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CIRCULAR POLYRIBONUCLEOTIDES ENCODING ANTIFUSOGENIC POLYPEPTIDES
Background
Delivery of polynucleotides and proteins is important for a wide variety of
therapeutic fields.
However, current delivery modalities are often ineffective. For example,
delivery of short polypeptides,
such as polypeptides encoding an antifusogenic polypeptide, often results in
short half-life and rapid
clearance of the polypeptides. Accordingly, a need exists for improved
compositions and methods for
delivering an antifusogenic polypeptide, e.g., to treat or prevent a viral
infection.
Summary of the Invention
The disclosure provides compositions and methods for producing, purifying, and
using circular
RNA encoding an antifusogenic polypeptide.
In one aspect, the invention features a circular polyribonucleotide that
includes a
polyribonucleotide cargo encoding the antifusogenic polypeptide. In some
embodiments, the
polyribonucleotide cargo includes an expression sequence encoding the
antifusogenic polypeptide.
In some embodiments, the polyribonucleotide cargo includes an expression
sequence encoding a
polypeptide of Table 1. In some embodiments, the polyribonucleotide cargo
includes an expression
sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%,
97%, 99%, or 100%)
sequence identity to a polypeptide of Table 1. In some embodiments, the
polyribonucleotide cargo
includes an expression sequence encoding a polypeptide having at least 85%
(e.g., at least 90%, 95%,
97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 1-324.
In some embodiments, the circular polyribonucleotide includes a splice
junction joining a 5' exon
fragment and a 3' exon fragment.
In some embodiments, the polyribonucleotide cargo includes an IRES operably
linked to the
expression sequence encoding the antifusogenic polypeptide. The circular
polyribonucleotide may further
include a spacer region between the IRES and the 3' exon fragment or the 5'
exon fragment. The spacer
region may be at least 5 ribonucleotides in length. For example, the spacer
region may be at least 10,
20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,
1,000, or more
ribonucleotides in length. In some embodiments, the spacer region is from 5 to
500 ribonucleotides in
length. The spacer region may include a polyA, a polyA-C, polyA-U, or polyA-G
sequence. The spacer
region may be a random sequence.
In some embodiments, the circular polyribonucleotide is at least 500
ribonucleotides in length.
For example, the circular polyribonucleotide may be at least 500, 600, 700,
800, 900, 1,000, 2,000, 3,000,
4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000,
30,000, 35,000, 40,000,
45,000, 50,000, or more polyribonucleotides. In some embodiments, the circular
polyribonucleotide is
from 500 to 20,000 ribonucleotides in length.
In another aspect, featured is a linear polyribonucleotide including, from 5'
to 3', (A) a 3' intron
fragment; (B) a 3' splice site; (C) a 3' exon fragment; (D) a
polyribonucleotide cargo encoding the
antifusogenic polypeptide; (E) a 5' exon fragment; (F) a 5' splice site; and
(G) a 5' intron fragment.
In some embodiments, the polyribonucleotide cargo includes an expression
sequence encoding
the antifusogenic polypeptide.
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In some embodiments, the polyribonucleotide cargo includes an IRES operably
linked to the
expression sequence encoding the antifusogenic polypeptide (e.g., a
polypeptide of Table 1). The
circular polyribonucleotide may further include a spacer region between the
IRES and the 3' exon
fragment or the 5' exon fragment. The circular polyribonucleotide may further
include a spacer region
between one or more of (A), (B), (C), (D), (E), (F), and (G).
The spacer region may be at least 5 ribonucleotides in length. For example,
the spacer region
may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,
600, 700, 800, 900, 1000, or
more ribonucleotides in length. In some embodiments, the spacer region is from
5 to 500 ribonucleotides
in length. The spacer region may include a polyA, a polyA-C, polyA-U, or polyA-
G sequence. The spacer
region may be a random sequence.
In some embodiments, the circular polyribonucleotide lacks an !RES. In some
embodiments, the
circular polyribonucleotide lacks one or both of a 5' cap and a polyA
sequence.
