucleotide as described herein.
Non-limiting examples of a non-carrier excipient include solvents, aqueous
solvents, non-aqueous
solvents, dispersion media, diluents, dispersions, suspension aids, surface
active agents, isotonic agents,
thickening agents, emulsifying agents, preservatives, polymers, peptides,
proteins, cells, hyaluronidases,
dispersing agents, granulating agents, disintegrating agents, binding agents,
buffering agents (e.g.,
phosphate buffered saline (PBS)), lubricating agents, oils, and mixtures
thereof. A non-carrier excipient
can be any one of the inactive ingredients approved by the United States Food
and Drug Administration
(FDA) and listed in the Inactive Ingredient Database that does not exhibit a
cell-penetrating effect. A non-
carrier excipient can be any inactive ingredient suitable for administration
to a non-human animal, for
example, suitable for veterinary use. Modification of compositions suitable
for administration to humans
in order to render the compositions suitable for administration to various
animals is well understood, and
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the ordinarily skilled veterinary pharmacologist can design and/or perform
such modification with merely
ordinary, if any, experimentation.
In some embodiments, the circular polyribonucleotide may be delivered as a
naked delivery
formulation, such as including a diluent. A naked delivery formulation
delivers a circular
polyribonucleotide, to a cell without the aid of a carrier and without
modification or partial or complete
encapsulation of the circular polyribonucleotide, capped polyribonucleotide,
or complex thereof.
A naked delivery formulation is a formulation that is free from a carrier and
wherein the circular
polyribonucleotide is without a covalent modification that binds a moiety that
aids in delivery to a cell or
without partial or complete encapsulation of the circular polyribonucleotide.
In some embodiments, a
circular polyribonucleotide without a covalent modification that binds a
moiety that aids in delivery to a cell
is a polyribonucleotide that is not covalently bound to a protein, small
molecule, a particle, a polymer, or a
biopolymer. A circular polyribonucleotide without covalent modification that
binds a moiety that aids in
delivery to a cell does not contain a modified phosphate group. For example, a
circular
polyribonucleotide without a covalent modification that binds a moiety that
aids in delivery to a cell does
not contain phosphorothioate, phosphoroselenates, boranophosphates,
boranophosphate esters,
hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl
phosphonates, or
phosphotriesters.
In some embodiments, a naked delivery formulation is free of any or all of:
transfection reagents,
cationic carriers, carbohydrate carriers, nanoparticle carriers, or protein
carriers. In some embodiments,
a naked delivery formulation is free from phtoglycogen octenyl succinate,
phytoglycogen beta-dextrin,
anhydride-modified phytoglycogen beta-dextrin, lipofectamine,
polyethylenimine, poly(trimethylenimine),
poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine, dideoxy-
diamino-b-cyclodextrin,
spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine),
poly(histidine),
poly(arginine), cationized gelatin, dendrimers, chitosan,l,2-Dioleoy1-3-
Trimethylammonium-
Propane(DOTAP), N-[ 1 -(2,3-dioleoyloxy)propyI]-N,N,N- trimethylammonium
chloride (DOTMA), l-[2-
(oleoyloxy)ethy1]-2-oley1-3-(2- hydroxyethyl)imidazolinium chloride (DOTIM),
2,3-dioleyloxy-N-
[2(sperminecarboxamido)ethy1]-N,N-dimethyl-l-propanaminium trifluoroacetate
(DOSPA), 3B-[N¨ (N\N'-
Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride (DC-Cholesterol HC1),
diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N- dimethylammonium
bromide (DDAB),
N-(l,2-dimyristyloxyprop-3-y1)-N,N-dimethyl-N- hydroxyethyl ammonium bromide
(DMRIE), N,N-dioleyl-
N,N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low-density
lipoprotein (LDL),
high- density lipoprotein (HDL), or globulin.
In certain embodiments, a naked delivery formulation includes a non-carrier
excipient. In some
embodiments, a non-carrier excipient includes an inactive ingredient that does
not exhibit a cell-
penetrating effect. In some embodiments, a non-carrier excipient includes a
buffer, for example PBS. In
some embodiments, a non-carrier excipient is a solvent, a non-aqueous solvent,
a diluent, a suspension
aid, a surface-active agent, an isotonic agent, a thickening agent, an
emulsifying agent, a preservative, a
polymer, a peptide, a protein, a cell, a hyaluronidase, a dispersing agent, a
granulating agent, a
disintegrating agent, a binding agent, a buffering agent, a lubricating agent,
or an oil.
In some embodiments, a naked delivery formulation includes a diluent. A
diluent may be a liquid
diluent or a solid diluent. In some embodiments, a diluent is an RNA
solubilizing agent, a buffer, or an
isotonic agent. Examples of an RNA solubilizing agent include water, ethanol,
methanol, acetone,
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formamide, and 2-propanol. Examples of a buffer include 2-(N-
morpholino)ethanesulfonic acid (MES),
Bis-Tris, 2-[(2-amino-2-oxoethyl)-(carboxymethyl)amino]acetic acid (ADA), N-(2-
Acetamido)-2-
aminoethanesulfonic acid (ACES), piperazine-N,N'-bis(2-ethanesulfonic acid)
(PIPES), 2-[[1,3-dihydroxy-
2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 3-(N-
morpholino)propanesulfonic acid
(MOPS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Tris,
Tricine, Gly-Gly, Bicine, or
phosphate. Examples of an isotonic agent include glycerin, mannitol,
polyethylene glycol, propylene
glycol, trehalose, or sucrose.
Carriers
In some embodiments, an immunogenic composition of the disclosure includes a
circular
polyribonucleotide and a carrier. In some embodiments, an immunogenic
composition of the disclosure
includes a linear polyribonucleotide and a carrier.
In certain embodiments, an immunogenic composition includes a circular
polyribonucleotide as
described herein in a vesicle or other membrane-based carrier. In certain
embodiments, an immunogenic
composition includes a linear polyribonucleotide as described herein in a
vesicle or other membrane-
based carrier.
In other embodiments, an immunogenic composition includes the circular
polyribonucleotide in or
via a cell, vesicle or other membrane-based carrier. In other embodiments, an
immunogenic composition
includes the linear polyribonucleotide in or via a cell, vesicle or other
membrane-based carrier. In one
embodiment, an immunogenic composition includes the circular
polyribonucleotide in liposomes or other
similar vesicles. In one embodiment, an immunogenic composition includes the
linear polyribonucleotide
in liposomes or other similar vesicles. Liposomes are spherical vesicle
structures composed of a uni- or
multilamellar lipid bilayer surrounding internal aqueous compartments and a
relatively impermeable outer
lipophilic phospholipid bilayer. Liposomes may be anionic, neutral, or
cationic. Liposomes are
biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug
molecules, protect their cargo
from degradation by plasma enzymes, and transport their load across biological
membranes and the
blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug
Delivery, vol. 2011, Article ID
469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).
Vesicles can be made from several different types of lipids; however,
phospholipids are most
commonly used to generate liposomes as drug carriers. Methods for preparation
of multilamellar vesicle
lipids are known in the art (see for example U.S. Pat. No. 6,693,086, the
teachings of which relating to
multilamellar vesicle lipid preparation are incorporated herein by reference).
Although vesicle formation
can be spontaneous when a lipid film is mixed with an aqueous solution, it can
also be expedited by
applying force in the form of shaking by using a homogenizer, sonicator, or an
extrusion apparatus (see,
e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID
469679, 12 pages, 2011.
doi:10.1155/2011/469679 for review). Extruded lipids can be prepared by
extruding through filters of
decreasing size, as described in Templeton et al., Nature Biotech, 15:647-652,
1997, the teachings of
which relating to extruded lipid preparation are incorporated herein by
reference.
In certain embodiments, an immunogenic composition of the disclosure includes
a circular
polyribonucleotide and lipid nanoparticles, for example lipid nanoparticles
described herein. In certain
embodiments, an immunogenic composition of the disclosure includes a linear
polyribonucleotide and
lipid nanoparticles. Lipid nanoparticles are another example of a carrier that
provides a biocompatible
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and biodegradable delivery system for a circular polyribonucleotide molecule
as described herein. Lipid
nanoparticles are another example of a carrier that provides a biocompatible
and biodegradable delivery
system for a linear polyribonucleotide molecule as described herein.
Nanostructured lipid carriers (NLCs)
are modified solid lipid nanoparticles (SLNs) that retain the characteristics
of the SLN, improve drug
stability and loading capacity, and prevent drug leakage. Polymer
nanoparticles (PNPs) are an important
component of drug delivery. These nanoparticles can effectively direct drug
delivery to specific targets
and improve drug stability and controlled drug release. Lipid¨polymer
nanoparticles (PLNs), a new type
of carrier that combines liposomes and polymers, may also be employed. These
nanoparticles possess
the complementary advantages of PNPs and liposomes. A PLN is composed of a
core¨shell structure;
the polymer core provides a stable structure, and the phospholipid shell
offers good biocompatibility. As
such, the two components increase the drug encapsulation efficiency rate,
facilitate surface modification,
and prevent leakage of water-soluble drugs. For a review, see, e.g., Li et al.
2017, Nanomaterials 7, 122;
doi:10.3390/nano7060122.
Additional non-limiting examples of carriers include carbohydrate carriers
(e.g., an anhydride-
modified phytoglycogen or glycogen-type material), protein carriers (e.g., a
protein covalently linked to the
circular polyribonucleotide or a protein covalently linked to the linear
polyribonucleotide), or cationic
carriers (e.g., a cationic lipopolymer or transfection reagent). Non-limiting
examples of carbohydrate
carriers include phtoglycogen octenyl succinate, phytoglycogen beta-dextrin,
and anhydride-modified
phytoglycogen beta-dextrin. Non-limiting examples of cationic carriers include
lipofectamine,
.. polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine),
polypropylenimine, aminoglycoside-
polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-
dimethylamino)ethyl
methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized
gelatin, dendrimers, chitosan, 1,2-
Dioleoy1-3- Trimethylammonium-Propane(DOTAP), N-[ 1 -(2,3-dioleoyloxy)propyI]-
N,N,N-
trimethylammonium chloride (DOTMA),1-[2-(oleoyloxy)ethy1]-2-oley1-3-(2-
hydroxyethyl)imidazolinium
chloride (DOTIM), 2,3-dioleyloxy-N- [2(sperminecarboxamido)ethy1]-N,N-dimethyl-
l-propanaminium
trifluoroacetate (DOSPA), 3B-[N¨ (N\N'-Dimethylaminoethane)-
carbamoyl]Cholesterol Hydrochloride
(DC-Cholesterol HC1), diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-
N,N-
dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yI)-N,N-dimethyl-N-
hydroxyethyl
ammonium bromide (DMRIE), and N,N-dioleyl-N,N-dimethylammonium chloride
(DODAC). Non-limiting
examples of protein carriers include human serum albumin (HSA), low-density
lipoprotein (LDL), high-
density lipoprotein (HDL), or globulin.
Exosomes can also be used as drug delivery vehicles for a circular RNA
composition or
preparation described herein. Exosomes can be used as drug delivery vehicles
for a linear
polyribonucleotide composition or preparation described herein. For a review,
see Ha et al. July 2016.
Acta Pharmaceutica Sinica B. Volume 6, Issue 4, Pages 287-296;
https://doi.org/10.1016/j.apsb.2016.02.001.
Ex vivo differentiated red blood cells can also be used as a carrier for a
circular RNA composition
or preparation described herein. Ex vivo differentiated red blood cells can
also be used as a carrier for a
linear polyribonucleotide composition or preparation described herein. See,
e.g., International Patent
Publication Nos. W02015/073587; W02017/123646; W02017/123644; W02018/102740;
W02016/183482; W02015/153102; W02018/151829; W02018/009838; Shi et al. 2014.
Proc Natl Acad
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Sci USA. 111(28): 10131-10136; US Patent 9,644,180; Huang et al. 2017. Nature
Communications 8:
423; Shi et al. 2014. Proc Natl Acad Sci USA. 111(28): 10131-10136.
Fusosome compositions, e.g., as described in International Patent Publication
No.
W02018/208728, can also be used as carriers to deliver a circular
polyribonucleotide molecule described
herein. Fusosome compositions, e.g., as described in W02018/208728, can also
be used as carriers to
deliver a linear polyribonucleotide molecule described herein.
Virosomes and virus-like particles (VLPs) can also be used as carriers to
deliver a circular
polyribonucleotide molecule described herein to targeted cells. Virosomes and
virus-like particles (VLPs)
can also be used as carriers to deliver a linear polyribonucleotide molecule
described herein to targeted
cells.
Plant nanovesicles and plant messenger packs (PMPs), e.g., as described in
International Patent
Publication Nos. W02011/097480, W02013/070324, W02017/004526, or W02020/041784
can also be
used as carriers to deliver the circular RNA composition or preparation
described herein. Plant
nanovesicles and plant messenger packs (PMPs) can also be used as carriers to
deliver a linear
polyribonucleotide composition or preparation described herein.
Microbubbles can also be used as carriers to deliver a circular
polyribonucleotide molecule
described herein. Microbubbles can also be used as carriers to deliver a
linear polyribonucleotide
molecule described herein. See, e.g., U57115583; Beeri, R. et al.,
Circulation. 2002 Oct 1;106(14):1756-
1759; Bez, M. et al., Nat Protoc. 2019 Apr; 14(4): 1015-1026; Hernot, S. et
al., Adv Drug Deliv Rev. 2008
Jun 30; 60(10): 1153-1166; Rychak, J.J. et al., Adv Drug Deliv Rev. 2014 Jun;
72: 82-93. In some
embodiments, microbubbles are albumin-coated perfluorocarbon microbubbles.
The carrier including the circular polyribonucleotides described herein may
include a plurality of
particles. The particles may have median article size of 30 to 700 nanometers
(e.g., 30 to 50, 50 to 100,
100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 100 to
500, 50 to 500, or 200 to
700 nanometers). The size of the particle may be optimized to favor deposition
of the payload, including
the circular polyribonucleotide into a cell. Deposition of the circular
polyribonucleotide into certain cell
types may favor different particle sizes. For example, the particle size may
be optimized for deposition of
the circular polyribonucleotide into antigen presenting cells. The particle
size may be optimized for
deposition of the circular polyribonucleotide into dendritic cells.
Additionally, the particle size may be
optimized for depositions of the circular polyribonucleotide into draining
lymph node cells.
Lipid Nanoparticles
The compositions, methods, and delivery systems provided by the present
disclosure may
employ any suitable carrier or delivery modality described herein, including,
in certain embodiments, lipid
nanoparticles (LNPs). Lipid nanoparticles, in some embodiments, comprise one
or more ionic lipids, such
as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids); one
or more conjugated lipids (such
as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5
of W02019217941;
incorporated herein by reference in its entirety); one or more sterols (e.g.,
cholesterol).
Lipids that can be used in nanoparticle formations (e.g., lipid nanoparticles)
include, for example
those described in Table 4 of W02019217941, which is incorporated by
reference¨e.g., a lipid-
containing nanoparticle can comprise one or more of the lipids in Table 4 of
W02019217941. Lipid
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nanoparticles can include additional elements, such as polymers, such as the
polymers described in
Table 5 of W02019217941, incorporated by reference.
In some embodiments, conjugated lipids, when present, can include one or more
of PEG-
diacylglycerol (DAG) (such as Kmonomethoxy-polyethyleneglycol)-2,3-
dimyristoylglycerol (PEG-DMG)),
.. PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a
pegylated
phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG)
(such as 4-0-(2',3'-
di(tetradecanoyloxy)propyl-I-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-
S-DMG)), PEG
dialkoxypropylcarbam, N- (carbonyl-methoxypoly ethylene glycol 2000)- 1 ,2-
distearoyl-sn-glycero-3-
phosphoethanolamine sodium salt, and those described in Table 2 of
W02019051289 (incorporated by
reference), and combinations of the foregoing.
In some embodiments, sterols that can be incorporated into lipid nanoparticles
include one or
more of cholesterol or cholesterol derivatives, such as those in W02009/127060
or US2010/0130588,
which are incorporated by reference. Additional exemplary sterols include
phytosterols, including those
described in Eygeris et al. (2020), dx.doi.org/10.1021/acs.nanolett.0c01386,
incorporated herein by
reference.
In some embodiments, the lipid particle comprises an ionizable lipid, a non-
cationic lipid, a
conjugated lipid that inhibits aggregation of particles, and a sterol. The
amounts of these components can
be varied independently and to achieve desired properties. For example, in
some embodiments, the lipid
nanoparticle comprises an ionizable lipid is in an amount from about 20 mol %
to about 90 mol % of the
total lipids (in other embodiments it may be 20-70% (mol), 30-60% (mol) or 40-
50% (mol); about 50 mol
% to about 90 mol % of the total lipid present in the lipid nanoparticle), a
non-cationic lipid in an amount
from about 5 mol % to about 30 mol % of the total lipids, a conjugated lipid
in an amount from about 0.5
mol % to about 20 mol % of the total lipids, and a sterol in an amount from
about 20 mol % to about 50
mol % of the total lipids. The ratio of total lipid to nucleic acid can be
varied as desired. For example, the
.. total lipid to nucleic acid (mass or weight) ratio can be from about 10: 1
to about 30: 1.
In some embodiments, the lipid to nucleic acid ratio (mass/mass ratio; w/w
ratio) can be in the
range of from about 1:1 to about 25:1, from about 10:1 to about 14:1, from
about 3:1 to about 15:1, from
about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about
9:1. The amounts of lipids and
nucleic acid can be adjusted to provide a desired N/P ratio, for example, N/P
ratio of 3, 4, 5, 6, 7, 8, 9, 10
or higher. Generally, the lipid nanoparticle formulation's overall lipid
content can range from about 5
mg/ml to about 30 mg/mL.
Some non-limiting example of lipid compounds that may be used (e.g., in
combination with other
lipid components) to form lipid nanoparticles for the delivery of compositions
described herein, e.g.,
nucleic acid (e.g., RNA (e.g., circular polyribonucleotide, linear
polyribonucleotide)) described herein
includes,
(i)
In some embodiments an LNP comprising Formula (i) is used to deliver a
polyribonucleotide
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
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(ii)
In some embodiments an LNP comprising Formula (ii) is used to deliver a
polyribonucleotide
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
0
N
(iii)
In some embodiments an LNP comprising Formula (iii) is used to deliver a
polyribonucleotide
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
===,
rH-c
(iv)
N
o---
(v)
In some embodiments an LNP comprising Formula (v) is used to deliver a
polyribonucleotide
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
(vi)
In some embodiments an LNP comprising Formula (vi) is used to deliver a
polyribonucleotide
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
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0
N
0 0 (vii)
0 0 (viii)
In some embodiments an LNP comprising Formula (viii) is used to deliver a
polyribonucleotide
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
=
L.,
(ix)
In some embodiments an LNP comprising Formula (ix) is used to deliver a
polyribonucleotide
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
.x1 N'tre o Y' R4
e.
Nrw
(x)
wherein
X1 is 0, NR1, or a direct bond, X2 is 02-5 alkylene, X3 is C(=0) or a direct
bond, R1 is H or Me, R3 is 01-3
alkyl, R2 is 01-3 alkyl, or R2 taken together with the nitrogen atom to which
it is attached and 1-3 carbon
atoms of X2 form a 4-, 5-, or 6-membered ring, or X1 is NR1, R1 and R2 taken
together with the nitrogen
atoms to which they are attached form a 5- or 6-membered ring, or R2 taken
together with R3 and the
nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y1
is 02-12 alkylene, Y2 is
selected from
0
-4 \ 0
(in either orientation), (in either orientation), (in either
orientation),
n is 0 to 3, R4 is 01-15 alkyl, Z1 is 01-6 alkylene or a direct bond,
0
Z2 is
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(in either orientation) or absent, provided that if Z1 is a direct bond, Z2 is
absent;
R5 is 05-9 alkyl or 06-10 alkoxy, R6 is 05-9 alkyl or 06-10 alkoxy, W is
methylene or a direct bond, and
R7 is H or Me, or a salt thereof, provided that if R3 and R2 are 02 alkyls, X1
is 0, X2 is linear 03 alkylene,
X3 is C(=0), Y1 is linear Ce alkylene, (Y2 )n-R4 is
, R4 is linear 05 alkyl, Z1 is 02 alkylene, Z2 is absent, W is methylene, and
R7 is H, then R5 and R6 are not
Cx alkoxy.
In some embodiments an LNP comprising Formula (xii) is used to deliver a
polyribonucleotide
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
0
(xi)
In some embodiments an LNP comprising Formula (xi) is used to deliver a
polyribonucleotide
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
w.f?
A
6P1
01'412 where R= (xii)
CFP23
_
Ho N f-I
HO
C10}121
(-2)0111 i
0 . OH
HO C H
(Xiii)
0
kkµ
"Ws
0
(xiv)
In some embodiments an LNP comprises a compound of Formula (xiii) and a
compound of
Formula (xiv).
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OH
OH
N
HO
OH
OH
(xv)
In some embodiments an LNP comprising Formula (xv) is used to deliver a
polyribonucleotide
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
PEI Cots
= osix)
HO n
C131127 (xvi)
In some embodiments an LNP comprising a formulation of Formula (xvi) is used
to deliver a
polyribonucleotide (e.g., a circular polyribonucleotide, a linear
polyribonucleotide) composition described
herein to cells.
C:X .
õ
,
0.
(xvii)
ye X s
6
9
=
X 44 amino sinlaurc where x=
(xviii)(a)
:
"s
*.=
========'s
r ,
(xviii)(b)
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,
N
N' (xix)
In some embodiments, a lipid compound used to form lipid nanoparticles for the
delivery of
compositions described herein, e.g., nucleic acid (e.g., RNA (e.g., circular
polyribonucleotide, linear
polyribonucleotide)) described herein is made by one of the following
reactions:
tiN
(xx)(a)
0
N = 0 .=
5O3 H N- ". 1,$
>"?.. Nit +
(xx)(b).
In some embodiments an LNP comprising Formula (xxi) is used to deliver a
polyribonucleotide
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells. In
some embodiments the LNP of Formula (xxi) is an LNP described by W02021113777
(e.g., a lipid of
Formula (1) such as a lipid of Table 1 of W02021113777).
Ri-Li
L3 ¨ R3
n
_
F2 (xxi)
wherein
each n is independently an integer from 2-15; Li and L3 are each independently
-0C(0)-* or -
.. C(0)0-*, wherein "*" indicates the attachment point to Ri or R3,
Ri and R3 are each independently a linear or branched 09-020 alkyl or 09-020
alkenyl, optionally
substituted by one or more substituents selected from a group consisting of
oxo, halo, hydroxy, cyano,
alkyl, alkenyl, aldehyde, heterocyclylalkyl, hydroxyalkyl, dihydroxyalkyl,
hydroxyalkylaminoalkyl,
aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,
(heterocyclyI)(alkyl)aminoalkyl, heterocyclyl, heteroaryl,
.. alkylheteroaryl, alkynyl, alkoxy, amino, dialkylamino,
aminoalkylcarbonylamino, aminocarbonylalkylamino,
(aminocarbonylalkyl)(alkyl)amino, alkenylcarbonylamino, hydroxycarbonyl,
alkyloxycarbonyl,
aminocarbonyl, aminoalkylaminocarbonyl, alkylaminoalkylaminocarbonyl,
dialkylaminoalkylaminocarbonyl, heterocyclylalkylaminocarbonyl,
(alkylaminoalkyl)(alkyl)aminocarbonyl,
alkylaminoalkylcarbonyl, dialkylaminoalkylcarbonyl, heterocyclylcarbonyl,
alkenylcarbonyl,
alkynylcarbonyl, alkylsulfoxide, alkylsulfoxidealkyl, alkyl sulfonyl, and
alkyl sulfonealkyl; and
R2 is selected from a group consisting of:
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4 14; =-/=-= 14 14 . ..1
,.... N I r-- p4.4: k.= NI'''s If - tr-
W / .5
, lir'N.1 4
it
'Ns,
.., t z
..=
,:= 1, IN-'"A=N. 1 i
I, '.=
,..
i,,.... A.
-,...
\le- =
1 : -,,
.õ....\iõ..õ. ....A..õ...., ,evi.r,,,
,./....A...õ,
. , . .
N .,.. ..s$
6:- 3 = i` 4
es¨ NI cll -.,µ rssl
# v. 3.3 ,c. ...!,-, t,. .:A,,,./
...),,,.. ..i !:...4- Nt. -%-e
iT 1
c 1 1 . r :¨. ..t.q
s.
,,.1
N ====;$\ \\µ 11 N' '',
.i
i
' \\
,
iZ.,.,,...0
A a \==
õ
'=\.. AAA,
INN
\ ,,..:µ-',,,,, = = /, *. is .)--õ Ni/
N"
i/ N.. Nr \"''''''' N' s'i=
.{,'"`N,.. "As\r Si . = , :$
, ail d
In some embodiments an LNP comprising Formula (xxii) is used to deliver a
polyribonucleotide
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells. In
some embodiments the LNP of Formula (xxii) is an LNP described by W02021113777
(e.g., a lipid of
Formula (2) such as a lipid of Table 2 of W02021113777).
Ri
0 --------,
0
n
0 0
----;:----'-
n
R2 y 0 R3
0 (xxii)
wherein
each n is independently an integer from 1-15;
Ri and R2 are each independently selected from a group consisting of:
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0 34,--,,,,,,-,..õ,,,---...õ,..,--,,.w,,,O.,,,,õ,"-=,...õ,..--
,..,õ,--1/4.õ,...-
sX:'----''''N.,=-"N=,-----'r=r=k.,-="'%w..,--"',-,...i",,....s,"- ,t:
...;
041.----,..õ...k '''',,....--",,...--"N\,...--"N---= ,-(N-2,,,;''
N-,..---- -N,...." =
;.
;
'''....,
---`- ----,....----õ,,x ,õ,,,,,,,,,,,,,,...0,....,,,......õ...,............x
3
:i.
-,....---N...-0
s ,
7 7 7
0
7 7
0
No)L.,'"'" ... ......,".....,=",cy. N.,...e."7::`,.
''')...."..'
0
7 7 7
Z
19 õ......-õ..Ø.,,
Y
0 (...k....õ.....,õ...;,.<
õ1. 0
-,...õ...,-,,......õ---......"-Neo ...õ..---...s---,.-..----,-IL,.-N=,..---
N,,,t-o
,....,
0 o
ii
7 7
.......O.T.P
k 1
0
,----,N,-----.
,
i
õ,,,, I, õ,... ....... ,..õ. ...... ,,, .....1.
7 7 7
õ.....õ....õ....,õ.õ.õ.õ.., ,,,,,,,,,..,,,
õ........ ,.
r. õõ..õ..,
r
ii
s'k.).\\,.. .. .---`'.N.,õ. . . = --"'N.,. . ..,"\,,,,,'" .1/2":1'N,-.."."\-
\,..."'"\--, .''''''N.,'\-\,,''' :k"\\,""\,,,,, fi.'
1 ) )
R3 is selected from a group consisting of:
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r =0====~N.,...":õ 0 '14NNI N.¨µ /0'.."'N\---"" \
.6 ,
, ,
r:rz:--µ..
N
\- \c- N -,--*---,- t'.1 0 fj ,,N \-.
,,,-
1 N ,,,:,....,,, \- -,õ,----..õ.....- - --,:-7
, ,
\
, and
. .
In some embodiments an LNP comprising Formula (xxiii) is used to deliver a
polyribonucleotide
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells. In
some embodiments the LNP of Formula (xxiii) is an LNP described by
W02021113777 (e.g., a lipid of
Formula (3) such as a lipid of Table 3 of W02021113777).
0 0
R1¨ X ..............õ...-......0)..............õ..-..õ ...õ..-..........)-
(0_,...-,......õõ X¨ R
N 1
F2 (xxiii)
wherein
X is se,loote,d from -0-, -S-, or -0C(0)-*, wherein * indicates the attachment
point to Ri;
R, is seiected from a group consisting of:
-.õ,,,,õ=-=
..-''''''''Nve-\'µ," ...,=-",......"N,....."
I
.õ.,...".c.,,,
Z.
,
,and
...c.,C"'
7;
and R2 is selected from a group consisting of:
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rrl til
-, N ers N e---N
n 4.¨ N Jr t4
4, 4,
\ = k õ'),
N ir N
ze
\,
A.
( :
...t. i\r'''' i ,,,% 1 i
........--\., 1
i IN .1/4--
"1.: ...-- ,..,
,
, , , , , , ,
itr. trN
J A_ 4,,, ,,,µ s .(1 1].i._ .1",
fiwN
--.." N --, =,:.=- -
:...
N = '-,
.,:.*54 1:?j4 V ( 1
1
,---"N,
5 5 5 '',.. 5 5 5
tVirin
...):
t
L"'sgN' `,)
st.,,,,p , '4,...,2/ 'iLii , µ===== tsis , and ,
In some embodiments, a composition described herein (e.g., a nucleic acid
(e.g., a circular
polyribonucleotide, a linear polyribonucleotide) or a protein) is provided in
an LNP that comprises an
5 ionizable lipid. In some embodiments, the ionizable lipid is heptadecan-9-
y18-((2-hydroxyethyl)(6-oxo-6-
(undecyloxy)hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of
US9,867,888
(incorporated by reference herein in its entirety). In some embodiments, the
ionizable lipid is 9Z,12Z)-3-
((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-
(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-
dienoate (LP01), e.g., as synthesized in Example 13 of W02015/095340
(incorporated by reference
herein in its entirety). In some embodiments, the ionizable lipid is Di((Z)-
non-2-en-1-y1) 9-((4-
dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g. as synthesized in
Example 7, 8, or 9 of
U52012/0027803 (incorporated by reference herein in its entirety). In some
embodiments, the ionizable
lipid is 1,1'-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2-
hydroxydodecyl) amino)ethyl)piperazin-1-
yOethyl)azanediyObis(dodecan-2-01) (C12-200), e.g., as synthesized in Examples
14 and 16 of
W02010/053572 (incorporated by reference herein in its entirety). In some
embodiments, the ionizable
lipid is Imidazole cholesterol ester (ICE) lipid (3S, 10R, 13R, 17R)-10, 13-
dimethy1-17- ((R)-6-
methylheptan-2-y1)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-
tetradecahydro-1H-
cyclopenta[a]phenanthren-3-y13-(1H-imidazol-4-yl)propanoate, e.g., Structure
(I) from W02020/106946
(incorporated by reference herein in its entirety).
In some embodiments, an ionizable lipid may be a cationic lipid, an ionizable
cationic lipid, e.g., a
cationic lipid that can exist in a positively charged or neutral form
depending on pH, or an amine-
containing lipid that can be readily protonated. In some embodiments, the
cationic lipid is a lipid capable
of being positively charged, e.g., under physiological conditions. Exemplary
cationic lipids include one or
more amine group(s) which bear the positive charge. In some embodiments, the
lipid particle comprises
a cationic lipid in formulation with one or more of neutral lipids, ionizable
amine-containing lipids,
biodegradable alkyne lipids, steroids, phospholipids including polyunsaturated
lipids, structural lipids (e.g.,
sterols), PEG, cholesterol and polymer conjugated lipids. In some embodiments,
the cationic lipid may be
an ionizable cationic lipid. An exemplary cationic lipid as disclosed herein
may have an effective pKa over
6Ø In embodiments, a lipid nanoparticle may comprise a second cationic lipid
having a different effective
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pKa (e.g., greater than the first effective pKa), than the first cationic
lipid. A lipid nanoparticle may
comprise between 40 and 60 mol percent of a cationic lipid, a neutral lipid, a
steroid, a polymer
conjugated lipid, and a therapeutic agent, e.g., a nucleic acid (e.g., RNA
(e.g., a circular
polyribonucleotide, a linear polyribonucleotide)) described herein,
encapsulated within or associated with
the lipid nanoparticle. In some embodiments, the nucleic acid is co-formulated
with the cationic lipid. The
nucleic acid may be adsorbed to the surface of an LNP, e.g., an LNP comprising
a cationic lipid. In some
embodiments, the nucleic acid may be encapsulated in an LNP, e.g., an LNP
comprising a cationic lipid.
In some embodiments, the lipid nanoparticle may comprise a targeting moiety,
e.g., coated with a
targeting agent. In embodiments, the LNP formulation is biodegradable. In some
embodiments, a lipid
nanoparticle comprising one or more lipid described herein, e.g., Formula (i),
(ii), (ii), (vii) and/or (ix)
encapsulates at least 1%, at least 5%, at least 10%, at least 20%, at least
30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at
least 95%, at least 97%, at
least 98% or 100% of an RNA molecule.
Exemplary ionizable lipids that can be used in lipid nanoparticle formulations
include, without
limitation, those listed in Table 1 of W02019051289, incorporated herein by
reference. Additional
exemplary lipids include, without limitation, one or more of the following
formulae: X of US2016/0311759;
lof US20150376115 or in US2016/0376224; I, II or III of US20160151284; I, IA,
II, or IIA of
US20170210967; I-c of US20150140070; A of US2013/0178541; 1 of US2013/0303587
or
US2013/0123338;lof US2015/0141678; II, Ill, IV, or V of US2015/0239926; 1 of
US2017/0119904; 1 or II
of W02017/117528; A of US2012/0149894; A of US2015/0057373; A of
W02013/116126; A of
US2013/0090372; A of US2013/0274523; A of US2013/0274504; A of US2013/0053572;
A of
W02013/016058; A of W02012/162210; lof US2008/042973; I, II, III, or IV of
US2012/01287670; 1 or II of
US2014/0200257; I, II, or III of US2015/0203446; 1 or III of US2015/0005363;
I, IA, IB, IC, ID, II, IIA, IIB,
IIC, IID, or III-XXIV of US2014/0308304; of US2013/0338210; I, II, III, or IV
of W02009/132131; A of
US2012/01011478;lor XXXV of US2012/0027796; XIV or XVII of US2012/0058144; of
US2013/0323269;lof US2011/0117125; I, II, or III of US2011/0256175; 1,11,111,
IV, V, VI, VII, VIII, IX, X,
XI, XII of US2012/0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII,
XIV, XV, or XVI of US2011/0076335; 1
or II of US2006/008378; 1 of US2013/0123338;lor X-A-Y-Z of US2015/0064242;
XVI, XVII, or XVIII of
US2013/0022649; I, II, or III of US2013/0116307; I, II, or III of
US2013/0116307;lor II of
US2010/0062967; I-X of US2013/0189351;lof US2014/0039032; V of
US2018/0028664;lof
US2016/0317458;lof US2013/0195920; 5, 6, or 10 of US10,221,127; 111-3 of
W02018/081480;1-5 or 1-8
of W02020/081938; 18 or 25 of US9,867,888; A of US2019/0136231; II of
W02020/219876; 1 of
US2012/0027803; OF-02 of US2019/0240349; 23 of US10,086,013; cKK-E12/A6 of
Miao et al (2020);
012-200 of W02010/053572; 701 of Dahlman et al (2017); 304-013 or 503-013 of
Whitehead et al; TS-
P402 of US9,708,628;lof W02020/106946; lof W02020/106946; and (1), (2), (3),
or (4) of
W02021/113777. Exemplary lipids further include a lipid of any one of Tables 1-
16 of W02021/113777.
In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,31Z)-heptatriaconta-
6,9,28,31-
tetraen-19-y1-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as
described in Example 9 of
W02019051289A9 (incorporated by reference herein in its entirety). In some
embodiments, the ionizable
lipid is the lipid ATX-002, e.g., as described in Example 10 of W02019051289A9
(incorporated by
reference herein in its entirety). In some embodiments, the ionizable lipid is
(13Z,16Z)-A,A-dimethy1-3-
nonyldocosa-13,16-dien-l-amine (Compound 32), e.g., as described in Example 11
of W02019051289A9
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(incorporated by reference herein in its entirety). In some embodiments, the
ionizable lipid is Compound
6 or Compound 22, e.g., as described in Example 12 of W02019051289A9
(incorporated by reference
herein in its entirety).
Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-
glycero-
phosphoethanolamine, distearoylphosphatidylcholine (DSPC),
dioleoylphosphatidylcholine (DOPC),
dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG),
dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine
(DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-
phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane- 1 - carboxylate
(DOPE-mal), dipalmitoyl
phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-
ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-0-
monomethyl PE), dimethyl-
phosphatidylethanolamine (such as 16-0-dimethyl PE), 18-1-trans PE,1-stearoy1-
2-oleoyl-
phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC),
egg
phosphatidylcholine (E PC), dioleoylphosphatidylserine (DOPS), sphingomyelin
(SM), dimyristoyl
phosphatidylcholine (DM PC), dimyristoyl phosphatidylglycerol (DM PG),
distearoylphosphatidylglycerol
(DSPG), dierucoylphosphatidylcholine (DEPC),
palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-
phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine,
lysolecithin,
lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,
sphingomyelin, egg
sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid,cerebrosides,
dicetylphosphate,
lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof.
It is understood that other
diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can
also be used. The acyl
groups in these lipids are preferably acyl groups derived from fatty acids
having C10-C24 carbon chains,
e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl. Additional exemplary
lipids, in certain embodiments,
include, without limitation, those described in Kim et al. (2020)
dx.doi.org/10.1021/acs.nanolett.0c01386,
incorporated herein by reference. Such lipids include, in some embodiments,
plant lipids found to
improve liver transfection with mRNA (e.g., DGTS).
Other examples of non-cationic lipids suitable for use in the lipid
nanoparticles include, without
limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine,
hexadecylamine, acetyl
palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate,
amphoteric acrylic polymers,
triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty
acid amides, dioctadecyl dimethyl
ammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic
lipids are described in
W02017/099823 or US patent publication U5201 8/0028664, the contents of which
is incorporated herein
by reference in their entirety.
In some embodiments, the non-cationic lipid is oleic acid or a compound of
Formula I, II, or IV of
US2018/0028664, incorporated herein by reference in its entirety. The non-
cationic lipid can comprise,
for example, 0-30% (mol) of the total lipid present in the lipid nanoparticle.
In some embodiments, the
non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid
present in the lipid nanoparticle.
In embodiments, the molar ratio of ionizable lipid to the neutral lipid ranges
from about 2:1 to about 8:1
(e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).
In some embodiments, the lipid nanoparticles do not comprise any
phospholipids.
In some aspects, the lipid nanoparticle can further comprise a component, such
as a sterol, to
provide membrane integrity. One exemplary sterol that can be used in the lipid
nanoparticle is cholesterol
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and derivatives thereof. Non-limiting examples of cholesterol derivatives
include polar analogues such as
5a-cholestanol, 53-coprostanol, cholestery1-(2-hydroxy)-ethyl ether,
cholestery1-(4.- hydroxy)-butyl ether,
and 6-ketocholestanol; non-polar analogues such as 5a-cholestane,
cholestenone, 5a-cholestanone, 5p-
cholestanone, and cholesteryl decanoate; and mixtures thereof. In some
embodiments, the cholesterol
derivative is a polar analogue, e.g., cholestery1-(4 '-hydroxy)-butyl ether.
Exemplary cholesterol
derivatives are described in PCT publication W02009/127060 and US patent
publication
US2010/0130588, each of which is incorporated herein by reference in its
entirety.
In some embodiments, the component providing membrane integrity, such as a
sterol, can
comprise 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%) of the
total lipid present in the
lipid nanoparticle. In some embodiments, such a component is 20-50% (mol) 30-
40% (mol) of the total
lipid content of the lipid nanoparticle.
In some embodiments, the lipid nanoparticle can comprise a polyethylene glycol
(PEG) or a
conjugated lipid molecule. Generally, these are used to inhibit aggregation of
lipid nanoparticles and/or
provide steric stabilization. Exemplary conjugated lipids include, but are not
limited to, PEG-lipid
conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates
(such as ATTA-lipid
conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In
some embodiments, the
conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy
polyethylene glycol)-
conjugated lipid.
Exemplary PEG-lipid conjugates include, but are not limited to, PEG-
diacylglycerol (DAG) (such
as1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-
dialkyloxypropyl
(DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated
phosphatidylethanoloamine (PEG-PE), PEG
succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-
di(tetradecanoyloxy)propy1-1-0-(w-
methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam,
N-(carbonyl-
methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-
phosphoethanolamine sodium salt, or a
mixture thereof. Additional exemplary PEG-lipid conjugates are described, for
example, in U55,885,6I3,
U56,287,591, U52003/0077829, U52003/0077829, U52005/01 75682, U52008/0020058,
U52011/0117125, U52010/0130588, U52016/0376224, U52017/0119904, and US/099823,
the contents
of all of which are incorporated herein by reference in their entirety. In
some embodiments, a PEG-lipid is
a compound of Formula III, III-a-2, Ill-b-1, Ill-b-2, or V of
US2018/0028664, the content of which is
incorporated herein by reference in its entirety. In some embodiments, a PEG-
lipid is of Formula II of
US20150376115 or US2016/0376224, the content of both of which is incorporated
herein by reference in
its entirety. In some embodiments, the PEG-DAA conjugate can be, for example,
PEG-dilauryloxypropyl,
PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl.
The PEG-lipid can be
one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-
disterylglycerol, PEG-
dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, PEG-
disterylglycamide, PEG-
cholesterol (1-[8.-(Cholest-5-en-3[beta]- oxy)carboxamido-3',6'-dioxaoctanyl]
carbamoyHomega]-methyl-
poly(ethylene glycol), PEG- DMB (3,4-Ditetradecoxylbenzyl- [omega]-methyl-
poly(ethylene glycol) ether),
and 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000]. In some
embodiments, the PEG-lipid comprises PEG-DMG, 1,2- dimyristoyl-sn-glycero-3-
phosphoethanolamine-
N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid
comprises a structure
selected from:
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0
o
fl
0
7
k
r,
H = 4.5
6
,and
0
In some embodiments, lipids conjugated with a molecule other than a PEG can
also be used in
place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates,
polyamide-lipid conjugates (such
5 as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates
can be used in place of or in
addition to the PEG-lipid.
Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-
lipid conjugates and
cationic polymer-lipids are described in the PCT and LIS patent applications
listed in Table 2 of
W02019051289A9, the contents of all of which are incorporated herein by
reference in their entirety.
10 In some embodiments, the PEG or the conjugated lipid can comprise 0-20%
(mol) of the total
lipid present in the lipid nanoparticle. In some embodiments, PEG or the
conjugated lipid content is 0.5-
10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. Molar
ratios of the ionizable lipid,
non-cationic-lipid, sterol, and PEG/conjugated lipid can be varied as needed.
For example, the lipid
particle can comprise 30-70% ionizable lipid by mole or by total weight of the
composition, 0-60%
15 cholesterol by mole or by total weight of the composition, 0-30% non-
cationic-lipid by mole or by total
weight of the composition and 1-10% conjugated lipid by mole or by total
weight of the composition.
Preferably, the composition comprises 30-40% ionizable lipid by mole or by
total weight of the
composition, 40-50% cholesterol by mole or by total weight of the composition,
and 10- 20% non-cationic-
lipid by mole or by total weight of the composition. In some other
embodiments, the composition is 50-
20 75% ionizable lipid by mole or by total weight of the composition, 20-
40% cholesterol by mole or by total
weight of the composition, and 5 to 10% non-cationic-lipid, by mole or by
total weight of the composition
and 1-10% conjugated lipid by mole or by total weight of the composition. The
composition may contain
60-70% ionizable lipid by mole or by total weight of the composition, 25-35%
cholesterol by mole or by
total weight of the composition, and 5-10% non-cationic-lipid by mole or by
total weight of the
25 composition. The composition may also contain up to 90% ionizable lipid
by mole or by total weight of the
composition and 2 to 15% non-cationic lipid by mole or by total weight of the
composition. The formulation
may also be a lipid nanoparticle formulation, for example comprising 8-30%
ionizable lipid by mole or by
total weight of the composition, 5-30% non-cationic lipid by mole or by total
weight of the composition,
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and 0-20% cholesterol by mole or by total weight of the composition; 4-25%
ionizable lipid by mole or by
total weight of the composition, 4-25% non-cationic lipid by mole or by total
weight of the composition, 2
to 25% cholesterol by mole or by total weight of the composition, 10 to 35%
conjugate lipid by mole or by
total weight of the composition, and 5% cholesterol by mole or by total weight
of the composition; or 2-
30% ionizable lipid by mole or by total weight of the composition, 2-30% non-
cationic lipid by mole or by
total weight of the composition, 1 to 15% cholesterol by mole or by total
weight of the composition, 2 to
35% conjugate lipid by mole or by total weight of the composition, and 1-20%
cholesterol by mole or by
total weight of the composition; or even up to 90% ionizable lipid by mole or
by total weight of the
composition and 2-10% non-cationic lipids by mole or by total weight of the
composition, or even 100%
cationic lipid by mole or by total weight of the composition. In some
embodiments, the lipid particle
formulation comprises ionizable lipid, phospholipid, cholesterol and a PEG-
ylated lipid in a molar ratio of
50: 10:38.5: 1.5. In some other embodiments, the lipid particle formulation
comprises ionizable lipid,
cholesterol and a PEG-ylated lipid in a molar ratio of 60:38.5: 1.5.
In some embodiments, the lipid particle comprises ionizable lipid, non-
cationic lipid (e.g.
phospholipid), a sterol (e.g., cholesterol) and a PEG-ylated lipid, where the
molar ratio of lipids ranges
from 20 to 70 mole percent for the ionizable lipid, with a target of 40-60,
the mole percent of non-cationic
lipid ranges from 0 to 30, with a target of 0 to 15, the mole percent of
sterol ranges from 20 to 70, with a
target of 30 to 50, and the mole percent of PEG-ylated lipid ranges from 1 to
6, with a target of 2 to 5.
In some embodiments, the lipid particle comprises ionizable lipid / non-
cationic- lipid / sterol /
conjugated lipid at a molar ratio of 50:10:38.5: 1.5.
In an aspect, the disclosure provides a lipid nanoparticle formulation
comprising phospholipids,
lecithin, phosphatidylcholine and phosphatidylethanolamine.
In some embodiments, one or more additional compounds can also be included.
Those
compounds can be administered separately, or the additional compounds can be
included in the lipid
nanoparticles of the invention. In other words, the lipid nanoparticles can
contain other compounds in
addition to the nucleic acid or at least a second nucleic acid, different than
the first. Without limitations,
other additional compounds can be selected from the group consisting of small
or large organic or
inorganic molecules, monosaccharides, disaccharides, trisaccharides,
oligosaccharides, polysaccharides,
peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics,
nucleic acids, nucleic acid
analogs and derivatives, an extract made from biological materials, or any
combinations thereof.
In some embodiments, the LNPs comprise biodegradable, ionizable lipids. In
some
embodiments, the LNPs comprise (9Z,I2Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-
((((3-
(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,I2-dienoate, also
called 3- ((4,4-
bis(octyloxy)butanoyl)oxy)-2-((((3-
(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,I2Z)-octadeca-
9,I2-dienoate) or another ionizable lipid. See, e.g, lipids of W02019/067992,
WO/2017/173054,
W02015/095340, and W02014/136086, as well as references provided therein. In
some embodiments,
the term cationic and ionizable in the context of LNP lipids is
interchangeable, e.g., wherein ionizable
lipids are cationic depending on the pH.
In some embodiments, the average LNP diameter of the LNP formulation may be
between lOs of
nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). In some
embodiments, the
average LNP diameter of the LNP formulation may be from about 40 nm to about
150 nm, such as about
40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm,
95 nm, 100 nm, 105
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nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.
In some
embodiments, the average LNP diameter of the LNP formulation may be from about
50 nm to about 100
nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from
about 50 nm to about 70
nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from
about 60 nm to about 90
nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from
about 70 nm to about 100
nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from
about 80 nm to about 100
nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In
some embodiments, the
average LNP diameter of the LNP formulation may be from about 70 nm to about
100 nm. In a particular
embodiment, the average LNP diameter of the LNP formulation may be about 80
nm. In some
embodiments, the average LNP diameter of the LNP formulation may be about 100
nm. In some
embodiments, the average LNP diameter of the LNP formulation ranges from about
I mm to about 500
mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from
about 20 mm to about
80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from
about 35 mm to
about 50 mm, or from about 38 mm to about 42 mm.
A LNP may, in some instances, be relatively homogenous. A polydispersity index
may be used to
indicate the homogeneity of a LNP, e.g., the particle size distribution of the
lipid nanoparticles. A small
(e.g., less than 0.3) polydispersity index generally indicates a narrow
particle size distribution. A LNP may
have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07,
0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20,
0.21, 0.22, 0.23, 0.24, or 0.25.
In some embodiments, the polydispersity index of a LNP may be from about 0.10
to about 0.20.
The zeta potential of a LNP may be used to indicate the electrokinetic
potential of the
composition. In some embodiments, the zeta potential may describe the surface
charge of an LNP. Lipid
nanoparticles with relatively low charges, positive or negative, are generally
desirable, as more highly
charged species may interact undesirably with cells, tissues, and other
elements in the body. In some
embodiments, the zeta potential of a LNP may be from about -10 mV to about +20
mV, from about -10
mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to
about +5 mV, from
about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV
to about +20 mV, from
about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5
mV to about +5 mV,
from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0
mV to about +15 mV,
from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5
mV to about +20 mV,
from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.
The efficiency of encapsulation of a protein and/or nucleic acid, describes
the amount of protein
and/or nucleic acid that is encapsulated or otherwise associated with a LNP
after preparation, relative to
the initial amount provided. The encapsulation efficiency is desirably high
(e.g., close to 100%). The
encapsulation efficiency may be measured, for example, by comparing the amount
of protein or nucleic
acid in a solution containing the lipid nanoparticle before and after breaking
up the lipid nanoparticle with
one or more organic solvents or detergents. An anion exchange resin may be
used to measure the
amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence
may be used to measure
the amount of free protein and/or nucleic acid (e.g., RNA) in a solution. For
the lipid nanoparticles
described herein, the encapsulation efficiency of a protein and/or nucleic
acid may be at least 50%, for
example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%,
98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at
least 80%. In some
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embodiments, the encapsulation efficiency may be at least 90%. In some
embodiments, the
encapsulation efficiency may be at least 95%.
A LNP may optionally comprise one or more coatings. In some embodiments, a LNP
may be
formulated in a capsule, film, or table having a coating. A capsule, film, or
tablet including a composition
described herein may have any useful size, tensile strength, hardness or
density.
Additional exemplary lipids, formulations, methods, and characterization of
LNPs are taught by
W02020/061457 and W02021/113777, each of which is incorporated herein by
reference in its entirety.
Further exemplary lipids, formulations, methods, and characterization of LNPs
are taught by Hou et al.
Lipid nanoparticles for mRNA delivery. Nat Rev Mater (2021).
doi.org/10.1038/s41578-021-00358-0,
which is incorporated herein by reference in its entirety (see, for example,
exemplary lipids and lipid
derivatives of Figure 2 of Hou et al.).
In some embodiments, in vitro or ex vivo cell lipofections are performed using
Lipofectamine
MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio).
In certain
embodiments, LNPs are formulated using the GenVoy ILM ionizable lipid mix
(Precision NanoSystems).
In certain embodiments, LNPs are formulated using 2,2-dilinoley1-4-
dimethylaminoethyl-[1,3]-dioxolane
(DLin-KC2-DMA) or dilinoleylmethy1-4-dimethylaminobutyrate (DLin-MC3-DMA or
MC3), the formulation
and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl
51(34):8529-8533
(2012), incorporated herein by reference in its entirety.
LNP formulations optimized for the delivery of CRISPR-Cas systems, e.g., Cas9-
g RNA RNP,
gRNA, Cas9 mRNA, are described in W02019067992 and W02019067910, both
incorporated by
reference, and are useful for delivery of circular polyribonucleotides and
linear polyribonucleotides
described herein.
Additional specific LNP formulations useful for delivery of nucleic acids
(e.g., circular
polyribonucleotides, linear polyribonucleotides) are described in US8158601
and US8168775, both
incorporated by reference, which include formulations used in patisiran, sold
under the name
ONPATTRO.
Exemplary dosing of polyribonucleotide (e.g., a circular polyribonucleotide, a
linear
polyribonucleotide) LNP may include about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5,
6, 8, 10, or 100 mg/kg (RNA).
Exemplary dosing of AAV comprising a polyribonucleotide (e.g., a circular
polyribonucleotide, a linear
polyribonucleotide) may include an MOI of about 1011, 1012, 1013, and 1014
vg/kg.
Adjuvants
An adjuvant enhances the immune responses (humoral and/or cellular) elicited
in a subject who
receives the adjuvant and/or an immunogenic composition including the
adjuvant. In some embodiments,
an adjuvant is administered to a subject as disclosed herein. In some
embodiments, an adjuvant is used
in the methods described herein to produce an immune response as described
herein. In a particular
embodiment, an adjuvant is used to promote an immune response in a subject
against an immunogen
expressed from a circular polyribonucleotide. In some embodiments, an adjuvant
and polyribonucleotide
are co-administered in separate compositions. In some embodiments, an adjuvant
is mixed or formulated
with a polyribonucleotide in a single composition and administered to a
subject. In some embodiments,
an adjuvant and circular or linear polyribonucleotide are co-administered in
separate compositions. In
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some embodiments, an adjuvant is mixed or formulated with a linear or circular
polyribonucleotide in a
single composition to obtain an immunogenic composition that is administered
to a subject.
An adjuvant may be a component of a circular or linear polyribonucleotide
(e.g., a
polyribonucleotide sequence), may be polypeptide adjuvant encoded by an
expression sequence of a
polyribonucleotide, may be a molecule (e.g., a small molecule, polypeptide, or
nucleic acid molecule) that
is not encoded by the polyribonucleotide. An adjuvant may be formulated with a
polyribonucleotide in the
same pharmaceutical composition. An adjuvant may be administered separately
(e.g., as a separate
pharmaceutical composition) in combination with a polyribonucleotide.
In some embodiments, the adjuvant is encoded by the circular or linear
polyribonucleotide. In
some embodiments, the circular or linear polyribonucleotide encodes more than
one adjuvant. For
example, the circular or linear polyribonucleotide encodes between 2 and 100
adjuvants. In some
embodiments, the circular or linear polyribonucleotide encodes between 2 and
10 adjuvants. In some
embodiments, the circular or linear polyribonucleotide encodes 2 adjuvants.
One or more of the
adjuvants encoded by a circular or linear polyribonucleotide may include an N-
terminal signal sequence,
e.g., that directs the expressed polypeptide adjuvant to the secretory
pathway. In some embodiments,
the polyribonucleotide encodes 3 adjuvants. In some embodiments, the
polyribonucleotide encodes 4
adjuvants. In some embodiments, the polyribonucleotide encodes 5 adjuvants. In
some embodiments,
the adjuvant is encoded by the same polyribonucleotide that encodes one or
more immunogens. The
adjuvant(s) and immunogen(s) may be co-delivered on the same
polyribonucleotide. In some
embodiments, the adjuvant encoded by the polyribonucleotide is a sequence
(e.g., a polyribonucleotide
sequence) that is an innate immune system stimulator. The innate immune system
stimulator sequence
may include at least 5, at least 10, at least 20, at least 50, at least 100,
or at least 500 ribonucleotides.
The innate immune system stimulator sequence may include between 5 and 1000,
between 10 and 500,
between 20 and 500, between 10 and 100, between 20 and 100, between 20 and 50,
between 100 and
500, between 500 and 1000, or between 10 and 1000 ribonucleotides. For
example, a sequence that is
an innate immune system stimulator may be selected from a GU-rich motif, an AU-
rich motif, a structured
region comprising dsRNA, or an aptamer.
Adjuvants may be a TH1 adjuvant and/or a TH2 adjuvant. Further adjuvants
contemplated by
this disclosure include, but are not limited to, one or more of the following:
Mineral-containing compositions. Mineral-containing compositions suitable for
use as adjuvants
in the disclosure include mineral salts, such as aluminum salts, and calcium
salts. The disclosure
includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates
(e.g. hydroxyphosphates,
orthophosphates), sulphates, etc., or mixtures of different mineral compounds,
with the compounds taking
any suitable form (e.g. gel, crystalline, amorphous, etc.). Calcium salts
include calcium phosphate (e.g.,
the "CAP"). Aluminum salts include hydroxides, phosphates, sulfates, and the
like.
Oil emulsion compositions. Oil-emulsion compositions suitable for use as
adjuvants in the
disclosure include squalene-water emulsions, such as MF59 (5% Squalene, 0.5%
Tween 80 and 0.5%
Span, formulated into submicron particles using a microfluidizer), A503 (a-
tocopherol, squalene and
polysorbate 80 in an oil-in-water emulsion), Montanide formulations (e.g.
Montanide ISA 51, Montanide
ISA 720), incomplete Freunds adjuvant (IFA), complete Freund's adjuvant (CFA),
and incomplete
Freund's adjuvant (IFA).
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Small molecules. Small molecules suitable for use as adjuvants in the
disclosure include
imiquimod or 847, resiquimod or R848, and gardiquimod.
Polymeric nanoparticles. Polymeric nanoparticles suitable for use as an
adjuvant in the
disclosure include poly(a-hydroxy acids), polyhydroxy butyric acids,
polylactones (including
polycaprolactones), polydioxanones, polyvalerolactone, polyorthoesters,
polyanhydrides,
polycyanoacrylates, tyrosine-derived polycarbonates, polyvinyl-pyrrolidinones
or polyester-amides, and
combinations thereof.
Saponin (i.e., a glycoside, polycyclic aglycones attached to one or more sugar
side chains).
Saponin formulations suitable for use as an adjuvant in the disclosure include
purified formulations, such
as 0S21, as well as lipid formulations, such as ISCOMs and ISCOMs matrix. 0S21
is marketed as
STIMULON (TM). Saponin formulations may also include a sterol, such as
cholesterol. Combinations of
saponins and cholesterols can be used to form unique particles called
immunostimulating complexes
(ISCOMs). ISCOMs typically also include a phospholipid such as
phosphatidylethanolamine or
phosphatidylcholine. Any known saponin can be used in ISCOMs. Preferably, the
ISCOM includes one
or more of QuilA, QHA & QHC. Optionally, the ISCOMS may be devoid of
additional detergent.