In some embodiments, the circular polyribonucleotide comprises a protein
translation initiation
site. In some embodiments, the protein translation initiation site comprises a
Kozak sequence.
In some embodiments, the linear polyribonucleotide is at least 500
ribonucleotides in length. For
example, the linear polyribonucleotide may be at least 500, 600, 700, 800,
900, 1,000, 2,000, 3,000,
4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 25,000,
30,000, 35,000, 40,000,
45,000, 50,000, or more polyribonucleotides. In some embodiments, the linear
polyribonucleotide is from
500 to 20,000 ribonucleotides in length.
In another aspect, featured is a DNA vector encoding a polyribonucleotide
(e.g., a linear or
circular polyribonucleotide) as described herein.
In another aspect, featured is a method of expressing an antifusogenic
polypeptide (e.g., a
polypeptide of Table 1) in a cell. The method includes providing a circular, a
linear polyribonucleotide, or
the DNA vector as described herein to the cell under conditions suitable to
express the antifusogenic
polypeptide.
In another aspect, featured is a method of producing a circular
polyribonucleotide from a linear
polyribonucleotide as described herein. The method includes providing the
linear polyribonucleotide
under conditions suitable for self-splicing of the linear polyribonucleotide
to produce the circular
polyribonucleotide.
In another aspect, featured is a pharmaceutical composition that includes the
circular
polyribonucleotide, the linear polyribonucleotide, or the DNA vector of any of
the above embodiments,
and a diluent, carrier, or excipient.
In another aspect, featured is a method of expressing the antifusogenic
polypeptide (e.g., a
polypeptide of Table 1) in a subject. The method includes administering a
first dose of the
pharmaceutical composition in an amount sufficient to produce a serum
concentration of at least 500
ng/mL (e.g., at least 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL,
1,100 ng/mL, 1,200
ng/mL, 1,300 ng/mL, 1,400 ng/mL, 1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800
ng/mL, 1,900 ng/mL,
2,000 ng/mL, 2,100 ng/mL, 2,200 ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL,
2,600 ng/mL, 2,700
ng/mL, 2,800 ng/mL, 2,900 ng/mL, 3,000 ng/mL, or more) of the antifusogenic
polypeptide (e.g., a
polypeptide of Table 1) in the subject.
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In some embodiments, the method may further include administering a second
dose of the
pharmaceutical composition. The method may further include administering a
third, fourth, fifth, sixth,
seventh, eighth, ninth, tenth, or more doses of the pharmaceutical
composition.
In some embodiments, the second dose is administered at least one hour (e.g.,
at least two
hours, three hours, four hours, five hours, six hours, seven hours, eight
hours, nine hours, 10 hours, 11
hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours,
19 hours, 20 hours, 21
hours, 22 hours, 23 hours, one day, two days, three days, four days, five
days, six days, one week, two
weeks, three weeks, one month, two months, three months, four months, five
months, six months, seven
months, eight months, nine months, ten months, eleven months, one year, or
longer) after the first dose
of the pharmaceutical composition.