Lipopolysaccharides. Adjuvants suitable for use in the disclosure include non-
toxic derivatives of
enterobacterial lipopolysaccharide (LPS). Such derivatives include
monophosphoryl lipid A (MPLA),
glucopyranosyl lipid A (GLA) and 3-0-deacylated MPL (3dMPL). 3dMPL is a
mixture of 3 De-O-acylated
monophosphoryl lipid A with 4, 5 or 6 acylated chains. Other non-toxic LPS
derivatives include
monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate
derivatives e.g. RC-529.
Liposomes. Liposomes suitable for use as an adjuvant in the disclosure include
virosomes and
CAF01.
Lipid nanoparticles. Adjuvants suitable for use in the disclosure include
lipid nanoparticles
(LNPs) and their components.
Lipopeptides (i.e., compounds including one or more fatty acid residues and
two or more amino
acid residues). Lipopeptide suitable for use as an adjuvant in the disclosure
include Pam2 (Pam2CSK4)
and Pam3 (Pam3CSK4).
Glycolipids. Glycolipids suitable for use as an adjuvant in the disclosure
include cord factor
(trehalose dimycolate).
Peptides and peptidoglycans derived from (synthetic or purified) gram-negative
or gram-positive
bacteria, such as MDP (N-acetyl-muramyl-L-alanyl-D-isoglutamine) are suitable
for use as an adjuvant in
the disclosure
Carbohydrates (carbohydrate containing) or polysaccharides suitable for use as
an adjuvant
include dextran (e.g., branched microbial polysaccharide), dextran-sulfate,
lentinan, zymosan, beta-
glucan, deltin, mannan, and chitin.
RNA based adjuvants. RNA based adjuvants suitable for use in the disclosure
are poly IC, poly
IC:LC, hairpin RNAs with or without a 5'triphosphate, viral sequences, polyU
containing sequence,
dsRNA natural or synthetic RNA sequences, and nucleic acid analogs (e.g.,
cyclic GMP-AMP or other
cyclic dinucleotides e.g., cyclic di-GMP, immunostimulatory base analogs e.g.,
C8-substituted and N7,C8-
disubstituted guanine ribonucleotides). In some embodiments, the adjuvant is
the linear
polyribonucleotide counterpart of the circular polyribonucleotide described
herein.
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DNA based adjuvants. DNA based adjuvants suitable for use in the disclosure
include CpGs,
dsDNA, and natural or synthetic immunostimulatory DNA sequences.
Proteins or peptides. Proteins and peptides suitable for use as an adjuvant in
the disclosure
include flagellin-fusion proteins, MBL (mannose-binding lectin), cytokines,
and chemokines.
Viral particles. Viral particles suitable for use as an adjuvant include
virosomes (phospholipid cell
membrane bilayer).
An adjuvant for use in the disclosure may be bacterial derived, such as a
flagellin, LPS, or a
bacterial toxin (e.g., enterotoxins (protein), e.g., heat-labile toxin or
cholera toxin). An adjuvant for use in
the disclosure may be a hybrid molecule such as CpG conjugated to imiquimod.
An adjuvant for use in
the disclosure may be a fungal or oomycete microbe-associated molecular
patterns (MAMPs), such as
chitin or beta-glucan. In some embodiments, an adjuvant is an inorganic
nanoparticle, such as gold
nanorods or silica-based nanoparticles (e.g., mesoporous silica nanoparticles
(MSN)). In some
embodiments, an adjuvant is a multi-component adjuvant or adjuvant system,
such as AS01, AS03, AS04
(MLP5 + alum), CFA (complete Freund's adjuvant: IFA + peptiglycan + trehalose
dimycolate), CAF01
(two component system of cationic liposome vehicle (dimethyl dioctadecyl-
ammonium (DDA)) stabilized
with a glycolipid immunomodulator (trehalose 6,6-dibehenate (TDB), which can
be a synthetic variant of
cord factor located in the mycobacterial cell wall).
Cytokines. An adjuvant may be a partial or full-length DNA encoding a cytokine
such as, a pro-
inflammatory cytokine (e.g., GM-CSF, IL-1 alpha, IL-1 beta, TGF-beta, TNF-
alpha, TNF-beta), Th-1
inducing cytokines (e.g., IFN-gamma, IL-2, IL-12, IL-15, IL-18), or Th-2
inducing cytokines (e.g., IL-4, IL-5,
IL-6, IL-10, IL-13).
Chemokines. An adjuvant may be a partial or full-length DNA or RNA (e.g.,
circRNA or mRNA)
encoding a chemokine such as, MCP-1, MIP-1 alpha, MIP-1 beta, Rantes, or TCA-
3.
An adjuvant may be a partial or full-length DNA encoding a costimulatory
molecule, such as CD80, 0D86,
CD4O-L, CD70, or 0D27.
An adjuvant may be a partial or full length DNA or RNA (e.g., circRNA or mRNA)
encoding for an
innate immune system stimulator (partial, full-length, or mutated) such as
TLR4, TLR3, TLR3, TLR9,
TLR7, TLR8, TLR7, RIG-I/DDX58, or MDA-5/IFIH1; or a constitutively active (ca)
innate immune
stimulator, such as caTLR4, caTLR3, caTLR3, caTLR9, caTLR7, caTLR8, caTLR7,
caRIG-I/DDX58, or
caMDA-5/IFIH1.
An adjuvant may be a partial or full-length DNA or RNA (e.g., circRNA or mRNA)
encoding for an
adaptor or signaling molecule, such as STING (e.g., caSTING), TRIF, TRAM,
MyD88, IPS1, ASC, MAVS,
MAPKs, IKK-alpha, IKK complex, TBK1, beta-catenin, and caspase 1.
An adjuvant may be a partial or full-length DNA or RNA (e.g., circRNA or mRNA)
encoding for a
transcriptional activator, such as a transcription activator that can
upregulate an immune response (e.g.,
AP1, NF-kappa B, IRF3, IRF7, IRF1, or IRF5). An adjuvant may be a partial or
full-length DNA encoding
for a cytokine receptor, such as IL-2beta, IFN-gamma, or IL-6.
An adjuvant may be a partial or full-length DNA or RNA (e.g., circRNA or mRNA)
encoding for a
bacterial component, such as flagellin or MBL.
An adjuvant may be a partial or full-length DNA or RNA (e.g., circRNA or mRNA)
encoding for
any component of the innate immune system.
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In some embodiments, a subject is administered a circular or linear
polyribonucleotide encoding
one or more immunogens in combination with an adjuvant (e.g., an adjuvant that
is a separate molecular
entity from the circular polyribonucleotide or an adjuvant that is encoded on
a separate
polyribonucleotide). The term "in combination with" as used throughout the
description includes any two
compositions administered as part of a therapeutic regimen. This may include,
for example, a
polyribonucleotide and an adjuvant formulated as a single pharmaceutical
composition. This also
includes, for example, a polyribonucleotide and an adjuvant administered to a
subject as separate
compositions according to a defined therapeutic or dosing regimen. An adjuvant
may be administered to
a subject before, at substantially the same time, or after the administration
of a polyribonucleotide. An
adjuvant may be administered within 1 day, 2 days, 5 days, 10 days, 20 days, 1
month, 2 months, 3
months, 4 months, 5 months, or 6 months before or after administration of a
polyribonucleotide. An
adjuvant may be administered by the same route of administration (e.g.,
intramuscularly, subcutaneously,
intravenously, intraperitoneally, topically, or orally) or a different route
than a polyribonucleotide.
Vaccines
In some embodiments of methods described herein, a second agent is also
administered to the
subject, e.g., a second vaccine is also administered to a subject. In some
embodiments, a composition
that is administered to a subject includes a circular polyribonucleotide
described herein and a second
vaccine. In some embodiments, a vaccine and circular polyribonucleotide are co-
administered in
separate compositions. The vaccine is simultaneously administered with the
circular polyribonucleotide
immunization, administered before the circular polyribonucleotide
immunization, or after the circular
polyribonucleotide immunization.
For example, in some embodiments, a subject is immunized with a non-circular
polyribonucleotide vaccine (e.g., protein subunit vaccine) and an immunogenic
composition including a
circular polyribonucleotide. In some embodiments, a subject is immunized with
a non-polyribonucleotide
vaccine for a first microorganism (e.g., pneumococcus) and an immunogenic
composition including a
circular polyribonucleotide as disclosed herein. A vaccine can be any
bacterial infection vaccine or viral
infection vaccine. In a particular embodiment, a vaccine is a pneumococcal
polysaccharide vaccine, such
as PCV13 or PPSV23. In some embodiments, the vaccine is an influenza vaccine.
In some
embodiments, the vaccine is an RSV vaccine (e.g., palivizumap).
In some embodiments, a composition that is administered to a subject includes
a linear
polyribonucleotide and a vaccine. In some embodiments, a vaccine and linear
polyribonucleotide are co-
administered in separate compositions. The vaccine is simultaneously
administered with the linear
polyribonucleotide immunization, administered before the linear
polyribonucleotide immunization, or after
the linear polyribonucleotide immunization.
For example, in some embodiments, a subject is immunized with a
polyribonucleotide (e.g., non-
linear polyribonucleotide) vaccine (e.g., protein subunit vaccine) and an
immunogenic composition
including a linear polyribonucleotide as disclosed herein including a sequence
encoding an immunogen.
In some embodiments, a subject is immunized with a non-polyribonucleotide
vaccine for a first
microorganism (e.g., pneumococcus) and an immunogenic composition including a
linear
polyribonucleotide as disclosed herein including a sequence encoding an
immunogen. A vaccine can be
any bacterial infection vaccine or viral infection vaccine. In a particular
embodiment, a vaccine is a
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pneumococcal polysaccharide vaccine, such as PCV13 or PPSV23. In some
embodiments, the vaccine
is an influenza vaccine. In some embodiments, the vaccine is an RSV vaccine
(e.g., palivizumap).
Other Embodiments
Various modifications and variations of the described compositions, methods,
and uses of the
invention will be apparent to those skilled in the art without departing from
the scope and spirit of the
invention. Although the invention has been described in connection with
specific embodiments, it should
be understood that the invention as claimed should not be unduly limited to
such specific embodiments.
Indeed, various modifications of the described modes for carrying out the
invention that are obvious to
those skilled in the art are intended to be within the scope of the invention.
All publications, patents, and patent applications are herein incorporated by
reference in their
entirety to the same extent as if each individual publication, patent, or
patent application was specifically
and individually indicated to be incorporated by reference in its entirety.
Examples
The following examples, which are intended to illustrate, rather than limit,
the disclosure, are put
forth to provide those of ordinary skill in the art with a description of how
the compositions and methods
described herein may be used, made, and evaluated. The examples are intended
to be purely exemplary
of the disclosure and are not intended to limit the scope of what the
inventors regard as their invention.
Example 1: Design of circular RNA encoding immunogens
This example describes the design of circular RNAs that encode immunogens. In
this example,
circular RNAs are designed to include an IRES, an ORF encoding an immunogen,
and two spacer
elements flanking the IRES-ORF. Circularization enables rolling circle
translation, multiple ORFs with
alternating stagger elements for discrete ORF expression and controlled
protein stoichiometry, and an
IRES that targets RNA for ribosomal entry. Exemplary immunogens that are
encoded by a circular RNA
are SARS-CoV-2 immunogens (RBD and Spike), influenza Hi Ni immunogens, HPV
immunogens, and
tumor neoantigens.
Example 2: Circular RNA generation and purification
In this example, circular RNAs are generated by one of two exemplary methods
and purified
again with the RNA purification system.
Exemplary Method 1: DNA-splint ligation
This exemplary method produces a circular RNA by splint-ligation. RppH-treated
linear RNA is
circularized using a splint DNA. Unmodified linear RNA is synthesized by in
vitro transcription using T7
RNA polymerase from a DNA segment. Transcribed RNA is purified with an RNA
purification system
(New England Biolabs), treated with RNA 5'phosphohydrolase (RppH) (New England
Biolabs, M0356)
following the manufacturer's instructions. Alternately or in addition, the RNA
was transcribed in an
excess of GMP over GTP.
Splint-ligation is performed as follows: circular RNA is generated by
treatment of the transcribed
linear RNA and a DNA splint between 10 and 40 nucleotides in length using an
RNA ligase. To purify the
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circular RNAs, ligation mixtures were resolved on 4% denaturing PAGE and RNA
bands corresponding to
each circular RNA were excised. Excised RNA gel fragments were crushed, and
RNA eluted with gel
elution buffer (0.5 M Sodium Acetate, 0.1% SDS, 1 mM EDTA) for one hour at 37
C. Alternately or in
addition, the circular RNA was purified by column chromatography. Supernatant
is harvested, and RNA is
eluted again by adding gel elution buffer to the crushed gel and incubated for
one hour. Gel debris is
removed by centrifuge filters and is precipitated with ethanol. Agarose gel
electrophoresis is used as a
quality control measurement for validating purity and circularization.
Exemplary Method 2: Circularization by self-splicing intron
This exemplary method produces a circular RNA by self-splicing. The circular
RNA is generated
in vitro. Unmodified linear RNA is in vitro transcribed from a DNA template
including all the motifs listed
above. In vitro transcription reactions included 1 g of template DNA T7 RNA
polymerase promoter, 10X
T7 reaction buffer, 7.5mM ATP, 7.5mM CTP, 7.5mM GTP, 7.5mM UTP, 10mM DTT, 40U
RNase
Inhibitor, and T7 enzyme. Transcription is carried out at 37 C for 4h.
Transcribed RNA is DNase treated
with 1U of DNase I at 37 C for 15min. To favor circularization by self-
splicing, additional GTP is added to
a final concentration of 2 mM, incubated at 55 C for 15 min. RNA is then
column purified and visualized
by UREA-PAGE.
Example 3: Multi-immunogen expression from circular RNA
This example describes expression of multiple immunogens from a circular RNA.
In this Example, one circular RNA is designed to include an IRES (SEQ ID NO:
1) followed by an
ORF encoding immunogen 1, corresponding to a portion of hemagglutinin (HA)
from a first strain of
Influenza A H1 N1, A/California/07/2009(H1N1) (SEQ ID NO: 2), a Stop codon, an
IRES (SEQ ID NO: 1),
another ORF encoding immunogen 2, corresponding to a portion of hemagglutinin
(HA) from a second
strain of Influenza A H1 N1, A/Puerto Rico/8/1934 (SEQ ID NO: 3), a Stop
codon, and a spacer (SEQ ID
NO: 4), see FIG. 1A The circular RNAs are generated either in vitro or in
cells according to the methods
described herein.
Briefly, the circular RNA is incubated for 1.5-3 h in rabbit reticulocyte
lysate (RRL; Promega,
Fitchburg, WI, USA) at 30 C. The final composition of the reaction mixture
includes 70% rabbit
reticulocyte lysate, 20 pM Amino Acid Mixture (Promega; L446A), and 0.8 U/pL
RNasine Ribonuclease
Inhibitor (Promega, N211A). Hemoglobin is removed by trichloroacetic acid
precipitation. After
precipitation and centrifugation, the supernatant is discarded and the pellet
is dissolved in 2x SDS sample
buffer (Thermo) and incubated at 70 C for 15 min. Samples are resolved on 4-
12% gradient
polyacrylamide/sodium dodecyl sulfate (SDS) gels (Thermo, NP0326BOX) followed
by Western blotting.
Proteins are electrotransferred to a polyvinylidene fluoride (PVDF) membrane
(Thermo) using a semi-dry
method, blotted, and probed with specific antibodies and visualized by
chemiluminescence on a C-Digit
scanner (LI-COR Biosciences). Image-Studio Lite (LI-COR Biosciences) is used
for quantification of
expression levels.
Additionally, immunogen 1 and immunogen 2 expression are measured by ELISA in
culture
supernatants from HeLa cells transfected with eRNA. Briefly, 0.1 pmol of eRNA
is transfected into 10,000
HeLa cells using MessengerMax (Invitrogen; LMRNA015) in Opti-MEM (Invitrogen;
31985062). Cell
supernatant is harvested at days 1 and 2. The ELISA is performed as follows: a
capture antibody is
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coated onto ELISA plates (MaxiSorp 442404, 96-well) overnight at 4 C in 100
I_ PBS. After washing
three times with TBS-T, the plates are blocked for 1 hour with blocking buffer
(TBS with 2% FBS and
0.05% Tween 20). Supernatant dilutions are then added to each well in 100 I_
blocking buffer and
incubated at room temperature for 1 hour. After washing three times with TBS-
T, plates are incubated
with HRP detection antibody for 1 hour at room temperature. Tetramethylbenzene
(Pierce 34021) is
added to each well, allowed to react for 5-15 minutes and then quenched with
2N sulfuric acid. The
optical density (OD) value will be determined at 450 nm.
Example 4: Circular RNA encoding an immunogen and an adjuvant
This example describes the ability of a circular RNA to express an immunogen
and to further
encode an adjuvant. The adjuvant may be expressed or may correspond to a non-
expressed nucleic acid
sequence, such as an innate immune stimulator sequence.
In a first instance, one circular RNA is designed to include an IRES followed
by an ORF encoding
an immunogen, a Stop codon, an IRES, another ORF encoding an adjuvant, a Stop
codon, and a spacer,
see FIG. 1A. The circular RNAs are generated either in vitro or in cells and
expressed according to the
methods described herein.
In a second instance, one circular RNA is designed to include an IRES followed
by an ORF
encoding an immunogen, a Stop codon, and further encodes a nucleic acid
sequence that is not
expressed and acts an adjuvant by stimulating the innate immune system (e.g.,
a GU-rich motif, an AU-
rich motif, a structured region comprising dsRNA, or an aptamer), see FIG. 2.
The circular RNAs are
generated either in vitro or in cells and expressed according to the methods
described herein.
Example 5: Circular RNA encoding a plurality of immunogens derived from
mutational variants
For this example, a circular RNA encodes two polypeptide immunogens that
mutational variants
of one another, such that they share at least 90% and less than 100% amino
acid sequence identity
(FIGs. 1A-16). The polypeptide immunogens each include one or more epitopes
corresponding to the
same protein. The polypeptide immunogens may be endogenously occurring
polypeptide immunogens
that are related by genetic drift or modified versions thereof. The circular
RNA is designed with a start-
codon, expression sequences, stagger element(s), and an IRES (FIG. 1B).
Circularization enables rolling
circle translation of multiple expression sequences separated by a stagger
element.
Mutational variants in the protein hemagglutinin associated with the influenza
virus are known to
those of skill in the art. Mutational variants are introduced into the
haemagglutinin protein as a result of
the low fidelity of the viral polymerase resulting in haemagglutinin proteins
that no longer have identical
sequences (Wong et al. (2011) J. Med. Virol., 83: 510-516). Therefore, a
plurality of immunogens
corresponding to the various mutational variants of haemagglutinin, where each
of the plurality of
immunogens shares at least 95% sequence identity is encoded by a circular RNA.
The circular RNA
includes a start-codon, a first ORF including a polypeptide immunogen derived
from the haemagglutinin
protein associated with head of the influenza virus, a stagger element, a
second ORF including a
polypeptide immunogen derived from a mutational variant of the hemagglutinin
protein, and an optional
!RES.
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Example 6: Circular RNA encoding a plurality of immunogens derived from the
same target
For this example, a circular RNA encodes two polypeptide immunogens derived
from two
different proteins but where both proteins identify the same target . The
circular RNA is designed with a
start-codon, expression sequences, stagger element(s), and an IRES (FIG. 1B).
Circularization enables
rolling circle translation of multiple expression sequences separated by a
stagger element.
Specifically, the circular RNA encodes a start codon, a first ORF including a
polypeptide
immunogen derived from HIV-1 envelope glycoprotein 120 (gp120), an optional
stagger element, a
second ORF including a polypeptide immunogen derived from HIV-1 envelope
glycoprotein 41 (gp41),
and an optional IRES, where the HIV-1 envelope protein is the target of the
polypeptide immunogens.
Three gp120s and three gp41s combine in a trimer of heterodimers where the
trimer of gp120s are the
head region and the trimer of gp41s are the tail region which together form
the envelope spikes of HIV-1.
Therefore, polypeptide immunogens derived from both gp120 are gp41 are
included in the circular RNA
to target the HIV-1 envelope protein.
Example 7: Circular RNA encoding a plurality of immunogens derived from
different targets
For this example, a circular RNA encodes two polypeptide immunogens derived
from two
different proteins that identify different targets from one another . The
circular RNA is designed with a
start-codon, expression sequences, stagger element(s), and an IRES (FIG. 1B).
Circularization enables
rolling circle translation of multiple expression sequences separated by a
stagger element.
A plurality of immunogens derived from different targets are encoded by the
circular RNA such
that is designed to have a start codon, a ORF encoding a polypeptide immunogen
derived from envelope
glycoprotein 1 (gP1) from Vance/la Zoster Virus, a stagger sequence, and a
polypeptide immunogen
derived from haemagglutinin. There are at least 6 envelope glycoproteins of
Vance/la Zoster Virus and
glycoproteins gP1, gP2, gP3 can induce the body to produce neutralizing
antibodies (Zweerink et al.
1981;31(1):436-444). Likewise, two envelope glycoproteins, haemagglutinin and
a fusion protein are
known immunogens of Morbiffivirus. Therefore, the circular RNA encoding a
polypeptide immunogen
derived from gP1 and a polypeptide immunogen derived from haemagglutinin both
targets Vance/la
Zoster Virus and Morbillivirus.
Example 8: Multi-immunogen administration from circular RNA
This example describes expression of multiple immunogens in a subject by
administrating
multiple circular RNA molecules.
In this Example, circular RNA 1 is designed to include an IRES (SEQ ID NO: 1)
followed by an
ORF encoding immunogen 1, corresponding to a portion of hemagglutinin (HA)
from a first strain of
Influenza A Hi Ni, A/California/07/2009 (SEQ ID NO: 2), a Stop codon and a
spacer (SEQ ID NO: 4), see
FIG. 2. Circular RNA 2 is designed to include an IRES (SEQ ID NO: 1) followed
by an ORF encoding
immunogen 2, corresponding to a portion of hemagglutinin (HA) from a second
strain of Influenza A
Hi Ni, A/Puerto Rico/8/1934 (SEQ ID NO: 3), a Stop codon, and a spacer (SEQ ID
NO: 4), see FIG. 3.
The circular RNAs are generated by in vitro transcription (Lucigen; AS3107)
and RNA ligation using a
RNA ligase as described by the methods provided herein.
Multiple circular RNAs encoding multiple different immunogens as described
herein are
formulated for administration to a mammalian subject.
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The circular RNAs are formulated in any of the formulations included herein.
These formulated
RNAs are injected via a suitable route, either intradermal, subcutaneous,
intramuscular, or intravenous
route at Day 0.
Secreted immunogen expression is evaluated in blood or tissues collected from
the mammalian
subjects. Blood samples are collected anti-coagulant free tubes, at 1, 2, 7,
14, and 21 days post-dosing.
Serum is isolated by centrifugation for 25 min at 1300 g at 4 C and secreted
protein expression is
measured by ELISA. Briefly, a capture antibody is coated onto ELISA plates
(MaxiSorp 442404, 96-well)
overnight at 40 in 100 I_ PBS. After washing three times with TBS-T, the
plates are blocked for 1 hour
with blocking buffer (TBS with 2% FBS and 0.05% Tween 20). Supernatant
dilutions are then added to
each well in 100 I_ blocking buffer and incubated at room temperature for 1
hour. After washing three
times with TBS-T, plates are incubated with HRP detection antibody for 1 hour
at room temperature.
Tetramethylbenzene (Pierce 34021) is added to each well, allowed to react for
5-15 minutes and then
quenched with 2N sulfuric acid. The optical density (OD) value is determined
at 450 nm.
Example 9: Co-administration of an immunogen and an adjuvant encoded by a
plurality of circular
RNAs
This example demonstrates administering a plurality of circular RNAs to a
subject to stimulate an
immune response. One of circular RNAs administered to a subject encodes a
polypeptide immunogen.
Another circular RNA administered to a subject encodes an adjuvant (FIG. 3).
In this example, circular RNA encoding a polypeptide immunogen is designed,
produced, purified,
and prepared as a formulation. To stimulate the immune response, a separate
circular RNA including a
polyU, which is known to act as an adjuvant by one of skill in the art is
designed, produced, purified, and
prepared as a pharmaceutical formulation. The formulations of the circular RNA
encoding the
polypeptide immunogen and the polyU adjuvant are administered to the subject
at the same time in
different formulations from one another.
Example 10: Co-administration of an immunogen encoded by a circular RNA and a
small molecule
adjuvant
This example demonstrates administering a circular RNA in combination with a
small molecule
adjuvant to a subject to stimulate an immune response.
In this example, circular RNA encoding a polypeptide immunogen is designed,
produced, purified,
and prepared as a formulation. To stimulate the immune response, a small
molecule adjuvant, such as
MF5 adjuvant, is administered to the subject. Both the formulation of
circular RNA encoding the
polypeptide immunogen and the small molecule adjuvant are administered to the
subject at the same
time to the subject.
Example 11: Co-administration of an immunogenic composition including a
plurality circular
RNAs each encoding a polypeptide immunogen corresponding to a different target
For this Example, a plurality of circular RNAs each encoding a polypeptide
immunogen are
administered to a subject (FIG. 3).
One circular RNA encoding a polypeptide immunogen derived from the envelope
protein
haemagglutinin as is known in the art to identify a Morbiffivirus target is
administered to a subject.
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Another circular RNA encoding a polypeptide immunogen derived from the
envelope protein glycoprotein
E that is known in the art to identify a Vance/la Zoster virus target is also
administered to a subject. Both
circular RNAs are designed, produced, purified, and prepared as a formulation.
The formulation including
both circular RNAs is administered to a subject.
Example 12: In vivo induction of an antibody against an immunogen in mammals
using circular
RNA
The circular polynucleotide encoding an immunogen as described herein is
formulated for
administration to the mammalian subject. The formulation is either in saline
or any of the formulations
taught herein. The vaccine containing the circular polynucleotide optionally
contains one or more
dendritic targeting agent or moieties. The vaccine comprising the
polynucleotide encoding the
immunogen is injected via a suitable route, either intradermal, subcutaneous,
intramuscular, or
intravenous route at Day 0. A polynucleotide encoding an immunostimulatory
agent or moiety can be co-
administered with the polynucleotide encoding the immunogen to stimulate
immune response. Additional
challenges of the vaccine containing the circular polynucleotide encoding the
immunogen are given on a
weekly, bi-weekly, every three week, every four week, every five week, every
six week, every seven
week, or every eight week basis until detection of an antibody against the
immunogen. Additional vaccine
challenges are administered to boost the production of immunogen specific
antibodies.
Example 13: Detecting Expression of a protein or immunogen from circular RNA
in mammalian
cells
To measure expression efficiency of non-secreted proteins or immunogens from
the RNA
constructs, circular RNA (0.1 picomole) encoding a protein or immunogen is
produced and purified
according to the methods described herein. Circular RNA is transfected into
HEK293 (10,000 cells per
well in a 96 well plate in serum-free media) using MessengerMax (Invitrogen,
LMRNA).
For a non-secreted protein or immunogen, protein expression is measured using
an immunogen-
specific ELISA at 24, 48, and 72 hours. To measure expression, cells are lysed
in each well at the
appropriate timepoint, using a lysis buffer and a protease inhibitor. The cell
lysate is retrieved and
centrifuged at 12,000 rpm for 10 minutes. Supernatant is collected.
For a secreted protein or immunogen, immunogen expression is detected using an
immunogen-
specific Western blot at 24, 48, and 72 hours. Briefly, 80 ut of supernatant
from mammalian cells is
taken from each well. Protein levels in harvested media is measured by BCA
protein assay method and
same amount of protein is resolved on 4%-12% gradient Bis-Tris gel (Thermo
Fisher Scientific) and
transferred to nitrocellulose membrane using by iBlot2 transfer system (Thermo
Fisher Scientific). Anti-
immunogen antibody (Sino Biological) is used to detect the immunogen. The
chemiluminescence signal
from protein bands is monitored by iBright FL1500 imaging system (Invitrogen).
Example 14: Expression of RBD immunogen from circular RNA in mammalian cells
This example demonstrates expression of RBD immunogens from circular RNA in
mammalian
cells.
In this example, circular RNAs encoding SARS-CoV-2 RBD immunogens were
produced and
purified according to the methods described herein.
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The expression of RBD-encoding circular RNA was tested by immunoprecipitation
coupled with
Western blot (IP-Western). Briefly, circular RNA encoding an RBD immunogen
(0.1 picomoles) was
transfected into BJ Fibroblasts and HeLa cells (10,000 cells) using
Lipofectamine MessengerMax
(ThermoFisher, LMRNA015). MessengerMax alone was used as a control.
Supernatant was collected at
24 hours and immunoprecipitation was performed with a rabbit antibody specific
to the SARS-CoV-2
RBD-Spike Glycoprotein (Sino Biologicals, Cat: 40592-T62) coupled to Protein G-
Dynabeads (Invitrogen,
10003D) and the same antibody was used to detect the immunoprecipitated
products resolved by PAGE.
A recombinant RBD (42 ng) Immunoprecipitation was used as control and to
quantify cell protein
expression. Membrane chemiluminescence was quantified using the Image StudioTM
Lite western blot
quantification software (Li-COR Biosciences).
RBD immunogen encoded by circular RNA was detected in BJ Fibroblast and HeLa
cell
supernatants and not in the controls (FIG. 4).
This example shows that SAR-CoV-2 RBD immunogens (which are secreted proteins)
were
expressed from circular RNA in mammalian cells.
Example 15: Immunogenicity of SARS-CoV-2 RBD immunogens in mouse model
The immunogenicity of a circular RNA encoding a SARS-CoV-2 RBD immunogen,
formulated
with a cationic polymer (e.g., protamine), was evaluated in a mouse model.
Production of antibodies to a
SARS-CoV-2 RBD immunogen, formulated with the cationic polymer, was also
evaluated in the mouse
model.
In this example, circular RNA was designed with an IRES and ORF encoding a
SARS-CoV-2
RBD immunogen and two spacer elements flanking the IRES-ORF. Circular RNAs
were generated as
follows. Unmodified linear RNA was synthesized by in vitro transcription with
an excess of guanosine 5'
monophosphate using T7 RNA polymerase from a DNA segment. Transcribed RNA was
purified with an
.. RNA purification system (New England Biolabs, Inc.) following the
manufacturer's instructions. Purified
linear RNA was circularized using a splint DNA.
Circular RNA was generated by split-ligation as follows: Transcribed linear
RNA and a DNA splint
were mixed and annealed and treated with an RNA ligase. To purify the circular
RNAs, ligation mixtures
were resolved by reverse-phase chromatography. Circular RNA was selectively
eluted from linear RNA
by increasing the organic content of the mobile phase. Eluted RNA was
fractionally collected and
assayed for circular RNA purity. Selected fractions were combined and buffer
exchanged to remove
mobile phase salts and solvents. Acrylamide gel electrophoresis was used as a
quality control
measurement for validating purity and circularization.
The purified circular RNA was diluted in pure water to a concentration of 1100
ng/pL. Protamine
sulfate was dissolved in Ringer's lactate solution (4000 ng/pL). While
stirring, the protamine-Ringer
lactate solution was added to half of the circular RNA solution until a weight
ratio of RNA:protamine is 2:1.
The solution was stirred for another 10 minutes to ensure the formation of
stable complexes. The
remaining circular RNA was then added (i.e., remaining circular RNA to
circular RNA:protamine solution)
and the solution stirred briefly. The final concentration of the mixture
(i.e., circular RNA mixture) was
adjusted using Ringer's lactate solution to obtain a circular RNA preparation
with a final RNA
concentration of 2 ug or 10 ug of RNA in 50 pL.
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Three mice per group were vaccinated intramuscularly or intradermally with a 2
ug or 10 ug dose
of the circular RNA preparation, or a protamine vehicle control at day 0 and
day 21. Addavax TM adjuvant
(Invivogen) was administered once to each mouse, intramuscularly or
intradermally, 24 hours after
administration of the circular RNA preparation at day 0 and day 21. Addavax TM
adjuvant was dosed at
50% in 1X PBS in 50 I_ following to the manufacturer's instructions.
Blood collection from each mouse was by submolar drawing. Blood was collected
into dry-
anticoagulant free-tubes, at day 7, 14, 21, 23, 28, 35, 41, 49, 56, 63, 69,
77, 84, 108 and 115 days post-
dosing of the circular RNA. Serum was separated from whole blood by
centrifugation at 1200g for 30
minutes at 4 C. The serum was heat-inactivated by heating at 56 C for 1 hour.
Individual heat-
inactivated serum samples were assayed for the presence of RBD-specific IgG by
enzyme-linked
immunosorbent assay (ELISA). ELISA plates (MaxiSorp 442404 96-well, Nunc) were
coated overnight at
4 C with SARS-CoV-2 RBD (Sino Biological, 40592-VO8B; 100 ng) in 100 I_ PBS.
The plates were then
blocked for 1 hour with blocking buffer (TBS with 2% FBS and 0.05% Tween 20).
Serum dilutions were
then added to each well in 100 I_ blocking buffer and incubated at room
temperature for 1 hour. After
washing three times with 1X Tris-buffered saline with Tween detergent (TBS-
T), plates were incubated
with anti-mouse IgG HRP detection antibody (Jackson 115-035-071) for 1 hour
followed by three washes
with TBS-T, then addition of tetramethylbenzene (Pierce 34021). The ELISA
plate was allowed to react
for 5 min and then quenched using 2N sulfuric acid. The optical density (OD)
value was determined at
450 nm.
The optical density of each serum sample was divided by that of the background
(plates coated
with RBD, incubated only with secondary antibody). The fold over background of
each sample was
plotted.
The results showed that anti-RBD responses were obtained at days 14, 21, 23,
28, 35, 41, 49,
56, 63, 69, 77, 84, 108 and 115 after injection with the circular RNA
preparations (FIG. 5). Anti-RBD
antibodies were not obtained after injection with the protamine vehicle. These
results showed that
circular RNA encoding the RBD immunogen induced an antigen-specific immune
response in mice.
A similar ELISA was used to assay serum samples for the presence of Spike-
specific IgG. ELISA
plates (MaxiSorp 442404 96-well, Nunc) were coated overnight at 4 C with SARS-
CoV-2 Spike (Sino
Biological, 40589-VO8B1; 100 ng) in 100 I_ PBS. The plates were then blocked
for 1 hour with blocking
buffer (TBS with 2% FBS and 0.05% Tween 20). Serum dilutions were then added
to each well in 100 I_
blocking buffer and incubated at room temperature for 1 hour. After washing
three times with 1X Tris-
buffered saline with Tween detergent (TBS-T), plates were incubated with anti-
mouse IgG HRP
detection antibody (Jackson 115-035-071) for 1 hour followed by three washes
with TBS-T, then addition
of tetramethylbenzene (Pierce 34021). The ELISA plate was allowed to react for
5 min and then
quenched using 2N sulfuric acid. The optical density (OD) value was determined
at 450 nm.
The results showed that anti-Spike antibodies were obtained at 35 days after
injection with the
circular RNA preparations (FIG. 6). Anti-Spike antibodies were not obtained
after injection with vehicle.
Serum antibodies at day 14 post-dosing were characterized using an assay to
measure relative
IgG1 vs IgG2a isotypes (FIG. 7), and the ability of serum antibodies to
neutralize the virus was
characterized using a PRNT neutralization assay. The results showed that 2 g
RBD circular RNA dosed
intramuscularly with adjuvant had neutralizing ability.
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Example 16: Formulation of circular RNA for administration to non-human animal
Circular RNAs encoding a SARS-CoV-2 immunogen or Gaussia Luciferase (GLuc), as
a model
immunogen, were designed and purified according to the methods described
herein. After purification,
the circular RNA or mRNA was formulated as follows:
A. circular RNA or mRNA was diluted in PBS to a final concentration of 2.5 or
25 picomoles in 50
L, to obtain a circular or a linear RNA preparation (unformulated).
B. circular RNA or mRNA was formulated with a lipid carrier (e.g., TransIT
(Mirus Bio)) and
mRNA Boost Reagent (Mirus Bio) according to the manufacturer's instructions
(15% TransIT, 5% Boost)
to obtain a final RNA concentration of 2.5 or 25 picomoles in 50 L, to obtain
a circular or a linear RNA
preparation.
C. circular RNA or mRNA was formulated with a cationic polymer (e.g.,
protamine). Briefly,
circular RNA was diluted in pure water. Protamine sulfate was dissolved in
Ringer lactate solution (4000
ng/ L). While stirring, the protamine-Ringer lactate solution was added to
half of the circular RNA or
mRNA solution until a weight ratio of RNA:protamine is 2:1. The solution was
stirred for another 10
minutes to ensure the formation of stable complexes. The remaining circular
RNA or mRNA was then
added, and the solution stirred briefly. The final concentration of the
solution was adjusted using Ringer
lactate solution such that the final concentration of circular RNA or linear
RNA preparation was 2.5 or 25
picomoles in per 50 L.
D. circular RNA or mRNA was formulated with a lipid nanoparticle. Briefly,
circular RNA or mRNA
was diluted in 25 mM acetate buffer pH=4 (filtered through 0.2 um filter) to a
concentration of 0.2 ug/uL.
Lipid nanoparticles (LNPs) were formulated by first dissolving the ionizable
lipid (e.g. ALC0315),
cholesterol, DSPC, and DMG-PEG2000 in ethanol (filtered through 0.2 um sterile
filter) in a molar ratio of
50/38.5/10/1.5 mol %. The final ionizable lipid / RNA weight ratio was 8/1
w/w. The lipid and RNA
solutions were mixed in a micromixer chip using microfluidics system with a
flow rate ratio of 3/1 buffer /
ethanol and a total flow rate of 1 ml/min. The LNPs were then dialyzed in PBS
pH=7.4 for 3 h to remove
ethanol. The RNA concentration inside the LNPs and the encapsulation
efficiency were measured using
Ribogreen assay. If necessary, the LNPs were concentrated down to the desired
RNA concentration
using Amicon centrifugation filters, 100 kDa cut off. The size, concentration,
and charge of the particles
were measured using Zetasizer Ultra (Malvern Pananaytical). The RNA
concentration was adjusted with
PBS to a final concentration of 0.1 or 0.2 ug/ul. For formulations containing
two RNA sequences the
RNAs were either mixed before formulating in LNPs or after each RNA was
formulated separately. For in
vivo experiments, the final RNA formulated in LNPs were filtered through
sterile 0.2 um regenerated
cellulose filters.
Example 17: Modulation of in vivo production of Gaussia Luciferase from
circular RNA in mice
using timed of adjuvant delivery
This example demonstrates the expression of proteins or immunogens from
circular RNA in vivo
whilst also delivering an adjuvant to stimulate an immune response.
In this example, circular RNA encoding GLuc was produced and purified
according to the
methods described herein. Circular RNAs were formulated as described in
Example 16 to obtain circular
RNA preparations (e.g., Trans-IT formulated, protamine formulated,
PBS/unformulated). Mice were
administered 50 L injections of each circular RNA preparation via either a
single intramuscular injection
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in a hind leg.. Another group of mice were administered a protamine formulated
circular RNA preparation
intradermally by single intradermal injection to the back.
To stimulate the immune response, AddavaxTM adjuvant (Invivogen), which is a
squalene-based
oil-in-water nano-emulsion with a formulation similar to MF59 adjuvant, was
injected into the mouse hind
leg at 0 hours (simultaneous delivery with a circular RNA preparation) or at
24 hours. AddavaxTM adjuvant
was dosed at 50 ut according to the manufacturer's instructions.
Blood samples (-25 L) were collected from each mouse by submolar drawing.
Blood was
collected into EDTA tubes, at 0, 6, 24 and 48 hours post-dosing of the
circular RNA. Plasma was isolated
by centrifugation for 30 minutes at 1300 g at 4 C and the activity of Gaussia
Luciferase, a secreted
enzyme, was tested using a Gaussia Luciferase activity assay (Thermo
Scientific Pierce). 50 .1_ of lx
GLuc substrate was added to 5 jL of plasma to carry out the GLuc luciferase
activity assay. Plates were
read immediately after mixing in a luminometer instrument (Promega).
This example demonstrated successful protein expression from circular RNA in
vivo for
prolonged periods of time using: (a) intramuscular injection of TransIT
formulated, protamine formulated
and unformulated circular RNA preparations without adjuvant (FIG. 8), and with
adjuvant delivered at 0
and 24 h (FIG. 9); and (b) intradermal injection of protamine formulated
circular RNA preparation without
adjuvant, and with adjuvant delivered at 24 h (FIG. 10).
Example 18: Characterization of a circular RNA preparation by assessing RNAse
H-produced
nucleic acid degradation products
This example demonstrates that assessment of a circular RNA preparation for
RNAse H-
produced nucleic acid degradation products can detect linear and
concatemerized versus circular
products.
RNA, when incubated with a ligase, can either not react or form an intra- or
intermolecular bond,
generating a circular (no free ends) or a concatemeric RNA (linear),
respectively. Treatment of each type
of RNA with a complementary DNA primer and RNAse H, a nonspecific endonuclease
that recognizes
DNA/RNA duplexes, is expected to produce a unique number of degradation
products of specific sizes
depending on the starting RNA material.
A ligated RNA may be shown to be circular RNA without concatemeric RNA
contamination or
circular RNA with concatemeric RNA contamination, based on the number and size
of RNAs produced by
RNAse H degradation. When the primer and RNase H are added to circular RNA, a
single primer
duplexes with the circular RNA and RNase H degrades the DNA/RNA duplex region
to result in a single
linear RNA product. When a primer and RNase H are added to a concatemer, at
least two primers
duplex with the concatemeric RNA and RNase H degrades the DNA/RNA duplexes to
result in three
.. products; one product is the RNA from the 5' end to the first primer
binding region, one product is the
RNA between the first primer binding region and the next primer binding region
which may include
multiple RNAs depending on the number of concatemers ligated together, and a
final product is the RNA
from the last primer binding region to the 3' end. When a primer and RNase H
are added to linear RNA, a
single primer duplexes with the linear RNA to result in one product for RNA
from the 5' end to the primer
binding region and another product for the primer binding region to the 3'
end. The left side cartoon of
FIG. 11 illustrates this strategy.
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In this example, circular RNA was generated as follows. Unmodified linear RNA
was synthesized
by in vitro transcription using T7 RNA polymerase from a DNA segment.
Transcribed RNA was purified
with an RNA purification system (New England Biolabs, Inc.), treated with RNA
5' Pyrophosphohydrolase
(RppH) (New England Biolabs, Inc., M0356) following the manufacturer's
instructions, and purified again
with the RNA purification system. Circular RNAs were designed to include an
IRES with an ORF
encoding Nanoluciferase (Nluc) and two spacer elements flanking the IRES-ORF.
To test circularization status of the RNA, 0.05 prnole/plof linear or circular
RNA preparation was
incubated with 0.25 U/plof RNAse H, an endoribonuclease that digests DNA/RNA
duplexes, and 0.3
prnole/ploligomer of 10 to 30 nucleic acids complementary to Nluc RNA at 37 C
for 30 min. After
incubation, the reaction mixture was analyzed by 6% denaturing PAGE. The gel
was stained with SYBR-
green and visualized by E-gel Imager. The band intensity on the visualized gel
was measured and
analyzed by ImageJ.
The right side of FIG. 11 shows the actual cleavage products in this
experiment. The number of
bands in the linear RNA lane incubated with RNAse H endonuclease produced two
bands as expected,
whereas a single band was detected in the circular RNA lane in the case of
lane A, indicating that the
circular RNA was in fact circular and not concatemeric. In the case of lane B
& lane C, bands from linear
and concatemer contamination were visible after RNase H treatment due to the
presence of multiple
smaller fragment bands appearing in the RNAse H lanes.
Example 19: Linear RNA in circular RNA preparations (standard curve)
This example demonstrates calculation of linear RNA in a circular RNA
preparation.
In this example, the circular RNA was generated as follows. Unmodified linear
RNA was
synthesized by in vitro transcription using T7 RNA polymerase from a DNA
segment. Transcribed RNA
was purified with an RNA purification system (New England Biolabs, Inc.),
treated with RNA 5'
Pyrophosphohydrolase (RppH) (New England Biolabs, Inc., M0356) following the
manufacturer's
instructions, and purified again with the RNA purification system. Circular
RNAs were designed to include
an IRES with an ORF encoding Nanoluciferase (Nluc) and two spacer elements
flanking the IRES-ORF.
Splint ligated circular RNA was generated by treatment of the transcribed
linear RNA and a DNA
splint with T4 DNA ligase 2 (New England Biolabs, Inc., M0239).
To purify the circular RNAs, ligation mixtures were resolved on 4% denaturing
PAGE and RNA
bands corresponding to each of the circular RNAs were excised. Excised RNA gel
fragments were
crushed, and RNA was eluted with gel elution buffer (0.5M Na0Ac, 1mM EDTA and
0.1% SDS) for an
hour at 37C. Supernatant was harvested, and RNA was eluted once again by
adding gel elution buffer
to the crushed gel and incubated for an hour. Gel debris was removed by
centrifuge filters and RNA was
precipitated with ethanol. Eluted circular RNA was analyzed by 6% denaturing
PAGE. The gel was
stained with SYBR-green and visualized by E-gel Imager.
The amount of RNA on the gel was determined by comparing the band intensity of
known amount
and same size of RNA (standard RNA). A standard curve was generated to
determine the amount of
unknown sample on the gel (FIG. 12). To generate a standard curve 1, 0.5, 0.2
and 0.05 pmoles of linear
counterpart of the circular RNA were loaded parallel with a circular RNA
preparation on a 6% denaturing
PAGE. The denaturing gel was stained with SYBR-green and visualized by E-gel
Imager. Then each
band intensity on the gel was measured and analyzed by ImageJ. The standard
curve for linear RNA
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was generated through analysis of band intensity of the RNA loaded in each of
the different lanes
(R2>0.98 in all cases), and the amount of linear RNA in the circular RNA
preparation was determined
based on the linear RNA standard curve.
The amount of linear RNA in the different samples was as follows for circular
RNA preparation A:
linear RNA was calculated to be approximately 0.31 mole/mole, or 115.99 ng/395
ng, or 30.2%. For
circular RNA preparation C: linear RNA was calculated to be approximately 0.45
mole/mole, or 260.52
ng/488 ng, or 49.2%.
Example 20: Linear RNA in circular RNA preparations (gel excision and
extraction)
This example demonstrates purification and quantification of circular RNA in a
preparation.
In this example, the circular RNA was generated as follows. Unmodified linear
RNA was
synthesized by in vitro transcription using T7 RNA polymerase from a DNA
segment. Transcribed RNA
was purified with an RNA purification system (New England Biolabs, Inc.),
treated with RNA 5'
Pyrophosphohydrolase (New England Biolabs, Inc., M0356) following the
manufacturer's instructions, and
purified again with the RNA purification system. Circular RNAs were designed
to include an IRES with an
ORF encoding Nanoluciferase (Nluc) and two spacer elements flanking the IRES-
ORF.
Splint ligated circular RNA was generated by treatment of the transcribed
linear RNA and a DNA
splint with T4 DNA ligase 2 (New England Biolabs, Inc., M0239).
To purify the circular RNAs, ligation mixtures were resolved on 6% denaturing
PAGE and RNA
bands corresponding to each of the circular RNAs were excised. Excised RNA gel
fragments were
crushed, and RNA was eluted with gel elution buffer (0.5M Na0Ac, 1mM EDTA and
0.1% SDS) for an
hour at 37 C. Supernatant was harvested, and RNA was eluted once again by
adding gel elution buffer to
the crushed gel and incubated for an hour. Gel debris was removed by
centrifuge filters and RNA was
precipitated with ethanol in the presence of 0.3M sodium acetate. Eluted
circular RNA was analyzed by
6% denaturing PAGE. The gel was stained with SYBR-green and visualized by E-
gel Imager (FIG. 13).
Visible bands were again excised and crushed using by gel breaker. To extract
RNA, gel elution buffer
(0.5M Na0Ac, 1mM EDTA, 0.1% SDS) was added to the crushed gel and incubated
for lhr at 37C.
Supernatant was harvested, and RNA was eluted once again by adding gel elution
buffer to the crushed
gel and incubated for an hour. Gel debris was removed by spin X column and
extracted RNA was
ethanol precipitated. Extracted RNA from individual band was measured by
0ubit3 fluorometer.
The extracted RNA from the different samples were quantified as follows:
(Preparation A)
approximately 1446 ng circular RNA and 176 ng linear RNA (89.1% circular RNA);
(Preparation B)
approximately 934 ng circular RNA and 270 ng linear RNA (77.5% circular RNA);
and (Preparation C)
approximately 320 ng circular RNA and 396 ng linear RNA (44.6% circular RNA).
Example 21: Linear RNA in circular RNA preparations
This example demonstrates production of 91% (w/w) pure circular RNA molecules
relative to the
total ribonucleotide molecules in the preparation and subsequent dosing in
mice to generate a biological
effect.
In this example, circular RNAs included an IRES, an ORF encoding Gaussia
Luciferase (GLuc),
and two spacer elements flanking the IRES-ORF.
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In this example, the circular RNA was generated in vitro. Unmodified linear
RNA was transcribed
in vitro from a DNA template including all the motifs listed above, as well as
a T7 RNA polymerase
promoter to drive transcription. Transcribed RNA was purified with an RNA
cleanup kit (New England
Biolabs, T2050), treated with RNA 5'phosphohydrolase (RppH) (New England
Biolabs, M0356) following
the manufacturer's instructions, and purified again with an RNA purification
column. RppH treated linear
RNA was circularized using a splint DNA between 10 and 40 nucleotides in
length and T4 RNA ligase 2
(New England Biolabs, M0239). Circular RNA was Urea-PAGE purified, eluted in a
buffer (0.5 M Sodium
Acetate, 0.1% SDS, 1 mM EDTA), ethanol precipitated and resuspended in RNA
storage solution
(ThermoFisher Scientific, cat# AM7000).
Linear polyribonucleotides remained in the final circular RNA product
preparation. The purity of
circular RNA and percentage of remaining linear RNA in the final product
preparation was quantified for
each batch by running final product preparations on 6% TBE-urea gels and
analyzing bands using
ImageJ. Purity was assessed by calculating the intensity of circular RNA
compared to the total RNA
intensity and noted as a percentage. Here, circular RNA was of 91% (w/w)
purity relative to the total RNA
in the preparation.
Prior to administration, PBS and 10% TransIT carrier were added to achieve the
desired final
circular RNA concentration of 0.25 pmole in a 100 ut final volume. Mice
received a single intravenous
tail-vein injection of 0.25 pmole of the circular RNA encoding the Gaussia
Luciferase ORF (100 L).
Blood samples (-25 L) was collected from each mouse by submolar drawing.
Blood was
collected into EDTA tubes, at 0, 6 hours, 1, 2, 3, 7, 14, 21, 28 and 35 days
post-dosing. Plasma was
isolated by centrifugation for 30 minutes at 1300 g at 4 C and the activity of
Gaussia Luciferase, a
secreted enzyme, was tested using a Gaussia Luciferase activity assay (Thermo
Scientific Pierce).
Briefly, 50 I_ of lx GLuc substrate was added to 5 I_ of plasma to carry out
the GLuc luciferase activity
assay. Plates were read immediately after mixing in a luminometer instrument
(Promega).
Gaussia Luciferase activity was detected in plasma at 6 hours and 1, 2, 3, 7,
14, and 21 days
post-dosing of circular RNA. Highest expression of circular RNA was observed
approximately 2 days
post-injection and high levels of expression were maintained for prolonged
periods of time and was still
detectable at 21 days. At all timepoints, these levels of activity were
greater than those observed for the
negative control (PBS vehicle only).
This example demonstrates that circular RNA of 91% (w/w) purity relative to
the total RNA in the
preparation was successfully produced, successfully delivered via intravenous
injection and was able to
express protein detectable in blood for prolonged periods of time.
Example 22: Quantification of nicked circular RNA in gel-purified circular RNA
This example demonstrates that circular RNA by purified by gel extraction
contains no more than
1.1% (w/w) nicked RNA relative to the total RNA molecules in the preparation.
In this example, RNAs included an IRES, an ORF encoding Gaussia Luciferase
(GLuc), and two
spacer elements flanking the IRES-ORF.
In this example, the circular RNA was generated in vitro. Unmodified linear
RNA was transcribed
in vitro from a DNA template including all the motifs listed above, as well as
a T7 RNA polymerase
promoter to drive transcription. Transcribed RNA was purified with an RNA
cleanup kit (New England
Biolabs, T2050), treated with RNA 5'phosphohydrolase (RppH) (New England
Biolabs, M0356) following
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the manufacturer's instructions, and purified again with an RNA purification
column. RppH-treated linear
RNA was circularized using a splint DNA between 10 and 40 nucleotides in
length and T4 RNA ligase 2
(New England Biolabs, M0239). This results in circularization of the RNA at
the ligation junction to
generate circular RNA. Circular RNA was Urea-PAGE purified on a 4% PAGE gel,
eluted in a buffer (0.5
M Sodium Acetate, 0.1% SDS, 1 mM EDTA), ethanol precipitated and resuspended
in RNA storage
solution (ThermoFisher Scientific, cat# AM7000). In this example, purified
circular RNA was evaluated to
have a purity of 80% (w/w) relative to the total RNA in the preparation.
In this example, the sequence of linear RNAs was assessed by next generation
sequencing. The
purified circular RNA preparation (80% purity) was prepared for the NGS
pipeline using the library
preparation method described in TruSeq Small RNA Workflow (IIlumina, RS-200-
0012). This method
preserved 3' end identity with high fidelity. Briefly, adapters were ligated
to any RNA molecules with
available 3' or 5' ends in the solution. Intact circular RNA will not undergo
this ligation ¨ as a result, this
step selected for non-circular RNA only. These products are then reverse
transcribed and amplified to
generate cDNA libraries which were subsequently purified, quality-controlled
and multiplexed. Libraries
then underwent sequencing on an IIlumina Miniseq machine.
In a similar manner to that described above, linear RNA product from in vitro
transcription after
RppH-treatment was processed for sequencing.
Sequencing results of both the linear RNA product of IVT and non-circular RNA
in the gel-purified
circular RNA preparation were compared by mapping reads back to the template
sequence used to
generate the circular RNA and evaluating the number of reads that map over the
ligation junction.
In this analysis, the non-circular RNA that remains is assumed to be a mixture
of nicked RNA and
residual linear RNA product from IVT. In this example, if the non-circular RNA
is assumed to comprise
only nicked RNA, the percentage of fragments that map over the ligation
junction is expected to be 50%.
If the non-circular RNA is assumed to comprise only residual linear RNA
product from IVT, the
percentage of fragments that maps over the ligation junction is expected to be
0%. Using these statistical
assumptions and control experiments, a standard curve was generated that
enabled quantification of
nicked RNA. This yielded a calculation of 5.4% of non-circular RNA as nicked
RNA.
This example demonstrates that 5.4% (w/w) of the non-circular RNA fraction of
a purified circular
RNA preparation (equivalent to 1.1% (w/w) of the total RNA) was nicked RNA.
Example 23: Reduction of linear RNA present in circular RNA preparations
increased expression
of the encoded protein
This example demonstrates that reducing linear RNA present in a predominantly
circular RNA
composition increased expression of the encoded protein in cells.
For this example, circular RNAs included an IRES, an ORF encoding Gaussia
Luciferase (GLuc),
and two spacer elements flanking the IRES-ORF.
Two batches of circular RNA were generated. In each case, the circular RNA was
generated in
vitro. Unmodified linear RNA was transcribed in vitro from a DNA template
including all the motifs listed
above, as well as a T7 RNA polymerase promoter to drive transcription.
Transcribed RNA was purified
with an RNA cleanup kit (New England Biolabs, T2050), treated with RNA
5'phosphohydrolase (RppH)
(New England Biolabs, M0356) following the manufacturer's instructions, and
purified again with an RNA
purification column. RppH treated linear RNA was circularized using a splint
DNA between 10 and 40
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nucleotides in length and T4 RNA ligase 2 (New England Biolabs, M0239).
Circular RNA was Urea-
PAGE purified, eluted in a buffer (0.5 M Sodium Acetate, 0.1% SDS, 1 mM EDTA),
ethanol precipitated,
and resuspended in RNA storage solution (ThermoFisher Scientific, cat#
AM7000).
Linear RNA remained in the final circular RNA product. The purity of circular
RNA and
percentage of remaining linear RNA in the final product was quantified for
each batch by running final
products on 6% TBE-urea gels and analyzing bands using ImageJ. Purity of
circRNA was assessed by
calculating the intensity of circRNA compared to the total RNA intensity and
noted as a percentage.
Here, batches were of 71% purity and 84% purity.
0.2 pmoles of each RNA batch was used for cell transfections. RNA was combined
with
Optimem and Messenger Max according to the manufacturer's recommendations. A
vehicle only control
was similarly prepared but did not contain any RNA. At time = 0, each
preparation was added to BJ
fibroblast cells.
The activity of Gaussia Luciferase was tested using a Gaussia Luciferase
Activity assay (Thermo
Scientific Pierce). Samples of 20 I_ of the cell supernatant were added to a
96 well plate (Corning 3990).
Samples were taken at 6, 24, 48, 72, 96 and 120 hours after transfection. In
brief, lx coelenterazine
substrate was added to each well. Plates were read immediately after substrate
addition and mixing in a
luminometer instrument (Promega).