In some embodiments, the second dose is administered from 1 hour to 1 year
(e.g., from 1 hour
to 1 day, e.g., one hour, two hours, three hours, four hours, five hours, six
hours, seven hours, eight
hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours,
16 hours, 17 hours, 18
hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or one day, e.g.,
from one day to one week,
e.g., two days, three days, four days, five days, six days, or one week, e.g.,
from one week to one month,
e.g., two weeks, three weeks, or one month, e.g., from one month to one year,
e.g., one month, two
months, three months, four months, five months, six months, seven months,
eight months, nine months,
ten months, eleven months, or one year) after the first dose of the
pharmaceutical composition. In some
embodiments, the second dose is administered from 1 days to 180 days (e.g.,
from 1 day to 90 days,
from 1 day to 45 days, from one day to 30 days, from 1 day to 14 days, from 1
day to 7 days, from 2 days
to 45 days, from 2 days to 30 days, from 2 days to 14 days, from 2 days to 7
days, from 3 days to 90
days, from 3 days to 45 days, from 3 days to 30 days, from 3 days to 14 days,
from 3 days to 7 days,
from 4 days to 90 days, from 4 days to 45 days, from 4 days to 30 days, from 4
days to 14 days, from 4
days to 7 days, from 5 days to 90 days, from 5 days to 45 days, from 5 days to
30 days, from 5 days to 14
days, from 5 days to 7 days, from 6 days to 90 days, from 6 days to 45 days,
from 6 days to 30 days,
from 6 days to 14 days, from 6 days to 7 days, from 7 days to 90 days, from 7
days to 45 days, from 7
days to 30 days, from 7 days to 14 days, from 14 days to 90 days, from 14 days
to 45 days, from 14 days
to 30 days, from 21 days to 90 days, from 21 days to 60 days, from 21 days to
45 days, from 21 days to
days, from 30 days to 90 days, from 30 days to 60 days, from 30 days to 45
days, from 45 to 180
30 days, from 45 to 120 days, form 45 to 100 days, from 45 to 90 days, from
45 to 60 days, from 60 to 180
days, from 60 to 120 days, from 60 to 100 days, from 60 to 90 days, from 90 to
100 days, from 90 to 120
days, or from 90 to 180 days) after the first dose of the pharmaceutical
composition.
In some embodiments, the third dose is administered at least one hour (e.g.,
at least two hours,
three hours, four hours, five hours, six hours, seven hours, eight hours, nine
hours, 10 hours, 11 hours,
12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19
hours, 20 hours, 21 hours, 22
hours, 23 hours, one day, two days, three days, four days, five days, six
days, one week, two weeks,
three weeks, one month, two months, three months, four months, five months,
six months, seven months,
eight months, nine months, ten months, eleven months, one year, or longer)
after the second dose of the
pharmaceutical composition.
In some embodiments, the third dose is administered from 1 hour to 1 year
(e.g., from 1 hour to 1
day, e.g., one hour, two hours, three hours, four hours, five hours, six
hours, seven hours, eight hours,
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nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16
hours, 17 hours, 18 hours, 19
hours, 20 hours, 21 hours, 22 hours, 23 hours, or one day, e.g., from one day
to one week, e.g., two
days, three days, four days, five days, six days, or one week, e.g., from one
week to one month, e.g., two
weeks, three weeks, or one month, e.g., from one month to one year, e.g., one
month, two months, three
months, four months, five months, six months, seven months, eight months, nine
months, ten months,
eleven months, or one year) after the second dose of the pharmaceutical
composition. In some
embodiments, the third dose is administered from 1 days to 180 days (e.g.,
from 1 day to 90 days, from 1
day to 45 days, from one day to 30 days, from 1 day to 14 days, from 1 day to
7 days, from 2 days to 45
days, from 2 days to 30 days, from 2 days to 14 days, from 2 days to 7 days,
from 3 days to 90 days,
from 3 days to 45 days, from 3 days to 30 days, from 3 days to 14 days, from 3
days to 7 days, from 4
days to 90 days, from 4 days to 45 days, from 4 days to 30 days, from 4 days
to 14 days, from 4 days to 7
days, from 5 days to 90 days, from 5 days to 45 days, from 5 days to 30 days,
from 5 days to 14 days,
from 5 days to 7 days, from 6 days to 90 days, from 6 days to 45 days, from 6
days to 30 days, from 6
days to 14 days, from 6 days to 7 days, from 7 days to 90 days, from 7 days to
45 days, from 7 days to 30
days, from 7 days to 14 days, from 14 days to 90 days, from 14 days to 45
days, from 14 days to 30 days,
from 21 days to 90 days, from 21 days to 60 days, from 21 days to 45 days,
from 21 days to 30 days,
from 30 days to 90 days, from 30 days to 60 days, from 30 days to 45 days,
from 45 to 180 days, from 45
to 120 days, form 45 to 100 days, from 45 to 90 days, from 45 to 60 days, from
60 to 180 days, from 60 to
120 days, from 60 to 100 days, from 60 to 90 days, from 90 to 100 days, from
90 to 120 days, or from 90
to 180 days) after the second dose of the pharmaceutical composition.