Gaussia Luciferase activity was detected in cells in experiments using
circular RNA of 84% purity
at 6, 24, 48, 72, 96, and 120 hours post-transfection (FIG. 12) and was higher
than the expression
afforded by the vehicle only control. Expression derived from circular RNA of
84% purity was significantly
higher than the expression derived from circular RNA of 71% purity. At 24
hours post-transfection, GLuc
activity was 397-fold greater when circular RNA of 84% purity was transfected
compared to when circular
RNA of 71% purity was transfected. GLuc activity was detected in cells in
experiments using circular
RNA of 71% purity at 6, 24, 48, and 72 hours post-transfection (FIG. 14) and
was higher than the
expression afforded by the vehicle only control.
This example demonstrated that circular RNA of greater purity (with reduced
linear RNA)
increases and prolongs expression of the encoded protein.
Example 24: Linear RNA present in circular RNA preparations affected innate
immune response in
a dose dependent manner in cells
This example demonstrates that linear RNA present in a circular RNA
preparation negatively
affected innate immune response in a dose dependent manner.
In this example, circular RNA and linear RNA were generated as follows.
Unmodified linear RNA
was synthesized by in vitro transcription using T7 RNA polymerase from a DNA
segment. Transcribed
RNA was purified with an RNA purification system (New England Biolabs, Inc.),
treated with RNA 5'
Pyrophosphohydrolase (RppH) (New England Biolabs, Inc., M0356) following the
manufacturer's
instructions, and purified again with the RNA purification system. Circular
RNAs were designed to include
an IRES with an ORF encoding Gaussia luciferase (GLuc) and two spacer elements
flanking the IRES-
ORF.
Splint ligated circular RNA was generated by treatment of the transcribed
linear RNA and a DNA
splint with T4 RNA ligase 2 (New England Biolabs, Inc., M0239).
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To purify the circular RNAs, ligation mixtures were resolved on 4% denaturing
PAGE and RNA
bands corresponding to each of the circular and linear RNAs were excised. The
linear RNAs were
purified using the same 4% denaturing PAGE gel. Excised RNA gel fragments
(linear or circular) were
crushed, and RNA was eluted with gel elution buffer (0.5M Na0Ac, 1mM EDTA and
0.1% SDS) for an
hour at 37C. Supernatant was harvested, and RNA was eluted once again by
adding gel elution buffer
to the crushed gel and incubated for an hour. Gel debris was removed by
centrifuge filters and RNA was
precipitated with ethanol.
The impact of varying levels of linear RNA counterparts in preparations of gel
purified circular
RNA was determined by monitoring circular RNA levels in BJ fibroblast cells.
Cells in a 96-well plate
were suspension (reverse) transfected with either a gel purified circular RNA
preparation, or an equal
amount of gel purified circular RNA preparation but supplemented with varying
levels of gel purified linear
RNA using a lipid-based transfection reagent (ThermoFisher Scientific
(LMRNA003). Immune genes
levels were analyzed by Q-PCR at 6 hrs and 1-5 days post-transfection. In
brief, cDNA was generated
from cell lysates by random priming using the Power SYBR Green cells to CT kit
(ThermoFisher Scientific,
cat # 4402953) and following manufacturer's instructions. Q-PCR was performed
using immune gene
specific primers, and relative RNA levels were calculated using the Pfaffl
method and 13-Actin as the
housekeeping gene.
Circular RNA in cells transfected with the gel purified circular RNA
preparation alone showed
limited increased expression of innate immune genes. Conversely cells
transfected with the combined
circular and linear RNAs, demonstrated upregulation of innate immune genes in
a dose dependent
manner (FIG. 15).
Example 25: Rolling circle translation of synthetic circular RNA produced
discrete protein
products in cells
This example demonstrates discrete protein or immunogen products were
translated via rolling
circle translation from synthetic circular RNA lacking a termination element
(stop codon), e.g., having a
stagger element in lieu of a termination element (stop codon), in cells.
Additionally, this example shows
that circular RNA with a stagger element expressed more protein or immunogen
product having the
correct molecular weight than its linear counterpart.
Circular RNAs were designed to include a nanoluciferase gene (nLUC) with a
stagger element in
place of a termination element (stop codon). Cells were transfected with
vehicle: transfection reagent
only; linear nLUC: EMCV IRES, stagger element (2A sequence), 3x FLAG tagged
nLuc sequences, and a
stagger element (2A sequence); or circular nLUC: EMCV IRES, stagger element
(2A sequence), 3x
FLAG tagged nLuc sequences, and a stagger element (2A sequence). As shown in
the FIG. 16, circular
RNA produced greater levels of protein having the correct molecular weight as
compared to linear RNA.
After 24hrs, cells were harvested by adding 100 I of RI PA buffer. After
centrifugation at 1400xg
for 5min, the supernatant was analyzed on a 10-20% gradient polyacrylamide/SDS
gel.
After being electrotransferred to a nitrocellulose membrane using dry transfer
method, the blot
was incubated with an anti-FLAG antibody and anti-mouse IgG peroxidase. The
blot was visualized with
an ECL kit and western blot band intensity was measured by ImageJ.
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As shown in FIG. 16, circular RNA translation product was detected in cells.
In particular, circular
RNA without a termination element (stop codon) produced higher levels of
discrete protein product having
the correct molecular weight than its linear RNA counterpart.
Example 26: Preparation of circular RNA with regulatory nucleic acid sites
This example demonstrates in vitro production of circular RNA with a
regulatory RNA binding site.
Different cell types possess unique nucleic acid regulatory machinery to
target specific RNA
sequences. Encoding these specific sequences in a circular RNA could confer
unique properties in
different cell types. As shown in the following example, circular RNA was
engineered to encode a
microRNA binding site.
In this example, circular RNA included a sequence encoding a WT EMCV IRES, a
mir692
microRNA binding site, and two spacer elements flanking the IRES-ORF.
The circular RNA was generated in vitro. Unmodified linear RNA was in vitro
transcribed from a
DNA template including all the motifs listed above, in addition to the T7 RNA
polymerase promoter to
drive transcription. Transcribed RNA was purified with an RNA cleanup kit (New
England Biolabs,
T2050), treated with RNA 5'-phosphohydrolase (RppH) (New England Biolabs,
M0356) following the
manufacturer's instructions, and purified again with an RNA purification
column. RppH treated RNA was
circularized using a splint DNA of 10 to 40 nucleotides in length and T4 RNA
ligase 2 (New England
Biolabs, M0239). Circular RNA was Urea-PAGE purified (FIG. 17), eluted in a
buffer (0.5M Sodium
Acetate, 0.1% SDS, 1mM EDTA), ethanol precipitated and resuspended in RNase
free water.
As shown in FIG. 17, circular RNA was generated with a miRNA binding site.
Example 27: Formulation of RNA for topical delivery
This example demonstrates formulation of RNA for topical delivery.
To determine topical effects of RNA, RNA was formulated for delivery to
epithelial tissues.
As described herein, RNA was formulated with a cell-penetrating agent
according to the
following:
lOng RNA linear or circular comprising an EGF ORF
5 pt 80% ethanol
35 pt PBS+glucose (4.5g/L)
Example 28: Topical delivery of circular RNA
This example demonstrates topical delivery of RNA.
To determine topical delivery effects, RNA was formulated and delivered to
epithelial tissues. As
described herein, RNA was formulated with a cell-penetrating agent and
delivered topically to ear tissue.
Samples of circular RNA were formulated as in Example 27 (50 L) and applied to
both ears of a
mouse. Ears were wiped with an isopropyl alcohol wipe prior to application of
the samples to the ears.
Samples were dried on the ears by exposing the ears briefly to a heat lamp and
fan in a sterilized hood.
Mice were placed back in cages under normal conditions.
At select timepoints (6 hrs, lday, 3 days or 12 days post application), ear
tissues (through a
single ear punch) were collected for each RNA sample.
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Example 29: Persistence of RNA after topical delivery
This example demonstrates RNA presence after topical delivery.
To determine RNA persistence, tissue samples were analyzed for delivered RNA.
As described
herein, ear punches were analyzed for persistence at varying timepoints after
topical delivery of the RNA.
Ear punch samples from Example 28 and untreated ear punch samples were
collected in an
RNA stabilization reagent and RNA was extracted using a standard RNA tissue
extraction kit (Maxwell
RSC simply RNA).
A volume of 200p1 of 1-Thioglycerol / Homogenization Solution was added to
each sample. A
working solution was prepared by adding 20p1 of 1-Thioglycerol per milliliter
of Homogenization Solution.
Alternatively, 600p1 of 1-Thioglycerol was added to the 30m1 bottle of
Homogenization Solution. Before
use, the 1-Thioglycerol/Homogenization Solution was chilled on ice or at 2-10
C.
The tissue samples were homogenized in 200p1 of chilled 1- Thioglycerol /
Homogenization
Solution with a handheld homogenizer and sterile pestle until no visible
tissue fragments remained. Each
sample was homogenized an additional 15-30 seconds for complete
homogenization.
To check for the presence of RNA at the different timepoints, the samples were
checked for RNA
via q-PCR. qPCR was used to measure the presence of both linear and circular
RNA in the ear punches.
To detect linear and circular RNA, primers that amplified the Nluc ORF were
used. To detect only circular
RNA, primers that amplified the 5'-3' junction allowed for detection of
circular but not linear RNA
constructs.
Linear and circular RNA was detected at 6 hrs, 24 hrs, and 72 hrs after
topical delivery. Higher
levels of circular RNA compared to linear RNA were detected in ears of mice at
3-days post-injection
(FIG. 18).
As shown in this example, linear and circular RNA administered topically were
detectable in vivo.
Example 30: Protein expression of mRNA after topical delivery
This example describes protein presence after topical delivery.
To determine if topical delivered RNA can be translated, tissue samples are
analyzed for protein
or immunogen expression at the different timepoints by western blot. Ear
punches are analyzed for
protein expression after topical delivery of the RNA.
In short, the ear punches are collected and stored in an RNA stabilization
reagent (Invitrogen).
The tissue is homogenized in RIPA buffer with micro tube homogenizer (Fisher
scientific) and protein is
extracted. Each sample is centrifuged at 14k x g for 15 mins.
The supernatant is removed, and the pellet is dissolved in 2x SDS sample
buffer (0.125 M Tris-
HC1, pH 6.8, 4% SDS, 30% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol
blue) at 70 C for
15 min.
A commercially available standard (BioRad) is used as the size marker. After
being
electrotransferred to a polyvinylidene fluoride (PVDF) membrane (Millipore)
using a semi-dry method, the
blot is visualized using a chemiluminescent kit (Rockland).
It is expected that the GFP protein is visualized in ear punch samples and is
detected in circular
RNA and linear RNA.
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Example 31: Topical administration of RNA results in RNA delivery to tissue
when ethanol is
included in the RNA solution
This example demonstrates the delivery of RNA to cells and tissues via topical
administration in
vivo when ethanol is included in the RNA solution.
In this example, circular RNA was designed with an EMCV IRES and ORF encoding
Nanoluciferase (NLuc).
The circular RNA was generated in vitro. Unmodified linear RNA was transcribed
in vitro from a
DNA template. Transcribed RNA was purified with an RNA cleanup kit (New
England Biolabs, T2050),
treated with RNA 5'phosphohydrolase (RppH) (New England Biolabs, M0356)
following the
manufacturer's instructions, and purified again with an RNA purification
column. RppH treated linear
RNA circularized using a splint DNA between 10 and 40 nucleotides in length
and T4 RNA ligase 2 (New
England Biolabs, M0239). Circular RNA was then Urea-PAGE purified, eluted in a
buffer (0.5 M Sodium
Acetate, 0.1% SDS, 1 mM EDTA), ethanol precipitated and resuspended in RNA
storage solution
(ThermoFisher Scientific, cat# AM7000). In this example, circular RNA was also
HPLC-purified.
In this example, linear mRNA was designed with an ORF encoding a Nano
Luciferase (NLuc). In
this example, modified linear mRNA was made in-house by in vitro
transcription. In this example, linear
RNA was fully substituted with Pseudo-Uridine and 5-Methyl-C, capped with
CleanCapTM AG, included 5'
and 3' human alpha-globin UTRs, and is polyadenylated.
RNA was then diluted in PBS/Glucose (4.5g/L) and ethanol (10% v/v) such that
total sample
volume for each sample was 50 L, and total RNA for each sample was 3.5
pmoles. All reagents are
brought to room temperature prior to mixing and mixtures are prepared
immediately prior to use.
At time = 0, 50 I_ dose of each sample was applied topically to the ear of a
BALB/c mouse
dropwise using a pipet tip. As a negative control, an untreated mouse was
used. Samples were dried by
exposing the ears briefly to a heat lamp and fan in a sterilized hood. Mice
were place back in cages
under normal conditions.
To determine RNA persistence in tissue, tissue samples were analyzed for RNA
at varying
timepoints after delivery using RT-qPCR. At 6 hours, 1, 3, and 12 days post-
administration, a 2 mm ear
punch was taken from each animal and stored in RNAlater solution (ThermoFisher
Scientific, cat#
AM7020). Total RNA was isolated from ear punches using trizol extraction. The
aqueous phase was
.. precipitated with isopropanol and the pellet was washed with 70% ETOH as
per manufacturer's
instructions. cDNA was synthesized from the total RNA and RT-PCR was performed
on cDNA templates
using primers specific to the NLuc ORF. All samples were assayed in triplicate
on the Bio-Rad CFX384
Thermal Cycler. RNA levels were then relativized to actin and to the untreated
negative control.
Circular RNA and linear RNA were detected in tissue samples at 6 hours and 1,
3 and 12 days
after topical administration and showed greater signal than the vehicle only
control (FIG. 19A & FIG.
19B).
This example demonstrates that circular RNA and linear RNA are successfully
delivered via
topical administration to the tissue when delivered with ethanol and persists
in tissue over prolonged
periods of time.
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Example 32: Topical administration of circular RNA results in RNA delivery to
tissue
This example demonstrates the delivery of circular RNA to cells and tissues
via topical
administration in vivo.
In this example, circular RNA was designed with an ORF encoding a protein or
immunogen. In
this case, the circular RNA encoded an erythropoietin protein (EPO).
The circular RNA was generated in vitro. Linear RNA was transcribed in vitro
from a DNA
template including all the motifs listed above, as well as a T7 RNA polymerase
promoter to drive
transcription. In this example, Cy5-UTP is used to generate Cy5-labeled RNA.
Transcribed RNA was
purified with an RNA cleanup kit (New England Biolabs, T2050), treated with
RNA 5'phosphohydrolase
(RppH) (New England Biolabs, M0356) following the manufacturer's instructions,
and purified again with
an RNA purification column. RppH treated linear RNA was circularized using a
splint DNA between 10
and 40 nucleotides and T4 RNA ligase 2 (New England Biolabs, M0239). Circular
RNA was Urea-PAGE
purified, eluted in a buffer (0.5 M Sodium Acetate, 0.1% SDS, 1 mM EDTA),
ethanol precipitated and
resuspended in RNA storage solution (ThermoFisher Scientific, cat# AM7000).
Circular RNA was diluted in PBS/glucose (4.5g/L) with 5% ethanol such that
total sample volume
for each sample was 25 L, and total RNA for each sample was 12 picomoles. All
reagents were brought
to room temperature prior to mixing and mixtures were prepared immediately
prior to use.
At time = 0, the ear of the mouse was wiped with an isopropyl alcohol wipe,
dried and a 25 I_
dose of each sample was applied topically to the ear of a BALB/c mouse
dropwise using a pipet tip.
Samples were dried by exposing the ears briefly to a heat lamp and fan in a
sterilized hood. Mice were
place back in cages under normal conditions.
To determine circular RNA delivery to tissue, tissue samples were analyzed by
fluorescence
microscopy at varying timepoints after administration. At 6 hours, 1 day, and
3 days post administration,
a 2 mm ear punch was taken from each animal and stored in ice-cold PBS. Tissue
samples were then
observed under an EVOS II fluorescent microscope. Images were then quantified
for fluorescence using
ImageJ.
Cy5 signal was detected in tissue samples at 6 hours, 1 and 3 days after
topical administration
and showed greater signal than the negative control which did not show any
fluorescence (FIG. 20 and
FIG. 21). This indicates that circular RNA is successfully delivered to the
tissue.
This example demonstrates that circular RNA is successfully delivered via
topical administration
when the skin and persists in tissue over prolonged periods of time.
Example 33: Topical administration of unmodified RNA to the nasal mucosal
epithelium results in
persistence of RNA in tissue
This example describes the delivery of unmodified RNA via topical
administration to the nasal
mucosal epithelium and achieve uptake of linear and circular RNA via topical
administration.
For this example, an IRES, an ORF encoding Nano Luciferase (NLuc), and two
spacer elements
flanking the IRES-ORF are included in the RNA.
The circular RNA is generated in vitro. Unmodified linear RNA is transcribed
in vitro from a DNA
template including all the motifs listed above, as well as a T7 RNA polymerase
promoter to drive
transcription. Transcribed RNA is purified with an RNA cleanup kit (New
England Biolabs, T2050),
treated with RNA 5'phosphohydrolase (RppH) (New England Biolabs, M0356)
following the
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manufacturer's instructions, and purified again with an RNA purification
column. RppH treated linear
RNA is circularized using a splint DNA between 10 and 40 nucleotides in length
and T4 RNA ligase 2
(New England Biolabs, M0239). Circular RNA is Urea-PAGE purified, eluted in a
buffer (0.5 M Sodium
Acetate, 0.1% SDS, 1 mM EDTA), ethanol precipitated and resuspended in RNaA
storage solution
(ThermoFisher Scientific, cat# AM7000). Linear RNA counterparts are generated
and included the same
NLuc ORF and coding components described above.
RNA is diluted to a concentration of 1 pmole/ L. 5 pmole of RNA in citrate
buffer is combined with
sterile PBS. Total sample volume to be used per application is 20 I_ Vehicle
only control samples are
prepared similarly but without RNA. All reagents are brought to room
temperature prior to mixing and
mixtures are prepared immediately prior to use.
In a sterilized hood, mice are placed in a hanging position by their ears. At
time = 0, a 20 I_
dose of each sample is gradually release into the nostrils of a BALB/c mouse
(10 I_ in each nostril) using
a micropipette. Mouth and alternate nostril are closed during application to
ensure uptake. Mice are kept
in the hanging position until breathing rate returned to normal. Mice are
placed back in cages under
normal conditions.
At 6, 24 and 48 hours post administration, mice are sacrificed, and nasal
tissue is taken from the
mouse. Each tissue sample (-2 mg) is placed in 200 I_ of chilled 1-
thioglycerol/Homogenization solution
and homogenized using a handheld homogenized and sterile pestle until no
visible tissue fragments
remain. Each sample is homogenized for an additional 15-30 seconds for
complete homogenization.
To determine RNA persistence in tissue, tissue samples are analyzed for RNA at
varying
timepoints after delivery using qPCR. qPCR is used to measure both linear and
circular RNA in the
extracted tissue. Primers that amplify the NLuc ORF are used. To detect
circular RNA only, primers that
amplify the 5'-3' junction allow for detection of circular but not linear RNA
constructs.
Example 34: Topical administration of circular RNA results in RNA delivery to
tissue when tissue
is wiped with an ethanol wipe prior to application
This example demonstrates the delivery of unmodified circular RNA to cells and
tissues via
topical administration in vivo.
In this example, circular RNA was designed with an IRES and ORF encoding
Nanoluciferase
(NLuc).
The circular RNA was generated in vitro. Unmodified linear RNA was transcribed
in vitro from a
DNA template including all the motifs listed above, as well as a T7 RNA
polymerase promoter to drive
transcription. Transcribed RNA was purified with an RNA cleanup kit (New
England Biolabs, T2050),
treated with RNA 5'phosphohydrolase (RppH) (New England Biolabs, M0356)
following the
manufacturer's instructions, and purified again with an RNA purification
column. RppH treated linear
RNA was circularized using a splint DNA having between 10 and 40 nucleotides
in length and T4 RNA
ligase 2 (New England Biolabs, M0239). Circular RNA was Urea-PAGE purified,
eluted in a buffer (0.5 M
Sodium Acetate, 0.1% SDS, 1 mM EDTA), ethanol precipitated and resuspended in
RNA storage solution
(ThermoFisher Scientific, cat# AM7000).
RNA was then diluted in either PBS only, or PBS with 10% (v/v) ethanol, such
that total sample
volume for each sample was 35 L, and total RNA for each sample was 20 pmoles.
As negative controls,
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vehicle only controls were prepared as described above but without RNA. All
reagents are brought to
room temperature prior to mixing and mixtures are prepared immediately prior
to use.
At time = 0, the ear of the mouse was wiped with an ethanol wipe, dried and a
35 I_ dose of
each sample was applied topically to the ear of a BALB/c mouse dropwise using
a pipet tip. Samples
.. were dried by exposing the ears briefly to a heat lamp and fan in a
sterilized hood. Mice were place back
in cages under normal conditions.
To determine RNA persistence in tissue, tissue samples were analyzed for RNA
at varying
timepoints after delivery using RT-qPCR. At 1 and 4 days post-administration,
a 2 mm ear punch was
taken from each animal and stored in RNAlater solution (ThermoFisher
Scientific, cat# AM7020). Total
.. RNA was isolated from ear punches by snap-cooling and homogenizing the
tissue in liquid nitrogen with a
glass mortar and pestle followed by trizol extraction (ThermoFisher Scientific
cat # 15596026). The
aqueous phase was precipitated with isopropanol and the pellet was washed with
70% ETOH as per
manufacturer's instructions. cDNA was synthesized from the total RNA using
Superscript IV (Thermo
Scientific, cat# 11766500). RT-PCR was performed on cDNA templates using iTaq
TM Universal
.. SYBROGreen Supermix (Bio-Rad, catalog #1725124) and primers specific to the
NLuc ORF. All samples
were assayed in triplicate on the Bio-Rad 0FX384 Thermal Cycler. RNA levels
were then relativized to
housekeeping gene (28s).
Circular RNA was detected in tissue samples at 1 and 4 days for circular RNA
in PBS only, and
at 1 days for circular RNA in PBS+10 /0Et0H after topical administration
following wiping the skin with an
ethanol wipe and showed greater signal than the relevant vehicle only control
(FIG. 22).
This example demonstrates that circular RNA is successfully delivered via
topical administration
when the skin is wiped with an ethanol wipe prior to administration and
persists in tissue over prolonged
periods of time.
.. Example 35: Topical administration of circular RNA results in RNA delivery
to tissue when tissue
is wiped with an isopropyl alcohol wipe prior to application
This example demonstrates the delivery of unmodified circular RNA to cells and
tissues via
topical administration in vivo.
In this example, circular RNA was designed with an IRES and ORF encoding
Nanoluciferase
(NLuc).
The circular RNA was generated in vitro. Unmodified linear RNA was transcribed
in vitro from a
DNA template including all the motifs listed above, as well as a T7 RNA
polymerase promoter to drive
transcription. Transcribed RNA was purified with an RNA cleanup kit (New
England Biolabs, T2050),
treated with RNA 5'phosphohydrolase (RppH) (New England Biolabs, M0356)
following the
manufacturer's instructions, and purified again with an RNA purification
column. RppH treated linear
RNA circularized using a splint DNA between 10 and 40 nucleotides in length
and T4 RNA ligase 2 (New
England Biolabs, M0239). Circular RNA Urea-PAGE purified, eluted in a buffer
(0.5 M Sodium Acetate,
0.1% SDS, 1 mM EDTA), ethanol precipitated and resuspended in RNA storage
solution (ThermoFisher
Scientific, cat# AM7000).
RNA was then diluted in PBS only such that total sample volume for each sample
was 35 L, and
total RNA for each sample was 20 pmoles. As negative controls, vehicle only
controls were prepared as
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described above but without RNA. All reagents are brought to room temperature
prior to mixing and
mixtures are prepared immediately prior to use.
At time = 0, the ear of the mouse was wiped with an isopropyl alcohol wipe
(CVS, 297584), dried
and a 35 I_ dose of each sample was applied topically to the ear of a BALB/c
mouse dropwise using a
pipet tip. As a negative control, an isopropyl alcohol wipe alone was used.
Samples were dried by
exposing the ears briefly to a heat lamp and fan in a sterilized hood. Mice
were place back in cages
under normal conditions.
To determine RNA persistence in tissue, tissue samples were analyzed for RNA
at varying
timepoints after delivery using RT-qPCR. At 1 day and 4 days post-
administration, a 2 mm ear punch
was taken from each animal and stored in RNAlater solution (ThermoFisher
Scientific, cat# AM7020).
Total RNA was isolated from ear punches by snap-cooling and homogenizing the
tissue in liquid nitrogen
with a glass mortar and pestle followed by trizol extraction (ThermoFisher
Scientific cat # 15596026). The
aqueous phase was precipitated with isopropanol and the pellet was washed with
70% ETOH as per
manufacturer's instructions. cDNA was synthesized from the total RNA using
Superscript IV (Thermo
Scientific, cat# 11766500). RT-PCR was performed on cDNA templates using iTaq
TM Universal
SYBROGreen Supermix (Bio-Rad, catalog #1725124) and primers specific to the
NLuc ORF. All samples
were assayed in triplicate on the Bio-Rad 0FX384 Thermal Cycler. RNA levels
were then relativized to
housekeeping gene (28s).
Circular RNA was detected in tissue samples at 1 day and 4 days after topical
administration and
showed greater signal than the vehicle only control (FIG. 23).
This example demonstrates that endless RNA is successfully delivered via
topical administration
to the tissue after wiping the skin with an isopropyl alcohol wipe and
persists in tissue over prolonged
periods of time.
Example 36: Topical administration of circular RNA results in protein
expression in tissue
This example demonstrates the delivery of unmodified circular RNA to cells and
tissues via
topical administration in vivo and achieve subsequent protein expression.
In this example, circular RNA was designed with an IRES and ORF encoding
Nanoluciferase
(NLuc).
The circular RNA was generated in vitro. Unmodified linear RNA was transcribed
in vitro from a
DNA template including all the motifs listed above, as well as a T7 RNA
polymerase promoter to drive
transcription. Transcribed RNA was purified with an RNA cleanup kit (New
England Biolabs, T2050),
treated with RNA 5'phosphohydrolase (RppH) (New England Biolabs, M0356)
following the
manufacturer's instructions, and purified again with an RNA purification
column. RppH treated linear
RNA was circularized using a splint DNA between 10 and 40 nucleotides in
length and T4 RNA ligase 2
(New England Biolabs, M0239). Circular RNA was Urea-PAGE purified, eluted in a
buffer (0.5 M Sodium
Acetate, 0.1% SDS, 1 mM EDTA), ethanol precipitated and resuspended in RNA
storage solution
(ThermoFisher Scientific, cat# AM7000).
RNA was then diluted in PBS with 10% (v/v) ethanol, such that total sample
volume for each
sample was 35 L, and total RNA for each sample was 20 pmoles. As negative
controls, vehicle only
controls were prepared as described above but without RNA. All reagents were
brought to room
temperature prior to mixing and mixtures were prepared immediately prior to
use.
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At time = 0, a 35 I_ dose of each sample was applied topically to the ear of
a BALB/c mouse
dropwise using a pipet tip. Samples were dried by exposing the ears briefly to
a heat lamp and fan in a
sterilized hood. Mice were place back in cages under normal conditions.
To determine RNA expression in tissue, tissue samples were analyzed for NLuc
activity at
varying timepoints after topical delivery. Ear punches were taken from the
mouse at 4 days after delivery.
Each tissue sample was crushed into fragments and was placed in 50 I_ of ice
cold NLuc Lysis Assay
Buffer with lx Protease Inhibitor Cocktail and placed on ice. Samples were
then incubated on an orbital
shaker for 5 minutes at 700 rpm, and then centrifuged at room temperature to
remove tissue debris. The
50 I_ supernatant was then transferred to a fresh tube without disturbing the
tissue pellet. 50 I_ of each
sample was transferred to a 96 well plate and Nano-Glo Luciferase Assay System
(Promega, #N1110)
assay was performed according to manufacturer's instruction. In brief, 1 I_
of furimazine substrate and
49 I_ of PBS were added to each well and mixed. Plates were incubated for 10
min after substrate
addition and mixing and then read in a luminometer instrument (Promega).
Nano Luciferase activity was detected in tissue samples at 4 days after
topical administration for
circular RNA in PBS with 10% ethanol (v/v) and was observed to be higher than
the relevant vehicle only
control (FIG. 24).
This example demonstrated that circular RNA was successfully delivered via
topical
administration and was able to express functional protein, detectable in
tissue for prolonged periods of
time.
Example 37: Topical administration of circular RNA results in RNA delivery to
tissue when tissue
is wiped with a povidone iodine prior to application
This example describes the delivery of unmodified circular RNA to cells and
tissues via topical
administration in vivo.
In this example, circular RNA is designed with an IRES and ORF encoding
Nanoluciferase
(NLuc).
The circular RNA is generated in vitro. Unmodified linear RNA is transcribed
in vitro from a DNA
template including all the motifs listed above, as well as a T7 RNA polymerase
promoter to drive
transcription. Transcribed RNA is purified with an RNA cleanup kit (New
England Biolabs, T2050), is
treated with RNA 5'phosphohydrolase (RppH) (New England Biolabs, M0356)
following the
manufacturer's instructions, and is purified again with an RNA purification
column. RppH treated linear
RNA is circularized using a splint DNA between 10 and 40 nucleotides in length
and T4 RNA ligase 2
(New England Biolabs, M0239). Circular RNA is Urea-PAGE purified, eluted in a
buffer (0.5 M Sodium
Acetate, 0.1% SDS, 1 mM EDTA), is ethanol precipitated and is resuspended in
RNA storage solution
(ThermoFisher Scientific, cat# AM7000).
RNA is then diluted in PBS only such that total sample volume for each sample
is 35 L, and total
RNA for each sample is 20 pmoles. As negative controls, vehicle only controls
are prepared as described
above but without RNA. All reagents are brought to room temperature prior to
mixing and mixtures are
prepared immediately prior to use.
At time = 0, the ear of the mouse is wiped with commercial povidone iodine
(10%), which is a
sterilizing agent. Excess povidone iodine is removed with a sterile cotton
swab and a 35 I_ dose of each
sample is applied topically to the ear of a BALB/c mouse dropwise using a
pipet tip. Samples are dried
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by exposing the ears briefly to a heat lamp and fan in a sterilized hood. Mice
are placed back in cages
under normal conditions.
To determine RNA persistence in tissue, tissue samples are analyzed for RNA at
varying
timepoints after delivery using RT-qPCR. At 1 day and 4 days post-
administration, a 2 mm ear punch is
taken from each animal and stored in RNAlater solution (ThermoFisher
Scientific, cat# AM7020). Total
RNA is isolated from ear punches by snap-cooling and homogenizing the tissue
in liquid nitrogen with a
glass mortar and pestle followed by trizol extraction (ThermoFisher Scientific
cat # 15596026). The
aqueous phase is precipitated with isopropanol and the pellet is washed with
70% ETOH as per
manufacturer's instructions. cDNA is synthesized from the total RNA using
Superscript IV (Thermo
Scientific, cat# 11766500). RT-PCR is performed on cDNA templates using iTaq
TM Universal
SYBROGreen Supermix (Bio-Rad, catalog #1725124) and primers specific to the
NLuc ORF. All samples
are assayed in triplicate on the Bio-Rad 0FX384 Thermal Cycler. RNA levels are
then be relativized to
housekeeping gene (28s).
Example 38: Topical administration of circular RNA results in RNA delivery to
tissue when tissue
is sprayed with a hydrogen peroxide spray prior to application
This example describes the delivery of unmodified circular RNA to cells and
tissues via topical
administration in vivo.
In this example, circular RNA is designed with an IRES and ORF encoding
Nanoluciferase
(NLuc).
The circular RNA is generated in vitro. Unmodified linear RNA is transcribed
in vitro from a DNA
template including all the motifs listed above, as well as a T7 RNA polymerase
promoter to drive
transcription. Transcribed RNA is purified with an RNA cleanup kit (New
England Biolabs, T2050),
treated with RNA 5'phosphohydrolase (RppH) (New England Biolabs, M0356)
following the
manufacturer's instructions, and purified again with an RNA purification
column. RppH treated linear
RNA will be circularized using a splint DNA between 10 and 40 nucleotides in
length and T4 RNA ligase 2
(New England Biolabs, M0239). Circular RNA is Urea-PAGE purified, eluted in a
buffer (0.5 M Sodium
Acetate, 0.1% SDS, 1 mM EDTA), ethanol precipitated and resuspended in RNA
storage solution
(ThermoFisher Scientific, cat# AM7000).
RNA is then diluted in PBS only such that total sample volume for each sample
is 35 L, and total
RNA for each sample is 20 pmoles. As negative controls, vehicle only controls
are prepared as described
above but without RNA. All reagents are brought to room temperature prior to
mixing and mixtures are
prepared immediately prior to use.
At time = 0, the ear of the mouse is sprayed with commercial hydrogen peroxide
(3%) , which is a
sterilizing agent, dried with a sterile cotton swab and a 35 I_ dose of each
sample is applied topically to
the ear of a BALB/c mouse dropwise using a pipet tip. Samples are dried by
exposing the ears briefly to a
heat lamp and fan in a sterilized hood. Mice are placed back in cages under
normal conditions.
To determine RNA persistence in tissue, tissue samples are analyzed for RNA at
varying
timepoints after delivery using RT-qPCR. At 1 day and 4 days post-
administration, a 2 mm ear punch is
taken from each animal and stored in RNAlater solution (ThermoFisher
Scientific, cat# AM7020). Total
RNA is isolated from ear punches by snap-cooling and homogenizing the tissue
in liquid nitrogen with a
glass mortar and pestle followed by trizol extraction (ThermoFisher Scientific
cat # 15596026). The
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aqueous phase is precipitated with isopropanol and the pellet is washed with
70% ETOH as per
manufacturer's instructions. cDNA is synthesized from the total RNA using
Superscript IV (Thermo
Scientific, cat# 11766500). RT-PCR is performed on cDNA templates using iTaq
TM Universal
SYBROGreen Supermix (Bio-Rad, catalog #1725124) and primers specific to the
NLuc ORF. All samples
are assayed in triplicate on the Bio-Rad 0FX384 Thermal Cycler. RNA levels are
then relativized to
housekeeping gene (28s).
Example 39: Detection of secreted immunogen in blood
Blood samples (-25 L) are collected from each mouse for analysis by submolar
drawing. Blood
is collected into EDTA tubes, at 0, 6 hours, 24, 48 hours and 7 days post-
dosing of the circular RNA.
Plasma is isolated by centrifugation for 30 minutes at 1300 g at 4 C.
Expression of secreted immunogen
is assessed using an ELISA or Western blot, e.g. for RBD immunogen, using
methods as described in
Example 14.
Example 40: Detection of antibodies to immunogen
This example describes how to determine the presence of antibodies to
immunogen.
An ELISA is used as described by Chen X et al. (medRxiv, doi:
doi.org/10.1101/2020.04.06.20055475 (2020)). Briefly, SARS-CoV-2 protein in
100 1_ PBS per well is
coated on ELISA plates overnight at 4 C. ELISA plates are then blocked for 1
hour with blocking buffer
(5% FBS plus 0.05% Tween 20). 10-fold diluted plasma is then added to each
well in 100 I_ blocking
buffer over 1 hour. After washing with 1X phosphate-buffered saline with Tween
detergent (PBST),
bound antibodies are incubated with anti-mouse IgG HRP detection antibody
(Invitrogen) for 30 mins,
followed by wash with PBST, then PBS, and addition of tetramethylbenzene. The
ELISA plate is allowed
to react for 5 min and then quenched using 1 M HCI Stop buffer. The optical
density (OD) value is
determined at 450 nm.
A. For antibodies to SARS-CoV-2 RBD immunogen, the SARS-CoV-2 protein used is
SARS-
CoV-2 RBD (Sino Biological, 40592-VO8B).
B. For antibodies to SARS-CoV-2 spike immunogen, the SARS-CoV-2 protein used
is SARS-
CoV-2 spike protein (Sino Biological, 40591-VO8H)
Example 41: Increased protein expressed from circular RNA
This example demonstrates synthetic circular RNA translation in cells.
Additionally, this example
shows that circular RNA produced more expression product of the correct
molecular weight than its linear
counterpart.
Linear and circular RNAs were designed to include a nanoluciferase gene (nLUC)
with a
termination element (stop codon). Cells were transfected with vehicle:
transfection reagent only; linear
nLUC: EMCV IRES, stagger element (2A sequence), 3x FLAG tagged nLuc sequences,
a stagger
element (2A sequence), and termination element (stop codon); or circular nLUC:
EMCV IRES, stagger
element (2A sequence), 3x FLAG tagged nLuc sequences, a stagger element (2A
sequence), and a
termination element (stop codon). As shown in the FIG. 25, circular RNA
produced greater levels of
protein having the correct molecular weight as compared to linear RNA.
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After 24hrs, cells were harvested by adding 100111 of RIPA buffer. After
centrifugation at 1400xg
for 5min, the supernatant was analyzed on a 10-20% gradient polyacrylamide/SDS
gel.
After being electrotransferred to a nitrocellulose membrane using dry transfer
method, the blot
was incubated with an anti-FLAG antibody and anti-mouse IgG peroxidase. The
blot was visualized with
an ECL kit and western blot band intensity was measured by ImageJ.
As shown in FIG. 25, circular RNA was translated into protein in cells. In
particular, circular RNA
produced higher levels of protein having the correct molecular weight as
compared to its linear RNA
counterpart.
Example 42: In vivo re-dosing of circular RNA
This example demonstrates the ability to drive expression from circular RNA in
vivo using two
doses of circular RNA.
For this example, circular RNAs included an EMCV IRES, an ORF encoding Gaussia
Luciferase
(GLuc), and two spacer elements flanking the IRES-ORF.
The circular RNA was generated in vitro. Unmodified linear RNA was in vitro
transcribed from a
DNA template including all the motifs listed above, as well as a T7 RNA
polymerase promoter to drive
transcription. Transcribed RNA was purified with a Monarch RNA cleanup kit
(New England Biolabs,
T2050), treated with RNA 5'-phosphohydrolase (RppH) (New England Biolabs,
M0356) following the
manufacturer's instructions, and purified again with a Monarch RNA cleanup
system. RppH treated RNA
was circularized using a splint DNA between 10 and 40 nucleotides in length
and T4 RNA ligase 2 (New
England Biolabs, M0239). Circular RNA was Urea-PAGE purified, eluted in a
buffer (0.5M Sodium
Acetate, 0.1% SDS, 1mM EDTA), ethanol precipitated and resuspended in RNA
storage solution
(ThermoFisher Scientific, cat# AM7000).
Mice received a single tail vein injection dose of 0.25 g of circular RNA
with the Gaussia
Luciferase ORF, or linear RNA as a control, both formulated in a lipid-based
transfection reagent (Mirus)
as a carrier at day 0, a second dose was administered at day 56.
Blood samples (50 I) were collected from the tail-vein of each mouse into
EDTA tubes, at 1, 2, 7,
11, 16, and 23 days post-dosing. Plasma was isolated by centrifugation for 25
min at 1300 g at 4 C and
the activity of Gaussia Luciferase, a secreted enzyme, was tested using a
Gaussia Luciferase activity
assay (Thermo Scientific Pierce). 50 ill of 1X GLuc substrate was added to 5
ill of plasma to carry out the
GLuc luciferase activity assay. Plates were read right after mixing in a
luminometer instrument
(Promega).
Gaussia Luciferase activity was detected in plasma at 1, 2, 7, 11, 16, and 23
days post-dosing of
the first dose of circular RNA (FIG. 26).
In contrast, Gaussia Luciferase activity was only detected in plasma at 1, and
2 days post-dosing
of modified linear RNA (FIG. 26).
Gaussia Luciferase activity was detected again in plasma at 2, 3, 8, and 15
days post-dosing of
the second dose of circular RNA (FIG. 26).
In contrast, Gaussia Luciferase activity was only detected in plasma at 1, 2,
3 days post-dosing of
modified linear RNA.
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This example demonstrated that circular RNA expressed protein in vivo for
prolonged periods of
time, with levels of protein activity in the plasma at multiple days post
injection. Additionally, it
demonstrates re-dosing of circular RNA results in a similar expression
profile.
Example 43: In vivo staggered dosing of circular RNA
This example demonstrates the ability to drive higher expression of a protein
or immunogen from
circular RNA in vivo using continuous staggered doses of circular RNA.
For this example, circular RNAs included an EMCV IRES, an ORF encoding Gaussia
Luciferase
(GLuc), and two spacer elements flanking the IRES-ORF.
The circular RNA was generated in vitro. Unmodified linear RNA was in vitro
transcribed from a
DNA template including all the motifs listed above, as well as a T7 RNA
polymerase promoter to drive
transcription. Transcribed RNA was purified with an RNA cleanup kit (New
England Biolabs, T2050),
treated with RNA 5'-phosphohydrolase (RppH) (New England Biolabs, M0356)
following the
manufacturer's instructions, and purified again with an RNA purification
column. RppH treated RNA was
.. circularized using a splint DNA between 10 and 40 nucleotides in length and
T4 RNA ligase 2 (New
England Biolabs, M0239). Circular RNA was Urea-PAGE purified, eluted in a
buffer (0.5M Sodium
Acetate, 0.1% SDS, 1mM EDTA), ethanol precipitated and resuspended in RNase
free water.
Mice received a tail vein injection dose of 0.25 pmol of circular RNA with the
Gaussia Luciferase
ORF, or linear RNA as a control, both formulated in a lipid-based transfection
reagent (Mirus) as a carrier
at day 0, day 2 and day 5.
Blood samples (50 I) were collected from the tail-vein of each mouse into
EDTA tubes, at 6
hours, 1, 2, 3, 5, 7, 14, 21, 28, 35, 42 days post-dosing. Plasma was isolated
by centrifugation for 25 min
at 1300 g at 4 C and the activity of Gaussia Luciferase, a secreted enzyme,
was tested using a Gaussia
Luciferase activity assay (Thermo Scientific Pierce). 50 I of 1X GLuc
substrate was added to 5 I of
plasma to carry out the GLuc luciferase activity assay. Plates were read right
after mixing in a
luminometer instrument (Promega).
Gaussia Luciferase activity was detected in plasma at 6 hours, 1, 2, 3, 5, 7,
14, 21, 28 days post-
dosing of a single dose of circular RNA (FIG. 27 and FIG. 28). Gaussia
Luciferase activity was detected
in plasma at 6 hours, 1, 2, 3, 5, 7, 14, 21, 28, 35 days post-dosing of the
first dose of circular RNA when
dosed with 3 doses (FIG. 27 and FIG. 28).
In contrast, Gaussia Luciferase activity was only detected in plasma at 6
hours, 1, 2, 3 days post-
dosing of modified linear RNA and expression levels never increased beyond its
initial dose. Enzyme
activity from linear RNA derived protein was not detected above background
levels at day x or beyond
even though additional linear RNA was dosed (FIG. 27 and FIG. 28).
This example demonstrated that circular RNA expressed protein in vivo for
prolonged periods of
time, with increased levels of protein activity in the plasma after multiple
injections. Additionally, it
demonstrates repeated dosing of circular RNA but not linear RNA results in
expression.
Example 44: Naked dose and redose of circular RNA via intramuscular injection
This example demonstrates the ability to drive expression of a protein or
immunogen from
circular RNA in vivo using two doses of circular administered intramuscularly.
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For this example, circular RNAs included an EMCV IRES, an ORF encoding Gaussia
Luciferase
(GLuc), and two spacer elements flanking the IRES-ORF.
The circular RNA and mRNA were generated according to the methods described
herein.
To generate unformulated RNA, circular RNA and mRNA were then diluted to a
final
concentration of 2.5 picomoles in 100 L of PBS.
Mice received a single intramuscular injection to the hind leg of dose of 2.5
picomoles of circular
RNA with the Gaussia Luciferase ORF. Injections were performed at day 0, and a
second dose was
administered at day 49. Vehicle only was used as control.
Blood samples (50 L) were collected by submental puncture into EDTA tubes, at
1, 2, 7, 11, 16,
and 23 days post-dosing. Plasma was isolated by centrifugation for 25 min at
1300 g at 4 C and the
activity of Gaussia Luciferase, a secreted enzyme, was tested using a Gaussia
Luciferase activity assay
(Thermo Scientific Pierce). 50 L of 1X GLuc substrate was added to 5 I of
plasma to carry out the
GLuc luciferase activity assay. Plates were read right after mixing in a
luminometer instrument
(Promega).
Gaussia Luciferase activity was detected in plasma at 1, 2, 7, 11, 16, and 23
days post-dosing of
the first dose of unformulated circular RNA. (FIG. 29)
In contrast, Gaussia Luciferase activity was only detected in plasma at 1, and
2 days post-dosing
of unformulated mRNA. (FIG. 29)
Gaussia Luciferase activity was detected again in plasma at 2, 3, 8, and 15
days post-dosing of
the second dose of unformulated circular RNA. (FIG. 29)
In contrast, Gaussia Luciferase activity was only detected in plasma at 1, 2,
3 days post-dosing of
unformulated modified mRNA. (FIG. 29)
In each case, Gaussia Luciferase activity was greater than the vehicle only
control.
This example demonstrated that circular RNA administered intramuscularly,
without a carrier,
expressed protein in vivo for prolonged periods of time, with levels of
protein activity in the plasma at
multiple days post injection. Additionally, it demonstrates re-dosing of
circular RNA results in a similar
expression profile.
Example 45: Carrier redose of circular RNA via intravenous injection repeated
five times, results
in expression of functional protein
This example demonstrates the ability to drive expression from circular RNA in
vivo using five
doses of circular RNA administered intravenously.
For this example, circular RNAs included an EMCV IRES, an ORF encoding Gaussia
Luciferase
(GLuc), and two spacer elements flanking the IRES-ORF.
The circular RNA and mRNA were generated according to the methods described
herein.
Circular RNA and mRNA were formulated using a cationic lipid carrier. In this
example, 10%
TransIT (Mirus Bio) and 5% Boost were complexed with the RNA according to the
manufacturer's
instructions.
Mice received a single tail vein injection dose of 0.25 picomoles of circular
RNA including the
Gaussia Luciferase ORF. Injections were performed at: day 0, day 71, day 120,
day 196, and day 359.
Vehicle only was used as control.
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Blood samples (50 L) were collected submental puncture into EDTA tubes, at
0.25, 1, 2, 3, 7,
14, 21, 28, and 35 days post-dosing. Plasma was isolated by centrifugation for
25 min at 1300 g at 4 C
and the activity of Gaussia Luciferase, a secreted enzyme, was tested using a
Gaussia Luciferase activity
assay (Thermo Scientific Pierce). 50 I_ of 1X GLuc substrate was added to 5
I_ of plasma to carry out
the GLuc luciferase activity assay. Plates were read right after mixing in a
luminometer instrument
(Promega).
When dosed with Trans-IT formulated circular RNA, Gaussia Luciferase activity
was detected in
plasma at: days 1, 2, 3, 7, 14, 21 and 28 post-doing of the first dose; days
1, 2, 3, 7, 14 and 21 post-
dosing of the second dose; 1, 2, 3, 7, 14 and 21 post-doing of the third dose;
days 1, 2, 3, 7, 14, 21 and
28 post-doing of the fourth dose; and, days 1, 2, 3, 7, 14 and 21 post-doing
of the fifth dose. (FIG. 30)
In contrast, when dosed with Trans-IT formulated modified mRNA, Gaussia
Luciferase activity
was detected in plasma at: days 0.25, 1 and 2 post-doing of the first dose;
days 0.25, 1 and 2 post-dosing
of the second dose; days 0.25, 1 and 2 post-doing of the third dose; days
0.25, 1 and 2 post-doing of the
fourth dose; and, days 0.25, 1 and 2 post-doing of the fifth dose. (FIG. 30)
In each case, Gaussia Luciferase activity and thus expression was greater for
circular RNA than
for the mRNA.
This example demonstrated that circular RNA administered intravenously,
expressed protein in
vivo for prolonged periods of time, with levels of protein activity in the
plasma at multiple days post
injection and could be redosed at least 5 times. Additionally, it demonstrates
extended re-dosing of
circular RNA results in a similar expression profile.
Example 46: Expression of multiple immunogens from circular RNAs in mammalian
cells
This example demonstrates expression of multiple immunogens from circular RNAs
in mammalian
cells.
Experiment 1
A first circular RNA encoding a SARS-CoV-2 RBD immunogen including an N-
terminal Gluc signal
sequence (Nucleic acid SEQ ID NO: 33; Amino acid SEQ ID NO: 32) and a second
circular RNA encoding
SARS-CoV-2 Spike immunogen (Nucleic acid SEQ ID NO: 31; Amino acid SEQ ID NO:
30) were designed
and purified according to the methods described herein. The first circular RNA
and the second circular
RNA were mixed together to obtain a mixture. The mixture (1 picomole of each
of the circular RNAs) was
transfected into HeLa cells (100,000 cells per well in a 24 well plate) using
Lipofectamine MessengerMax
(ThermoFisher, LMRNA015). As controls, the first circular RNA and the second
circular RNA were also
separately transfected into HeLa cells using MessengerMax.
RBD immunogen expression was measured at 24 hours using a SARS-CoV-2 RBD
immunogen-
specific ELISA. Spike immunogen expression was measured at 24 hours by flow
cytometry.
From the transfection with the mixture, SARS-Co-V-2 RBD immunogen was detected
in the HeLa
cell supernatant and SARS-CoV-2 Spike immunogen was detected on the cell
surface of the HeLa cells.
From the transfection with the first circular RNA, SARS-CoV-2 RBD immunogen
was detected, but SARS-
CoV-2 Spike immunogen was not detected. From the transfection with the second
circular RNA, SARS-
CoV-2 Spike immunogen was detected, but SARS-CoV-2 RBD immunogen was not
detected. This
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demonstrates that both SAR-CoV-2 RBD and SARS-CoV-2 Spike immunogens were
expressed in
mammalian cells from a combination mixture of circular RNAs.
Experiment 2
A first circular RNA encoding a SARS-CoV-2 RBD immunogen including an N-
terminal Gaussia
Luciferase (Gluc) signal sequence (Nucleic acid SEQ ID NO: 33; Amino acid SEQ
ID NO: 32) and a second
circular RNA encoding a GLuc polypeptide (Nucleic acid SEQ ID NO: 37; Amino
acid SEQ ID NO: 36) were
designed and produced according to the methods described herein. The first
circular RNA and the second
circular RNA were separately complexed with Lipofectamine MessengerMax
(ThermoFisher, LMRNA015),
and then mixed together to obtain a mixture. The mixture (0.1 picomoles of
each circular RNAs) was
transfected into HeLa cells (20,000 cells per well in a 96 well plate). As
controls, the first circular RNA and
the second circular RNA were also separately transfected into HeLa cells using
MessengerMax.
RBD immunogen expression was measured at 24 hours using a SARS-CoV-2 RBD
immunogen-
specific ELISA. GLuc activity was measured at 24 hours using a Gaussia
Luciferase activity assay (Thermo
Scientific Pierce).
From the transfection with the mixture, SARS-CoV-2 RBD immunogen and GLuc
activity were
detected in the HeLa cell supernatant at 24 hrs. From the transfection with
the first circular RNA, SARS-
CoV-2 RBD immunogen was detected, but GLuc activity was not detected. From the
transfection with the
second circular RNA, GLuc activity was detected, but SARS-CoV-2 RBD immunogen
was not detected.
This demonstrates that both SAR-CoV-2 RBD and GLuc immunogens were expressed
in mammalian
cells from a combination mixture of circular RNAs.
Experiment 3
A first circular RNA encoding a SARS-CoV-2 RBD immunogen including an N-
terminal Gluc signal
sequence (Nucleic acid SEQ ID NO: 33; Amino acid SEQ ID NO: 32) and a second
circular RNA encoding
hemagglutinin (HA) immunogen from Influenza A Hi Ni, A/California/07/2009
(Nucleic acid SEQ ID NO:
35; Amino acid SEQ ID NO: 34), were designed and produced according to the
methods described herein.
The first circular RNA and the second circular RNA were mixed together to
obtain a mixture. The mixture
(1 picomoles of each circular RNA) was transfected into HeLa cells (100,000
cells per well in a 24 well
plate) using Lipofectamine MessengerMax (ThermoFisher, LMRNA015). As controls,
the first circular RNA
and the second circular RNA were also separately transfected into HeLa cells
using MessengerMax.
RBD immunogen expression was measured at 24 hours using a SARS-CoV-2 RBD
immunogen-
specific ELISA. HA immunogen expression was measured at 24 hours using
immunoblot. Briefly, for
immunoblot, 24 hours after transfection, cells were lysed and Western blot was
performed to detect the
HA immunogen using Influenza A Hi Ni HA (A/California/07/2009) monoclonal
antibody (MA5-29920
(Thermo Fisher)) as the primary antibody and goat anti-mouse IgG H&L (HRP) as
the secondary antibody
(Abcam, ab 97023). For loading control alpha tubulin was used with alpha
tubulin (DM1A) mouse
antibody as the primary antibody (Cell Signaling Technology, CST #3873) and
goat anti-mouse IgG H&L
(HRP) as the secondary antibody (Abcam, ab 97023).
From the transfection with the mixture, both SARS-CoV-2 RBD and Influenza HA
immunogens
were detected. From the transfection with the first circular RNA, SARS-CoV-2
RBD was detected, but
Influenza HA immunogen was not detected. From the transfection with the second
circular RNA,
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Influenza HA immunogen was detected, but SARS-CoV-2 RBD immunogen was not
detected. This
demonstrates that both SAR-CoV-2 RBD and Influenza HA immunogens were
expressed in mammalian
cells from a combination mixture of circular RNAs.
Experiment 4
A first circular RNA encoding a SARS-CoV-2 Spike immunogen (Nucleic acid SEQ
ID NO: 31;
Amino acid SEQ ID NO: 30) and a second circular RNA encoding hemagglutinin
(HA) from Influenza A
Hi Ni, A/California/07/2009 (Nucleic acid SEQ ID NO: 35; Amino acid SEQ ID NO:
34), were designed and
produced according to the methods described herein. The first circular RNA and
the second circular RNA
were mixed together to obtain a mixture. The mixture (1 picomoles of each
circular RNAs) was transfected
into HeLa cells (100,000 cells per well in a 24 well plate) using
Lipofectamine MessengerMax
(ThermoFisher, LMRNA015). As controls, the first circular RNA and the second
circular RNA were also
separately transfected into HeLa cells using MessengerMax.
Spike immunogen expression was measured at 24 hours by flow cytometry. HA
immunogen
expression was measured at 24 hours by immunoblot as described above in
Experiment 3.
From the transfection with the mixture, both SARS-CoV-2 Spike immunogen and
Influenza HA
immunogen were detected. From the transfection with the first circular RNA,
SARS-CoV-2 Spike
immunogen was detected, but Influenza HA immunogen was not detected. From the
transfection with the
second circular RNA, Influenza HA immunogen was detected, but SARS-CoV-2 Spike
immunogen was not
detected. This demonstrates that both SAR-CoV-2 Spike and Influenza HA
immunogens were expressed
in mammalian cells from a combination mixture of circular RNAs.
This example shows that multiple immunogens were expressed in mammalian cells
from circular
RNA preparations comprising different combinations of circular RNAs.
Example 47: Multi-immunogen expression from circular RNA
This example demonstrates expression of multiple immunogens from a circular
RNA in mammalian
cells.
Experiment 1
In this example, a circular RNA was designed to include an IRES followed by an
ORF encoding a
GLuc polypeptide, a stop codon, a spacer, an IRES, an ORF encoding a SARS-CoV-
2 RBD immunogen,
and a stop codon. The circular RNA was produced and purified according to the
methods described herein.
As controls, the following circular RNAs were produced as described above: (i)
a circular RNA with an IRES
and ORF encoding a SARS-CoV-2 RBD immunogen; (ii) a circular RNA with an IRES
and ORF encoding
GLuc polypeptide.
The circular RNAs (0.1 picomoles) were transfected into HeLa cells (10,000
cells per well in a 96
well plate) using Lipofectamine MessengerMax (ThermoFisher, LMRNA015).
RBD immunogen expression was measured at 24 hours using a SARS-CoV-2 RBD
immunogen-
specific ELISA. GLuc activity was measured at 24 hours using a Gaussia
Luciferase activity assay (Thermo
Scientific Pierce).
RBD immunogen expression was detected from circular RNAs encoding a SARSs-CoV-
2 RBD
immunogen and GLuc polypeptide (FIG. 31A). GLuc activity was detected from
circular RNAs encoding
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a SARSs-CoV-2 RBD immunogen and GLuc (FIG. 31B). This demonstrates that both
SAR-CoV-2 RBD
and GLuc immunogens were expressed in mammalian cells from a circular RNA
encoding both SARS-
CoV-2 RBD and GLuc immunogens.
Experiment 2
In this example, a circular RNA designed to include an IRES followed by an ORF
encoding a SARS-
CoV-2 RBD immunogen, a stop codon, a spacer, an IRES, an ORF encoding a Middle
Eastern Respiratory
Syndrome (MERS) RBD immunogen, and a stop codon. The circular RNA is produced
and purified
according to the methods described herein.
The circular RNAs are transfected at various concentrations into HeLa cells
(10,000 cells per well
in a 96 well plate) using Lipofectamine MessengerMax (ThermoFisher, LMRNA015).
SARS-CoV-2 RBD immunogen expression is measured at 24 hours using a SARS-CoV-2
RBD
immunogen-specific ELISA. MERS RBD immunogen expression is measured at 24
hours using a MERS
RBD immunogen specific antibody capable of detection.
Example 48: Immunogenicity of multiple immunogens from circular RNAs in mouse
model
This example describes expression of multiple immunogens in a subject by
administrating multiple
circular RNA molecules.
Experiment 1
The immunogenicity of a circular RNA preparation comprising (a) a circular RNA
encoding a SARS-
CoV-2 RBD immunogen and (b) a circular RNA encoding GLuc polypeptide as a
model immunogen,
formulated in lipid nanoparticles, was evaluated in a mouse model. Production
of antibodies to the SARS-
CoV-2 RBD immunogen and GLuc activity were also evaluated in the mouse model.
A first circular RNA encoding a SARS-CoV-2 RBD immunogen including an N-
terminal Gluc signal
sequence (Nucleic acid SEQ ID NO: 33; Amino acid SEQ ID NO: 32) and a second
circular RNA encoding
GLuc polypeptide (Nucleic acid SEQ ID NO: 37; Amino acid SEQ ID NO: 36) were
designed and purified
according to the methods described herein. The first circular RNA and the
second circular RNA were mixed
together to obtain a mixture. This mixture was then formulated with lipid
nanoparticles as described in
Example 16 to obtain a first circular RNA preparation. The first circular RNA
and the second circular RNA
were also separately formulated with lipid nanoparticles as described in
Example 16, and then mixed
together to obtain a second circular RNA preparation.
Three mice were vaccinated intramuscularly with the first circular RNA
preparation (for a total dose
of 10 ug RBD + 10 ug GLuc) at day 0 and with the second circular RNA
preparation (for a total dose of 10
ug RBD + 10 ug GLuc) at day 12. Additional mice (3 or 4 per group) were also
vaccinated intramuscularly
at day 0 and day 12 with: (i) a 10 ug dose of the first circular RNA
formulated with lipid nanoparticles; (ii) a
10 ug dose of the second circular RNA formulated with lipid nanoparticles; or
(iii) PBS.
Blood collection from each mouse was by submandibular drawing. Blood was
collected into dry-
anticoagulant free-tubes, at 2 and 17, days post-priming with the first
circular RNA preparation. Serum
was separated from whole blood by centrifugation at 1200g for 30 minutes at 4
C. Individual serum
samples were assayed for the presence of RBD-specific IgG by enzyme-linked
immunosorbent assay
(ELISA). ELISA plates (MaxiSorp 442404 96-well, Nunc) were coated overnight at
4 C with SARS-CoV-2
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RBD (Sino Biological, 40592-VO8B; 100 ng) in 100 uL of 1X coating buffer
(Biolegend, 421701). The
plates were then blocked for 1 hour with blocking buffer (TBS with 2% BSA and
0.05% Tween 20).
Serum dilutions (1:500, 1:1500, 1:4500, and 1:13,500) were then added to each
well in 100 uL blocking
buffer and incubated at room temperature for 1 hour. After washing three times
with 1X Tris-buffered
saline with Tweene detergent (TBS-T), plates were incubated with anti-mouse
IgG HRP detection
antibody (Abcam, ab97023) for 1 hour followed by three washes with TBS-T, then
addition of
tetramethylbenzene (Biolegend, 421101). The ELISA plate was allowed to react
for 10-20 minutes and
then quenched using 0.2N sulfuric acid. The optical density (0.D.) value was
determined at 450 nm.
The optical density of each serum sample was divided by that of the background
(plates coated
.. with RBD, incubated only with secondary antibody). The fold over background
of each sample was
plotted.
The activity of GLuc was tested using a Gaussia Luciferase activity assay
(Thermo Scientific
Pierce). 50 uL of lx GLuc substrate was added to 10 uL of serum to carry out
the GLuc luciferase activity
assay. Plates were read immediately after mixing in a luminometer instrument
(Promega).
The results showed that anti-RBD antibodies were obtained at 17 days post
prime (i.e., 17 days
after injection with the first circular RNA preparation) (FIG. 32A) and GLuc
activity was detected at 2 days
post prime (i.e. 2 days after injection with the first circular RNA
preparation) (FIG. 32B).
These results showed that circular RNA preparations comprising two circular
RNAs encoding
different immunogens induced immunogen-specific immune responses.
Experiment 2
The immunogenicity of a circular RNA preparation comprising (a) a circular RNA
encoding a SARS-
CoV-2 RBD immunogen and (b) a circular RNA encoding an Influenza hemagglutinin
(HA) immunogen,
formulated in lipid nanoparticles, was evaluated in a mouse model. Production
of antibodies to the SARS-
CoV-2 RBD and Influenza HA immunogens were also evaluated in the mouse model.
A first circular RNA encoding a SARS-CoV-2 RBD immunogen including an N-
terminal Gluc signal
sequence (Nucleic acid SEQ ID NO: 33; Amino acid SEQ ID NO: 32) and a second
circular RNA encoding
hemagglutinin (HA) from Influenza A Hi Ni, A/California/07/2009 (Nucleic acid
SEQ ID NO: 35; Amino acid
SEQ ID NO: 34), were designed and produced according to the methods described
herein. The first circular
RNA and the second circular RNA were mixed together to obtain a mixture. This
mixture was then
formulated with lipid nanoparticles as described in Example 16 to obtain a
first circular RNA preparation.
The first circular RNA and the second circular RNA were also separately
formulated with lipid nanoparticles
as described in Example 16, and then mixed together to obtain a second
circular RNA preparation.
Three mice were vaccinated intramuscularly with the first circular RNA
preparation (for a total dose
of 10 ug RBD + 10 ug HA) at day 0 and with the second circular RNA preparation
(for a total dose of 10 ug
RBD + 10 ug HA) at day 12. Additional mice (3 or 4 per group) were also
vaccinated intramuscularly at
day 0 and day 12 with: (i) a 10 ug dose of the first circular RNA formulated
with lipid nanoparticles; (ii) a 10
ug dose of the second circular RNA formulated with lipid nanoparticles; or
(iii) PBS.
Blood collection was as described in Experiment 1. The presence of RBD-
specific IgG by ELISA
was determined as described in Experiment 1.
Individual serum samples were assayed for the presence of HA-specific IgG by
ELISA. ELISA
plates were coated overnight at 4 C with HA recombinant protein (Sino
Biological, 11085-VO8B; 100 ng)
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and plates were processed as described in Experiment 1. The optical density of
each serum sample was
divided by that of the background (plates coated with HA, incubated only with
secondary antibody). The
fold over background of each sample was plotted.
The results showed that anti-RBD and anti-HA antibodies were obtained at 17
days post prime
(i.e., 17 days after injection with the first circular RNA preparation (FIG.
33A and FIG. 33B).
The results also showed that circular RNA preparations comprising two circular
RNAs encoding
different immunogens were expressed in vivo and induced immunogen-specific
immune responses.
Experiment 3
The immunogenicity of a circular RNA preparation comprising (a) a circular RNA
encoding a SARS-
CoV-2 Spike immunogen and (b) a circular RNA encoding an Influenza
hemagglutinin (HA) immunogen,
formulated in lipid nanoparticles, was evaluated in a mouse model. Production
of antibodies to the SARS-
CoV-2 Spike and Influenza HA immunogens were also evaluated in the mouse
model.
A first circular RNA encoding a SARS-CoV-2 Spike immunogen (Nucleic acid SEQ
ID NO: 31;
Amino acid SEQ ID NO: 30) and a second circular RNA encoding hemagglutinin
(HA) from Influenza A
Hi Ni, A/California/07/2009 (Nucleic acid SEQ ID NO: 35; Amino acid SEQ ID NO:
34), were designed and
produced according to the methods described herein. The first circular RNA and
the second circular RNA
were mixed together to obtain a mixture. This mixture was then formulated with
lipid nanoparticles as
described in Example 16 to obtain a first circular RNA preparation. The first
circular RNA and the second
circular RNA were also separately formulated with lipid nanoparticles as
described in Example 16, and
then mixed together to obtain a second circular RNA preparation.
Three mice were vaccinated intramuscularly with the first circular RNA
preparation (for a total dose
of 10 ug Spike + 10 ug HA) at day 0 and with the second circular RNA
preparation (for a total dose of 10
ug Spike + 10 ug HA) at day 12. Additional mice (3 or 4 per group) were also
vaccinated intramuscularly
at day 0 and day 12 with: (i) a 10 ug dose of the first circular RNA
formulated with lipid nanoparticules; (ii)
a 10 ug dose of the second circular RNA formulated with lipid nanoparticles;
or (iii) PBS.
Blood collection was as described in Experiment 1. Serum was separated from
whole blood by
centrifugation at 1200g for 30 minutes at 400. Individual serum samples were
assayed for the presence
of RBD (i.e., RBD of Spike)-specific IgG by ELISA as described in Experiment
1.
Individual serum samples were assayed for the presence of HA-specific IgG by
ELISA. ELISA
plates were coated overnight at 4 C with HA recombinant protein (Sino
Biological, 11085-VO8B; 100 ng)
and plates were processed as described in Experiment 1. The optical density of
each serum sample was
divided by that of the background (plates coated with HA, incubated only with
secondary antibody). The
fold over background of each sample was plotted.
The results showed that anti-RBD antibodies and anti-HA antibodies were
obtained at 17 days
post prime (i.e., 17 days after injection with the first circular RNA
preparation (FIG. 34A and FIG. 34B).
The results also showed that circular RNA preparations comprising two circular
RNAs encoding
different immunogens induced immunogen-specific immune responses in mice.
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Example 49: Immunogenicity of a circular RNA comprising multiple immunogens in
a mouse
model
This example describes the immunogenicity of a circular RNA comprising
multiples immunogens.
This example also describes production of antibodies in a mouse model to
multiple immunogens encoded
by a single circular RNA.
Experiment 1
This example describes the immunogenicity of a circular RNA comprising
multiple immunogens.
This example also describes production of antibodies in a mouse model to
multiple immunogens encoded
by a single circular RNA.
In this experiment, a first circular RNA was designed to include an IRES
followed by an ORF
encoding Gluc polypeptide, a stop codon, a spacer, an IRES, an ORF encoding
SARS-CoV-2 RBD
immunogen, and a stop codon, produced and purified as described in Example 47.
The following circular
RNAs were also produced as described above: (i) a second circular RNA with an
IRES and ORF encoding
Gluc polypeptide; and (ii) a third circular RNA with an IRES and ORF encoding
a SARS-CoV-2 RBD
immunogen.
The first circular RNA and the second circular RNA were each formulated with
lipid nanoparticles
as described in Example 16 to obtain a first circular RNA preparation and a
second circular RNA
preparation, respectively. The second circular RNA and the third circular RNA
were also separately
formulated with lipid nanoparticles, and then mixed together to obtain a third
circular RNA preparation.
Three mice per group were vaccinated intramuscularly at day 0 and day 28 with:
(i) the first circular
RNA preparation (40 ug dose per injection); or (ii) the second circular RNA
preparation (10 ug dose per
injection); or (iii) the third circular RNA preparation (10 ug Gluc + 10 ug
RBD dose per injection).
Blood collection from each mouse was by submandibular drawing. Blood was
collected from each
mouse into dry-anticoagulant free-tubes at day 2, 14, 28, 35, 43, 49, 61, 62
and 63 post-priming of the
circular RNAs. Serum was separated from whole blood by centrifugation at 1200g
for 30 minutes at 4 C.
Individual serum samples were assayed for the presence of RBD-specific IgG by
enzyme-linked
immunosorbent assay (ELISA). ELISA plates (MaxiSorp 442404 96-well, Nunc) were
coated overnight at
4 C with SARS-CoV-2 RBD (Sino Biological, 40592-VO8B; 100 ng) in 100 uL of 1X
coating buffer
(Biolegend, 421701). The plates were then blocked for 1 hour with blocking
buffer (TBS with 2% BSA and
0.05% Tween 20). Serum was serially diluted (4-fold, 8 times: 1:500 to 1:
8192000) and then added to
each well in 100 uL blocking buffer and incubated at room temperature for 1
hour. After washing three times
with 1X Tris-buffered saline with Tweene detergent (TBS-T), plates were
incubated with anti-mouse IgG
HRP detection antibody (Abcam, ab97023) for 1 hour followed by three washes
with TBS-T, then addition
of TMB ELISA substrate (Pierce, 34021). The ELISA plate was allowed to react
for 10-20 minutes and then
stopped using 2N sulfuric acid. The optical density (0.D.) value was
determined at 450 nm. Serum titer was
defined as the reciprocal of the highest serum dilution that gives an O.D.
value 5-fold above background.
The activity of Gluc was tested using a Gaussia Luciferase activity assay
(ThermoScientific,
16159). 50 uL of lx Gluc substrate was added to 10 uL of serum to carry out
the Gluc luciferase activity
assay. Plates were read immediately after mixing in a luminometer instrument
(Promega).
The results showed that Gluc activity was detected at 2 days post prime (i.e.,
2 days after injection
with the first, second or third circular RNA preparation) (FIG. 36), and that
anti-RBD antibodies were
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obtained at 14 days post prime (i.e., 14 days after injection with the first,
second or third circular RNA
preparation) (FIG. 37A). Anti-RBD antibodies were also obtained at 28, 35, 43,
49, 61, 62 and 63 days
post-priming of the circular RNA (data not shown).
Serum antibodies at day 63 post-dosing were characterized using an assay to
measure relative
.. IgG1 vs IgG2a isotypes, and the ability of serum antibodies to neutralize
the virus was characterized using
a Plaque Reduction Neutralization Test assay. The results showed that
neutralizing antibody was obtained
in mice dosed with the first circular RNA preparation and the third circular
RNA preparation (FIG. 37B).
These results show that a single circular RNA encoding two immunogens can
prime an
immunogen-specific immune response. In addition, the results show that the
circular RNAs comprising an
.. ORF encoding a SARS-CoV-2 RBD immunogen had neutralizing ability.
Experiment 2
The immunogenicity of a circular RNA preparation comprising a circular RNA
designed to include
an IRES followed by an ORF encoding a SARS-CoV-2 RBD immunogen, a stop codon,
a spacer, an
IRES, an ORF encoding a MERS RBD immunogen, and a stop codon, formulated in
lipid nanoparticles, is
evaluated in a mouse model. Production of antibodies to the SARS-CoV-2 RBD and
MERS RBD
immunogens are also evaluated in the mouse model.
This circular RNA is then formulated with lipid nanoparticles as described in
Example 16 to
obtain a circular RNA preparation.
Mice are vaccinated intramuscularly or intradermally with the circular RNA
preparation with
amounts of 5 g, 10 g, 20 g, or 50 g at day 0 and again at least one day
after the initial administration.
Blood collection is as described in Experiment 1. The presence of SARS-CoV-2
RBD-specific
and MERS RBD-specific IgGs by ELISA is determined as described in Experiment
1.
Individual serum samples are assayed for the presence of anti-SARS-CoV-2 RBD
binding
.. antibodies, anti-MERS RBD binding antibodies, neutralizing antibodies
against the SARS-CoV-2 RBD
immunogen, neutralizing antibodies against the MERS RBD immunogen, a cellular
response to the
SARS-CoV-2 immunogen, and a cellular response to the MERS RBD immunogen.
Example 50: Evaluation of T cell responses
.. Experiment 1
An ELISpot assay is used to detect the presence of SARS-CoV-2 Spike or RBD-
specific T cells
or Influenza HA-specific T cells. This assay is performed on the following
groups of mice from Example
48:
1. RBD
2. GLuc
3. HA
4. Spike
5. RBD+HA
6. Spike+HA
7. PBS
Mice spleens are harvested on day 30 post boost (i.e., 30 days after injection
with the first circular
RNA preparation), and processed into a single cell suspension. Splenocytes are
plated at 0.5M cells per
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well on IFN-gamma or IL-4 ELISpot plates (ImmunoSpot). Splenocytes are either
left unstimulated or
stimulated with SARS CoV-2 and HA peptide pools (JPT, PM-WCPV-SRB and PM-IFNA
HACal).
ELISPOT plates are processed according to manufacturer's protocol.
Experiment 2
An ELISpot assay was used to detect the presence of SARS-CoV-2 RBD-specific T
cells. This
assay was performed on the groups of mice from Example 49.
Mice spleens were harvested on day 63 post prime (i.e., 63 days after
injection with the first,
second or third circular RNA preparation), and processed into a single cell
suspension. Splenocytes were
plated at 0.5M cells per well on IFN-y (Mabtech). Splenocytes were either left
unstimulated or stimulated
with SARS CoV-2 RBD peptide pool (JPT, PM-WCPV-SRBD). ELISPOT plates were
processed
according to manufacturer's protocol.
The results showed that a preparation of circular RNA comprising of a single
circular RNA
encoding different two immunogens induced immunogen specific T cell responses
(FIG. 38)
Example 51: Evaluation of antibody response in mice administered circular RNA
encoding
multiple immunogens
This example demonstrates an antibody response resulting from administration
of a circular RNA
encoding the expression of the multiple immunogens.
A hemagglutination inhibition assay (HAI) was used to measure anti-Influenza
HA antibodies that
prevent hemagglutination in serum from mice. Mice were administered a
preparation of circular RNA
each of which was designed and produced the methods described herein, and
which encode for the
expression of: a SARS-CoV-2 RBD immunogen, a SARS-CoV-2 Spike immunogen, an
Influenza HA
immunogen, a SARS-CoV-2 RBD immunogen and an Influenza HA immunogen, a SARS-
CoV-2 RBD
immunpgen and a GLuc polypeptide, or a SARS-CoV-2 RBD immunogen and a SARS-CoV-
2 Spike
immunogen. Blood collection was as described in Example 47, Experiment 1 and
was performed on day
2 and day 17 after injection.
Two-fold serial dilutions of the collected sample from mice on day 2 and day
17 were prepared.
A fixed amount of influenza virus with known hemagglutinin (HA) titer was
added to every well of a 96-
well plate, to a concentration equivalent to 4 hemagglutinin units, with the
exception of the serum control
wells, where no virus was added. The plate was allowed to stand at room
temperature for 60 minutes,
after which the red blood cell samples were added and allowed to incubate at 4
C for 30 minutes. The
highest serum dilution that prevented hemagglutination was determined to be
the HAI titer of the serum.
The sample collected on day 17 showed HAI titer in samples that were
administered circular RNA
preparations encoding the Influenza HA immunogen when it was administered
alone or when
administered in combination with SARS-CoV-2 immunogens e.g. RBD or Spike (FIG.
35). HAI titers on
day 17 were not seen from samples where HA immunogen had not been administered
e.g. the SARS-
CoV-2 RBD immunogen alone or SARS-CoV-2 Spike immunogen alone.
Example 52: Expression of an adjuvant from circular RNA in mammalian cells
This example demonstrates expression of a polypeptide adjuvant from circular
RNA in
mammalian cells.
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In this example, a circular RNA was designed to include an IRES, an ORF
encoding the adjuvant
IL-12 (Nucleic acid SEQ ID NO: 38; Amino acid SEQ ID NO: 39), and two spacer
elements flanking the
IRES-ORF. As a control, a circular RNA including an IRES, an ORF encoding a
SARS-CoV-2 RBD
immunogen, and two spacer elements flanking the IRES-ORF was used. The
circular RNAs were
produced and purified according to the methods described herein.
Purified circular RNAs (0.1 and 1 picomoles) were transfected into HeLa cells
(10,000 cells per
well) using Lipofectamine MessengerMax (Invitrogen LMRNA001) according to the
manufacturer's
instructions.
IL-12 expression was measured using an IL-12 specific ELISA (ThermoFisher,
BM56004) in cell
culture supernatant. Data are shown as Mean and SEM values of two replicates.
The results showed that IL-12 encoded by circular RNA was expressed by HeLa
cells and not in
the control (FIG. 39). This example shows that the IL-12 adjuvant was
expressed from circular RNA in
mammalian cells.
Example 53: In vivo expression of an adjuvant from circular RNA in mouse model
This example demonstrates in vivo expression of a polypeptide adjuvant from a
circular RNA.
In this example, the following circular RNAs were produced and purified
according to the methods
described herein: (i) a first circular RNA with an IRES and an ORF encoding an
IL-12 adjuvant (Nucleic
acid SEQ ID NO: 38; Amino acid SEQ ID NO: 39); and (ii) a second circular RNA
with an IRES and ORF
encoding a SARS-CoV-2 RBD immunogen with an N-terminal Gluc signal sequence
(Nucleic acid SEQ
ID NO: 33; Amino acid SEQ ID NO: 32). The first circular RNA and the second
circular RNA were each
separately formulated with lipid nanoparticles as described in Example 16 and
then mixed together to
obtain a first circular RNA preparation.
Three mice were vaccinated intramuscularly at day 0 with the first circular
RNA preparation (10
ug dose). As controls, three mice per group were vaccinated intramuscularly
with: (i) the second circular
RNA formulated with lipid nanoparticles (10 ug dose) (i.e., the second
circular RNA preparation); or (ii)
PBS. Blood collection from each mouse was by submandibular drawing. Blood was
collected from each
mouse into dry-anticoagulant free-tubes at 2 days after administration of
circular RNA or PBS. Serum
was separated from whole blood by centrifugation at 1200g for 30 minutes at 4
C. Individual serum
samples were assayed for the presence of IL12 using a cytokine bead array
(Biolegend, 749622). Data
are shown as Mean and SEM values of three replicates.
The results showed that IL-12 expression was detected in serum at 2 days after
injection with the
first circular RNA preparation but was not detected after injection with
either of the controls (FIG. 40A).
To determine if the expressed IL12 is functional, IFN-y (directly downstream
of IL12 signaling)
production in the serum was measured 2 days after injection with the circular
RNA preparations. IFN-y
production was detected in serum in the same cytokine bead array assay
described herein (Biolegend,
749622). Data are shown as Mean and SEM values of 3 replicates.
The results showed an increase in serum IFN-y, indicating that circular RNA
expressed IL12 is
functional (FIG. 40B).
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Example 54: Induction of immunogenicity in a mouse model by co-administration
of an
immunogen and an adjuvant encoded by a plurality of circular RNAs
This example demonstrates the immunogenicity induced by administration of a
plurality of circular
RNAs to a subject. One circular RNA administered encodes an immunogen. Another
circular RNA
administered encodes a polypeptide adjuvant.
In this example, the following circular RNAs were produced and purified
according to the methods
described herein: (i) a first circular RNA with an IRES and an ORF encoding an
IL-12 adjuvant (Nucleic
acid SEQ ID NO: 38; Amino acid SEQ ID NO: 39); and (ii) a second circular RNA
with an IRES and ORF
encoding a SARS-CoV-2 RBD immunogen with an N-terminal Gluc signal sequence
(Nucleic acid SEQ
ID NO: 33; Amino acid SEQ ID NO: 32). The first circular RNA and the second
circular RNA were each
separately formulated with lipid nanoparticles as described in Example 16 and
then mixed together to
obtain a first circular RNA preparation.
Three mice were vaccinated intramuscularly at day 0 and day 14 with the first
circular RNA
preparation (2.5 ug dose per circular RNA per injection). As controls, three
mice per group were
vaccinated intramuscularly with: (i) the second circular RNA formulated with
lipid nanoparticles (2.5 ug
dose per injection) (i.e., the second circular RNA preparation); or (ii) PBS.
Mice were euthanized 22 days
post the first dose and splenocytes processed into a single cell suspension.
Splenocytes were either left
unstimulated or stimulated with RBD peptide pool (JPT, PM-WCPV-S-RBD-2) for 1
hour. Protein
transport inhibitors (Monensin, BD 554724) and Brefeldin A, BD 555029)) were
then added to the media
followed by 5 more hours of culture. The cells were stained using the BD
fixation and permeabilization kit
(BD, 554714) according to the manufacturer's protocol. Antibodies used:
viability dye (ThermoFisher,
L1011), CD8 (ThermoFisher, MA5-16759) and 0D44 (Biolegend, 103026). Stained
cells were analyzed
by flow cytometry.
The data shows that the first circular RNA preparation increased the number of
RBD specific CD4
T cells relative to the control, i.e., the second circular RNA preparation
(FIG. 41A) and that no changes in
RBD specific CD8 T cells were observed (FIG. 41B). In addition, the first
circular RNA preparation
increased the amount of IFN-y production by both CD4 and CD8 T cells (FIGS.
41C, 410). This shows
that circular RNA expressing IL12 is acting as an adjuvant boosting cellular
immune response elicited by
circular RNA expressing an immunogen. Data shown as mean and SEM of 3
replicates. FIG. 41A:
Asterisks denotes statistical significance as determined by a two-way RM ANOVA
protected Tukey's post
hoc test. FIGS. 41C, 410: Asterisks denotes statistical significance as
determined by unpaired t-test.
Example 55: Evaluation of T cell responses after co-administration of an
immunogen and an
adjuvant encoded by a plurality of circular RNAs
In this experiment, the following circular RNAs were produced and purified
according to the
methods described herein: (i) a first circular RNA including an IRES, an ORF
encoding Ovalbumin (OVA)
(Nucleic acid SEQ ID NO: 40; Amino acid SEQ ID NO: 41), and two spacer
elements flanking the IRES-
ORF; and (ii) a second circular RNA including an IRES, an ORF encoding the
adjuvant IL-12 (Nucleic
acid SEQ ID NO: 38; Amino acid SEQ ID NO: 39), and two spacer elements
flanking the IRES-ORF. The
first circular RNA and the second circular RNA were mixed together and
complexed with Lipofectamine
MessengerMax (Invitrogen LMRNA001) to obtain a mixture. As a control, a third
circular RNA including
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an IRES, an ORF encoding a Gluc polypeptide, and two spacer elements flanking
the IRES-ORF was
also produced.
The following were transfected into EL4 cells (10,000 cells per well; EL4
cells were obtained from
ATCC) using Lipofectamine MessengerMax according to the manufacturer's
instructions:
1) Mixture of the first circular RNA and second circular RNA complexed with
Lipofectamine
MessengerMax (0.1 picomoles eRNA OVA + 0.1 picomoles eRNA IL12);
2) first circular RNA (0.1 picomoles, eRNA OVA);
3) second circular RNA (0.1 picomoles, eRNA IL12);
4) third circular RNA (0.1 picomoles, eRNA Gluc);
5) transfection reagent alone (Blank transfection).
The transfected EL4 cells were cocultured with OT-I CD8 T cells (OVA specific
T cells; isolated
from OT-I mice from The Jackson Laboratory) for 2 days. The activation status
of the T cells was
monitored by the production of IFN-y. Supernatant was harvested after 2 days
and IFN-y detected using
a cytokine bead array (Biolegend, 741044). Data shown as mean and SEM of 3
replicates. Asterisks
denotes statistical significance as determined by a one-way ANOVA protected
Tukey's post hoc test.
FIG. 42 shows that circular RNA expressing OVA combined with circular RNA
expressing IL12
induces a stronger CD8 T cell response when compared to the control (circular
RNA expressing OVA
only). This demonstrates that circular RNA expressing IL12 acts as an
adjuvant.
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Sequences referenced in the Examples
SEQ ID NO: 1
acgttactg gccg aagccgcttgg a ataag g ccg g tg
tgcgtttgtctatatgttattttccaccatattgccgtcttttg gcaatgtg ag ggcccgg aaa
cctggccctgtcttcttg acg ag cattcctag g g g tctttcccctctcg cc aaag g
aatgcaaggtctgttg aatgtcgtg a ag g aagcagttcctctgg
aagcttcttg aag acaaacaacg tctg tag cg accctttgcaggcagcgg a accccccacctg g cg
acagg tgcctctgcggccaaaagccacg
tgtataag atacacctgcaaaggcggcacaaccccagtgccacgttgtg agttgg atagttgtgg aaag
agtcaaatg gctctcctcaagcgtattc
aacaaggggctg aagg atgcccag aaggtaccccattgtatgg g atctg atctggg
gcctcggtgcacatgctttacatgtgtttagtcg aggttaaa
aaacgtctaggccccccg aaccacgggg acgtg gttttcctttg aaaaacacg atg ataata
SEQ ID NO: 2
atg a aag cg attctggtggtgctgctgtatacctttgcg accgcg aacgcg g
ataccctgtgcattggctatcatgcg aacaacagcaccg ataccgt
ggataccgtgctggaaaaaaacgtgaccgtgacccatagcgtgaacctgctggaagataaacataacggcaaactgtgc
aaactgcgcggcgt
ggcgccgctgcatctgg gcaaatgcaacattgcgg gctgg attctgg gcaacccgg aatgcg aaagcctg
ag caccgcg agcagctgg agctat
attgtgg aaaccccg agcagcg ataacggcacctgctatccgg gcg attttattg attatg aag a actg
cg cg aacagctg agcagcgtg agcag
ctttg aacgctttg aaatttttccg aaaaccagcagctggccg aaccatg atagcaacaaaggcgtg
accgcggcgtgcccgcatgcgggcgcg a
aaagcttttataaaaacctgatttggctggtgaaaaaaggcaacagctatccgaaactgagcaaaagctatattaacga
taaaggcaaagaagtg
ctggtgctgtggg gcattcatcatccg agcaccagcgcgg atcagcag agcctgtatcag aacgcgg
atgcgtatgtgtttgtg ggcagcagccgct
atagcaaaaaatttaaaccg g aaattgcg attcgcccg aaagtgcgcg atcagg aaggccgcatg
aactattattgg accctggtg g aaccgggc
g ataaaattacctttg aagcg accg gcaacctggtggtgccgcgctatgcg tttgcg atgg
aacgcaacgcgggcagcggcattattattagcg ata
ccccggtgcatg attgcaacaccacctgccag accccg aaaggcgcg attaacaccagcctgccgtttcag
aacattcatccg attaccattggca
aatgcccg aaatatgtg aaaagcaccaaactgcgcctggcg accggcctgcgcaacattccg agcattcag ag
ccgcggcctgtttggcgcg att
gcgggctttattg aag gcggctg g accggcatggtg g atggctgg tatgg ctatcatcatcag aacg
aacaggg cagcggctatgcggcgg atctg
aaaagcacccag aacgcg attg atg aaattaccaacaaagtg aacagcgtg attg aaaaaatg
aacacccagtttaccgcggtg ggcaaag aa
tttaaccatctg g aaaaacgcattg aaaacctg aacaaaaaagtgg atg atggctttctgg atatttgg
acctataacgcgg aactgctggtgctgct
gg aaaacg aacgcaccctgg attatcatg atagcaacgtg aaaaacctgtatg
aaaaagtgcgcagccagctg aaaaacaacgcg aaag aaa
ttggcaacggctgctttg aattttatcataaatgcg ataacacctgcatgg aaagcgtg
aaaaacggcacctatg attatccg aaatatagcg aag a
agcg aaactg aaccgcg aag aaattg atggcgtg aaactgg a aag cacccg catttatcag
attctggcg atttatagcaccgtggcg ag c ag cc
tggtgctggtg gtg agcctgggcgcg attagcttttg g atgtgcagcaacg
gcagcctgcagtgccgcatttgcatt
SEQ ID NO: 3
atg a aag cg aacctgctggtgctgctgtgcgcgctggcggcggcg g atgcg
gataccatttgcattggctatcatg cg aacaacagcaccg atacc
gtgg ataccgtgctg g aaaaaaacgtg accgtg acccatagcgtg aacctg ctgg aag
atagccataacggcaaactgtgccgcctg aaaggc
attgcgccgctgcagctgggcaaatgcaacattgcgggctggctgctgg gcaacccgg aatgcg
atccgctgctgccggtgcgcagctgg agcta
tattgtgg a aaccccg aacagcg a aaacg g catttg ctatccg g g cg attttattg attatg
aag a actg cg cg aacagctg agcagcgtg agcag
ctttg aacgctttg aaatttttccg aaag aaagcagctg gccg aaccataacaccaacggcgtg
accgcggcgtgcagccatg aaggcaaaagc
agcttttatcgcaacctgctgtg gctg accg aaaaag aaggcagctatccg aaactg aaaaacagctatgtg
aacaaaaaaggcaaag aagtgc
tggtgctgtgg ggcattcatcatccgccg aacagcaaag aacagcag aacctgtatcag aacg
aaaacgcgtatgtg agcgtggtg accagcaa
ctataaccgccgctttaccccgg aaattgcgg aacgcccg aaagtgcgcg atcag gcgggccgcatg
aactattattgg accctgctg aaaccgg
gcg ataccattatttttg aagcg aacggcaacctg attgcgccg atg tatg cgtttgcgctg
agccgcggctttg gcagcggcattattaccagcaacg
cg agcatgcatg aatgcaacaccaaatgccag accccgctgggcgcg attaacagcagcctgccgtatcag a
acattcatccg g tg accattggc
g aatgcccg aaatatgtgcgcagcgcg aaactgcgcatgg tg accggcctg cgcaacattccg
agcattcag agccgcggcctgtttggcgcg at
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tgcgggctttattg aaggcggctgg accg g catg attg atg g ctg g tatg g ctatcatcatcag
aacg a acag g g cag cg g ctatg cg g cg g atca
gaaaagcacccagaacgcgattaacggcattaccaacaaagtgaacaccgtgattgaaaaaatgaacattcagtttacc
gcggtgggcaaaga
atttaacaaactggaaaaacgcatggaaaacctgaacaaaaaagtgg
atgatggctttctggatatttggacctataacgcggaactgctggtgctg
ctgg aaaacg aacgcaccctgg attttcatg atagcaacgtg aaaaacctg tatg aaaaag tg
aaaagccagctg aaaaacaacgcg aaag a
aattggcaacggctgctttgaattttatcataaatgcgataacgaatgcatggaaagcgtgcgcaacggcacctatgat
tatccgaaatatagcgaa
gaaagcaaactgaaccgcgaaaaagtggatggcgtgaaactggaaagcatgggcatttatcagattctggcgatttata
gcaccgtggcgagca
gcctggtgctgctggtgagcctgggcgcgattagcttttggatgtgcagcaacggcagcctgcagtgccgcatttgcat
t
SEQ ID NO: 4
aaaaaacaaaaaacaaaacggctattaatagccg
aaaaacaaaaaacaaaaaaaacaaaaaaaaaaccaaaaaaacaaaacaca
SEQ ID NO: 30
M FVF LVLL P LVSSQCVN LTTRTQL P PAYTNSFTRG VYYP D KV FRSSVLHSTQDL F L P F
FSNVTW FHAI HVS
GTNGTKRFDN PVL PFN DGVYFASTEKSN II RGW 1 FGTTLDSKTQSLLIVN NATNVVIKVCE FQFCN
DPFLGV
YYHKNNKSWM ESE FRVYSSANNCTFEYVSQPFLM DLEGKQG N FKNLR EFVFKN 1 DGYFKIYSKHTPI
NLV
RDLPQG FSAL E PLVDL PIG IN ITRFQTLLALH RSYLTPG DSSSGWTAGAAAYYVGYLQPRTFLLKYN
ENGTI
TDAVDCAL D PLS ETKCTLKS FTV E KG IYQTSN FRVQPTESIVRFPN ITN LC PFG EV FNATR
FASVYAWN RK
RISNCVADYSVLYNSASFSTFKCYGVSPTKLN DLCFTNVYADSFVI RG D EV RQIAPGQTG KIADYNYKLPD
D FTGCV !AWN SN N L DSKVG G NYNYLYR L FRKSN LK P F ER DI STE IYQAGSTPCNGVEG
FNCYFPLQSYG F
QPTNGVGYQPYRVVVLSFELLHAPATVCG PKKSTN LVKNKCVN FN FNG LTGTGVLT ESN KKFLPFQQFG
R D IADTTDAV R D PQTL El L D ITPCSFGG VSV ITPGTNTSNQVAVLYQ DVNCT EV PVAI
HADQLTPTWRVYS
TGSNV FQTRAG CL IGAEHVN N SYEC D I PI GAG I CASYQTQTNSP R
RARSVASQSIIAYTMSLGAENSVAYS
NNSIAIPTN FTISVTTE 1 LPVSMTKTSVDCTMYICG DSTECSNLLLQYGSFCTQLN RALTG IAVEQDKNTQE
V FAQVKQ IYKTP P I KD FGG FN FSQIL P D PSK PSKRSF I E DLL FN KVTLADAG F I KQYG
DCLG D IAAR DL I CAQ
KFNG LTVL P PL LTD E M IAQYTSALLAGTITSGWTFGAGAALQI PFAMQMAYRFNG IGVTQNVLYE
NQKL IA
NQFNSAIG KIQDSLSSTASALG KLQDVVNQNAQALNTLVKQLSSN FGAISSVLN D I LS RL D P P
EAEVQI DRL
ITG RLQSLQTYVTQQL I RAAE I RASAN LAATKMSECVLGQSKRVDFCG KGYHLMSFPQSAPHGVVFLHVT
YVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYE PQ 1 ITTDNTFVSG NC DVV IG IVNNTV
YDPLQ P EL DSFKE E L D KYFKN HTS PDVDLG D 1 SG INASVVN IQKE 1 DRLN EVAKNLN ESL
1 DLQ E LG KYEQY
IKWPWYIWLG FIAG LIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDED DS E PVLKGVKLHYT
SEQ ID NO: 31
atgtttgtttttcttgttttattgccactagtctctagtcagtgtgttaatcttacaaccagaactcaattaccccctg
catacactaattctttcacacgtggtgttt
attaccctgacaaagttttcagatcctcagttttacattcaactcaggatttgttcttacctttcttttccaatgttac
ttggttccatgctatacatgtctctggg a
ccaatg g tactaag ag g tttg ataaccctg tcctaccatttaatg atg g tg tttattttg
cttccactg ag a ag tctaacataataag ag g ctg g atctttg g
tactactttagattcgaag
acccagtccctacttattgttaataacgctactaatgttgttattaaagtctgtgaatttcaattttgtaatgatccat
ttttgggtgt
ttattaccacaaaaaca acaaaag ttg g atg g aaag tg ag ttcag ag tttattctag tg cg
aataattg cacttttg a atatg tctctcag ccttttcttatg
g accttg aag g a aaacag g g taatttcaaaaatcttag g g
aatttgtgtttaagaatattgatggttattttaaaatatattctaagcacacgcctattaatt
tag tg cg tg atctccctcag g g tttttcg g ctttag aaccattg g tag atttg ccaatag g
tattaacatcactag g tttcaaactttacttg ctttacatag aa
gttatttgactcctggtgattcttcttcaggttggacagctggtgctgcagcttattatgtgggttatcttcaacctag
gacttttctattaaaatataatg aaaa
tgg a accattacag atg ctg tag actg tg cacttg accctctctcag aaacaaag tg tacg ttg
aaatccttcactg tag aa aaag g a atctatca aa
cttctaactttagagtccaaccaacagaatctattgttagatttcctaatattacaaacttgtgcccttttggtgaagt
ttttaacgccaccagatttgcatccg
190
CA 03193746 2023-03-02
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tgtatgcttgg aacag g aag ag aatcagcaactgtgttgctg attattctg
tcctatataattccgcatcattttccacttttaagtgttatgg agtg tctccta
ctaaattaaatg atctctgctttactaatgtctatgcag attcatttgtaattag aggtg atg a ag tcag
acaaatcgctccagggcaaactgg aaag att
gctg attataattataaattaccag atg attttacaggctgcgttatagcttgg aattctaacaatcttg
attctaaggttggtg g taattataattacctg tat
ag attgtttagg aagtctaatctcaaaccttttg ag ag ag atatttcaactg aaatctatcag g ccg g
tag cacaccttg taatg g tg ttg aag gttttaatt
gttactttcctttacaatcatatggtttccaacccactaatggtgttg gttaccaaccatacag ag tag tag
tactttcttttg aacttctacatgcaccag ca
actgtttgtgg
acctaaaaagtctactaatttggttaaaaacaaatgtgtcaatttcaacttcaatggtttaacaggcacaggtgttctt
actg agtctaac
aaaaagtttctgcctttccaacaatttggcag ag acattgctg acactactg atgctgtccgtg atccacag
acacttg ag attcttg acattacaccatg
ttcttttggtggtgtcagtgttataacaccagg aacaaatacttctaaccaggttgctgttctttatcag g
atgttaactgcacag aagtccctgttgctattc
atgcag atcaacttactcctacttg gcgtgtttattctacag gttctaatg
tttttcaaacacgtgcaggctgtttaatag gggctg aacatg tcaacaactc
atatg agtgtg acatacccattggtgcaggtatatgcgctagttatcag actcag actaattctcctcg g cg
g g cacg tag tg tag ctag tca atccatc
attgcctacactatgtcacttgg tgcag aaaattcag
ttgcttactctaataactctattgccatacccacaaattttactattag tgttaccacag aaattct
accagtgtctatg accaag acatcag tag attgtacaatgtacatttgtgg tg attcaactg
aatgcagcaatcttttg ttg caatatg g cag tttttg tac a
caattaaaccgtgctttaactgggatagctgttgaacaagacaaaaacacccaagaagtttttgcacaagtcaaacaaa
tttacaaaacaccacca
attaaag attttggtggttttaatttttcacaaatattaccag atccatcaaaaccaagcaag
aggtcatttattg aag atctacttttcaacaaagtg aca
cttgcag atgctggcttcatcaaacaatatggtg attgccttg gtg atattgctgctaggg
acctcatttgtgcacaaaagtttaacggccttactg ttttgc
cacctttgctcacag atg aaatg attgctcaatacacttctgcactg ttagcgggtacaatcacttctg
gttgg acctttggtgcaggtgctgcattacaaa
taccatttgctatgcaaatggcttataggtttaatg gtattgg agttacacag aatgttctctatg ag
aaccaaaaattg attgccaaccaatttaatagtg
ccattggcaaaattcaag actcactttcttccacagcaagtgcacttgg a a aacttcaag
atgtggtcaaccaaaatgcacaagctttaaacacgctt
gttaaacaacttagctccaattttggtgcaatttcaagtg ttttaaatg atatcctttcacgtcttg
accctcccg aggctg aagtgcaaattg atag gttg at
cacaggcag acttcaaagtttgcag acatatg tg actcaacaattaattag agctgcag aaatcag
agcttctgctaatcttgctgctactaaaatgtc
ag agtgtg tacttg g acaatcaaaaag agttg atttttgtgg aaag g g ctatcatcttatg
tccttccctcag tcag cacctcatg g tg tag tcttcttg cat
gtg acttatgtccctgcacaag aaaag aacttcacaactgctcctgccatttgtcatg atgg
aaaagcacactttcctcgtg aaggtgtctttgtttcaaat
ggcacacactggtttgtaacacaaagg aatttttatg a accacaaatcatt actacag
acaacacatttgtgtctgg taactgtg atg ttgtaatagg aa
ttgtcaacaacacagtttatg atcctttgcaacctg aattag actcattcaag g agg agttag
ataaatattttaag aatcatacatcaccag atgttg att
tag g tg acatctctggcattaatgcttcagttgtaaacattcaaaaag aaattg accgcctcaatg
aggttgccaag aatttaaatg aatctctcatcg at
ctccaag aacttg g aaagtatg agcagtatataaaatggccatg
gtacatttggctaggttttatagctggcttg attgccatagtaatggtg acaattat
gctttgctgtatg accag ttg ctg tag ttg tctcaag g g ctg ttg ttcttg tgg
atcctgctgcaaatttg atg aag acg actctg agccagtgctcaaagg
agtcaaattacattacaca
SEQ ID NO: 32
MG VKVL FAL I C I AVA EAKR VQ PT ESI V R F PN ITN LC PFG EV FN AT R FASVYAWN
RK R I SNCVADYSVLYN S
AS FST FKCYGVS PTKLN D LC FTNVYA DS FV I RG D EV RQIA PG QTG K I ADYN YKL P D
D FTG CV !AWN SN N L
DSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV
VV LS F E L L H A PATVCG PKKSTN LVKN KCVN F
SEQ ID NO: 33
atgg g agtcaaagttctgtttgccctg atctgcattgctgtggccg ag g cc aagag agtccaaccaacag
aatctattgttag atttcctaatattacaa
acttgtgcccttttg gtg a ag tttttaacg ccaccag atttgcatccgtgtatgcttgg aacagg aag
ag aatcagcaactgtgttgctg attattctgtcct
atataattccgcatcattttccacttttaagtgttatg g agtgtctcctactaaattaaatg
atctctgctttactaatgtctatgcag attcatttgtaattag ag
gtg atg aagtcag acaaatcgctccagggcaaactg g aaag attgctg attataattataaattaccag
atg attttacaggctgcgttatagcttgg a
attctaacaatcttg attctaaggttg gtggtaattataattacctgtatag attgtttagg
aagtctaatctcaaaccttttg ag ag ag atatttcaactg aa
atctatcag g ccg g tag cacaccttg taatg g tg ttg
aaggttttaattgttactttcctttacaatcatatggtttccaacccactaatggtgttggttaccaa
191
CA 03193746 2023-03-02
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ccatacag ag tag tag tactttcttttg aacttctacatgcaccagcaactgtttgtgg
acctaaaaagtctactaatttggttaaaaacaaatgtg tcaatt
tc
SEQ ID NO: 34
MKA 1 LVVLLYT FATANA DTLC IG YHAN N ST DTV DTVL EKNVTVTHSVNLLE DKH N G KLCKL
RGVA P LH LG K
CN IAG W 1 LG N P EC ESLSTASSWSYIVETPSSDNGTCYPG D F 1 DYE
ELREQLSSVSSFERFEIFPKTSSW PN
H DSN KG VTAAC PHAGAKSFYKNLIWLVKKGNSYPKLSKSYIN D KG KEVLVLWGIHH PSTSADQQSLYQN
A DAYV FVGSS RYSKKFK P E 1AI R P KV R DQ EG R MNYYWTLVE PG DKITF EATGN LVV P
RYA FAM E RNAGS
GIIISDTPVH DCNTTCQT P KG AI NTSL PFQN 1 H P IT IG KC PKYVKSTKL RLATG L RN 1 PS
IQS RG L FGA IAG Fl E
GGWTG MV DG WYG YH HQ N EQGSGYAADLKSTQNAI DE ITN KVN SVI E KM NTQ FTAVG
KEFNHLEKR 1 EN L
NKKVDDG F L D IWTYNAE L LVL L EN ERTLDYH DSNVKN LYE KVRSQ LKN NAK E IG NGCF
EFYH KC D NTCM
ESVKNGTYDYPKYSEEAKLN RE El DGVKLESTRIYQI LA IYSTVASSLVLVVSLG AI S FW MCSNG
SLQC RICI
SEQ ID NO: 35
ATGAAAG CAATACTAGTAG TTCTTCTATATACATTTG CAAC CG CTAACG CTGATACATTG TG TATAG GA
TATCACGCGAACAACTCCACCGATACAGTAGATACAGTACTAGAGAAGAACGTAACAGTAACACATT
CTG TTAATCTTCTAG AAG ACAAG CATAACGG CAAACTGTGCAAACTAAG AG G TG TAGCCCCATTGCA
TCTAG GAAAG TG TAATATAG CTG G CTG G ATTTTG G G AAATCCAG AG TG TG AATCATTAAGTAC
AG CAA
GCTCCTGGTCCTATATAGTGGAAACACCTAGTAGTGATAACGGAACGTGTTACCCAGGAGATTTTATA
GATTACG AG GAG CTAAG AG AG CAGCTGTCGTCAG TATCATCATTTG AAAGG TTTGAAATTTTCCCG AA
AACATCCTCCTGGCCCAATCACGATAGTAACAAAGGAGTAACAGCAGCCTGTCCTCACGCTGGAGCA
AAAAGCTTCTATAAAAATTTAATCTGGCTAGTGAAGAAGGGAAATTCATATCCAAAGCTAAGTAAAAGT
TATATAAACGATAAGGGCAAGGAAGTACTCGTACTGTGGGGCATTCATCATCCATCTACTAGTGCTG
ATCAAC AAAGTTTATATC AAAACG CAG ACG CATACG TTTTTG TG G G G TCAAGTAGATATAG CAAG
AAA
TTTAAACCAGAAATAGCAATAAGACCTAAAGTAAGGGATCAAGAAGGCAGAATGAACTATTATTGGAC
ACTAG TAG AACCG GG AG ATAAAATAACTTTTG AAG CAACAG GAAATCTAG TGG TTCCCAG GTACG
CA
TTTGCAATGGAAAGAAACGCTGGATCAGGCATCATTATATCTGATACACCAGTCCACGATTGTAATAC
AACTTGTCAAAC ACCTAAAG GAG CTATAAAC ACC AG CTTACCATTTCAAAATATTCATCCTATC AC AAT
TG GAAAGTGTCCAAAATACG TAAAAAGTACAAAATTG AG ATTG G CCACAG G ATTACG AAATATTCCAT
CAATTCAATCTAG AGG ACTTTTTG GTGCAATTG CAGG TTTCATAGAAGG AG G CTG GACTG GG ATGG
T
AGACGGCTGGTACGGTTATCATCATCAAAACGAACAGGGAAGTGGATACGCAGCTGATCTTAAAAGT
ACACAAAACGCAATTGACGAGATTACTAATAAAGTAAATTCTGTAATTGAAAAAATGAATACTCAGTTT
ACAG C AG TAG G GAAAGAG TTTAACC ACCTG G AAAAAAG AATAG AAAATTTAAATAAAAAAGTAG
ACGA
CGGATTTCTTGACATTTGGACTTATAACGCCGAACTATTGGTATTACTAGAAAACGAAAGAACTCTAG
ATTATCACGATTCAAACGTAAAAAATTTATACGAAAAAGTAAGAAGCCAACTTAAAAATAACGCAAAAG
AAATAGGAAACGGCTGTTTTGAATTTTATCACAAGTGTGATAATACCTGCATGGAAAGTGTTAAAAAC
GG GACATACGATTATCCAAAATACTC AG AAG AAG CAAAATTAAATAGAGAAG AAATAG ACG GCG TAA
AATTAGAATCAACAAGGATATATCAAATATTAGCAATATATTCAACTGTCGCTTCTTCATTGGTACTGG
TAGTTTCTCTAG GTG CAATATCATTTTG GATG TG CTCTAACG G CTCCCTAC AGTGTAG AATTTG TATA
SEQ ID NO: 36
192
CA 03193746 2023-03-02
WO 2022/051629
PCT/US2021/049077
MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTR
GCL I CLSH I KCTPKM KKF I PG RCHTYEG DKESAQGG I G EAIV DI PEI PG FK DL EPM
EQFIAQVDLCVDCTTG
CLKG LANVQCS DLL KKWL PQRCATFASKIQGQVDKI KGAGG D
SEQ ID NO: 37
ATGGGAGTCAAAGTTCTGTTTGCCCTGATCTGCATCGCTGTGGCCGAGGCCAAGCCCACCGAGAAC
AACGAAGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCGACCACGGATCTCGATGCTGACCGC
GGGAAGTTGCCCGGCAAGAAGCTGCCGCTGGAGGTGCTCAAAGAGATGGAAGCCAATGCCCGGAA
AGCTGGCTGCACCAGGGGCTGTCTGATCTGCCTGTCCCACATCAAGTGCACGCCCAAGATGAAGAA
GTTCATCCCAGGACGCTGCCACACCTACGAAGGCGACAAAGAGTCCGCACAGGGCGGCATAGGCG
AGGCGATCGTCGACATTCCTGAGATTCCTGGGTTCAAGGACTTGGAGCCCATGGAGCAGTTCATCG
CACAGGTCGATCTGTGTGTGGACTGCACAACTGGCTGCCTCAAAGGGCTTGCCAACGTGCAGTGTT
CTGACCTGCTCAAGAAGTGGCTGCCGCAACGCTGTGCGACCTTTGCCAGCAAGATCCAGGGCCAGG
TGGACAAGATCAAGGGGGCCGGTGGTGACTAA
SEQ ID NO: 38
atggctccccaaaagctgaccatcagctggttcgccatcgtgctgctcgtg
agccccctgatggccatgtgggagctggagaaggacgtgtacgtg
gtcgaggtgg actgg acccccgacgcccccggcgaaacagtgaacctgacctgcgacacccccgaggaag
acgatatcacctggacaagcg
accag cg g cacg g cg tg atcg g cag cg g caag accctg accatcaccg tg a ag g
agttcctgg acgccggccagtacacctgccacaagggc
gg ag aaacactg agccacagccacctgctgttgcacaag aaag ag aacggcatctgg agcaccg ag
atcctcaagaatttcaagaacaagac
cttcctgaagtgcgaggcccccaactacagcggccggttcacctgcagctggctggtgcagcgg
aacatggacctgaagttcaacatcaagagct
ccagctctagccccgacagccgggccgtgacctgcggcatggccagcctgagcgccgagaaggtg accctgg
accagcgggactacgagaa
g tacag cg tg ag ctg ccag g ag g acg tg acctg ccctaccg ctg ag g a aacactg
cccatcg ag ctg g ccctg g ag g cccg g cag ca aaaca
agtacg agaactacagcaccagcttctttatccggg
acatcattaagcccgacccccctaagaacctgcagatgaagcccctgaagaacagcca
ggtggaggtgagctgggagtaccccgacagctggagcaccccccacagctacttcagcctgaagttcttcgtgcggatc
cagcggaagaaggag
aagatgaagg aaacag ag g agg gctgcaaccag aagggcgccttcctggtg g aaaag
acaagcacagaggtgcagtgcaagggcggcaa
cgtgtgcgtgcaggcccaggaccggtactataacagcagctgcagcaagtgggcctgcgtgccctgccgggtgcggagt
gtgcctggcgtcggc
gtgcccggcgtgggccgggtgatccccgtgagcggccccgcccggtgcctgagccagagccggaacctgctg
aagaccacagacgacatggt
gaagaccgcccgggag aagctg
aagcactacagctgcaccgccgaggacatcgaccacgaggacatcacccgggaccagaccagcaccct
gaagacctgcctccccctggagctgcacaagaacg
agagctgcctggccacccgggaaacaagctctaccacacggggcagctgcctgcctcc
tcagaagaccagcctgatgatgaccctgtgcctgggcagcatctacgaggacctgaagatgtaccagaccgagttccag
gccatcaacgccgctc
tgcag a accacaaccaccag cag atcatcctg g acaag g g catg ctg g tg g ccatcg acg ag
ctg atg cag ag cctg aaccacaacg g cg aa
acactgcggcag aagcccccagtgggcgaggccgacccctaccgggtgaag
atgaagctgtgcatcctgctgcacgccttcagcacccgggtg
g tg accatcaaccg g g tg atg g g ctacctg ag cag cg cc
SEQ ID NO: 39
MC PQKLT I SW FAIVLLVS P LMAMW EL E KDVYVV EV DWT P DA PG ETVN LTC DT PE E D D
ITWTS DQR HGV I
G SG KTLT ITVK E F L DAGQYTCH KG G ETLSHSH LLLH KK E NG IWST E I LKN FKNKT
FLKC EA PNYSG RFTCS
WLVQRNM DLKFN IKSSSSSP DS RAVTCG MASLSAEKVTL DQR DYE KYSVSCQ E DVTC PTAE ETLP
I ELAL
EARQQNKYENYSTSF Fl R DI IKP DP PKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKF FV R I QRKK
E K
MK ETE EGCNQKGAFLVE KTST EVQC KG G NVCVQAQ D RYYNSSCSKWACV PC RVRSV PGVG VPG
VG R
VI PVSG PARCLSQSRNLLKTTDDMVKTAR EKLKHYSCTAE DI DH E D IT R DQTSTL KTCL P L E
LH KN ESC LA
193
CA 03193746 2023-03-02
WO 2022/051629
PCT/US2021/049077
TRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQS
LNHNGETLRQKPPVG EADPYRVKMKLCILLHAFSTRVVTIN RVMGYLSSA
SEQ ID NO: 40
atgggcagcatcggcgccgctagcatggagttctgctttgacgtcttcaaagagctgaaggtgcaccacgccaacgaga
acatcttctactgcccta
ttgccatcatgagcgccctggccatggtgtacctgggcgccaaggacagcacccggacccagatcaacaaggtggtccg
gttcg acaagctgcc
cggcttcggcgacagcatcgaggcccagtgcggcaccagcgtg
aacgtgcacagctccctgcgggacatcctgaaccagatcaccaagcccaa
cgacgtgtacagcttcagcctggccagccggctgtacgccgaggaacggtaccccatcctgcccgagtacctgcagtgc
gtg aaggagctgtacc
ggggcggactggagcccatcaacttccagaccgccgccgaccaggcccggg
agctgatcaacagctgggtggagagccagaccaacggcat
cattcggaacgtgctgcagcccagcagcgtgg
acagccagaccgccatggtgctggtgaacgccatcgtgttcaagggcctgtgggagaaggcc
ttcaaggacgaggacacccaggccatgcccttccgggtgaccgagcaggag
agcaagcccgtgcagatgatgtaccagatcggcctgttccgg
gtggccagcatggccagcg agaagatg aag atcctgg agctgcccttcgccagcggcaccatg agcatgctg
gtgctgctccccg acg aggtg a
gcggcctggagcagctggagagcatcatcaacttcgagaagctgaccg
agtggaccagctctaacgtgatggaggagcggaagatcaaggtgt
acctgccccggatgaagatggaggagaagtacaacctgaccagcgtgctgatggccatgggcatcaccgacgtgttcag
cagcagcgccaacc
tgagcggcatcagctccgccgagagcctgaagatcagccaggccgtgcacgccgcacacgccgagatcaacgaggccgg
ccgggaggtggt
gggcagcgccgaggccggcgtgg acgccgccagcgtgagcg
aggagttccgggccgaccaccccttcctgttctgcatcaagcatattgccacc
aacgccgtgctgttctttggccggtgcgtgagcccc
SEQ ID NO: 41
MGSIGAASM EFCFDVFKELKVHHAN EN I FYC PIAIMSALAMVYLGAKDSTRTQI NKVVR F DKLPG FG
DSI E
AQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQA
R ELI NSWV ESQTNG I I RNVLQ PSSV DSQTAMVLVNAIVFKGLWEKAFKD EDTQAM PF RVTEQ
ESKPVQM
MYQIGLFRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYL
PRMKMEEKYNLTSVLMAMGITDVFSSSANLSG ISSAESLKISQAVHAAHAE IN EAG R EVVGSAEAGV DAA
SVSEEFRADHPFLFCIKHIATNAVLFFGRCVSP
194