In some embodiments, the second dose is administered before a serum
concentration of the
antifusogenic polypeptide is less than about 500 ng/mL in serum of the
subject.
In some embodiments, the method maintains a serum concentration of at least
500 ng/mL (e.g.,
at least 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL, 1,100 ng/mL,
1,200 ng/mL, 1,300
ng/mL, 1,400 ng/mL, 1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800 ng/mL, 1,900
ng/mL, 2,000 ng/mL,
2,100 ng/mL, 2,200 ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL, 2,600 ng/mL,
2,700 ng/mL, 2,800
ng/mL, 2,900 ng/mL, 3,000 ng/mL, or more) of the antifusogenic polypeptide in
the subject, e.g., for at
least one hour (e.g., at least two hours, three hours, four hours, five hours,
six hours, seven hours, eight
hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours,
16 hours, 17 hours, 18
hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days,
three days, four days, five
days, six days, one week, two weeks, three weeks, one month, two months, three
months, four months,
five months, six months, seven months, eight months, nine months, ten months,
eleven months, one
year, or longer).
In some embodiments, the method treats or prevents a viral infection in the
subject. For
example, the pharmaceutical composition may be administered to the subject in
an amount and for a
duration sufficient to treat or prevent a viral infection. The pharmaceutical
composition may be
administered to the subject to reduce the risk of a viral infection.
In some embodiments, the method treats or prevents a human immunodeficiency
virus (HIV)
infection.
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In some embodiments, the method treats or prevents a coronavirus infection
(e.g., a
betacoronavirus infection, e.g., SARS-CoV-2 infection, such as a SARS-CoV-2
infection that produces
symptoms of COVID-19).
In some embodiments, the method treats or prevents a Hepatitis C Virus (HCV)
infection.
In some embodiments, a circular polynucleotide encoding the antifusogenic
polypeptide (e.g., a
polypeptide of Table 1) is used for reducing viral entry.
In another aspect, featured is a circular polyribonucleotide that includes a
polyribonucleotide
cargo encoding multiple antifusogenic polypeptides. The polyribonucleotide
cargo may include
expression sequences encoding the antifusogenic polypeptides. In some
embodiments, the
antifusogenic polypeptides are directed to the same virus. Alternatively, the
antifusogenic polypeptides
may be directed to more than one virus.
Definitions
To facilitate the understanding of this disclosure, a number of terms are
defined below. Terms
defined herein have meanings as commonly understood by a person of ordinary
skill in the areas relevant
to the disclosure. Terms such as "a", "an," and "the" are not intended to
refer to only a singular entity but
include the general class of which a specific example may be used for
illustration. The term "or" is used
to mean "and/or" unless explicitly indicated to refer to alternatives only or
the alternative are mutually
exclusive, although the disclosure supports a definition that refers to only
alternatives and "and/or." The
terminology herein is used to describe specific embodiments, but their usage
is not to be taken as
limiting, except as outlined in the claims.
As used herein, any values provided in a range of values include both the
upper and lower
bounds, and any values contained within the upper and lower bounds.
As used herein, the term "about" refers to a value that is within 10% of a
recited value.
As used herein, the term "carrier" is a compound, composition, reagent, or
molecule that
facilitates the transport or delivery of a composition (e.g_, a circular
polyribonucleotide) into a cell by a
covalent modification of the circular polyribonucleotide, via a partially or
completely encapsulating agent,
or a combination thereof. Non-limiting examples of carriers include
carbohydrate carriers (e.g., an
anhydride-modified phyto glycogen or glycogen-type material), nanoparticles
(e.g., a nanoparticle that
encapsulates or is covalently linked binds to the circular
polyribonucleotide), liposomes, fusosomes, ex
vivo differentiated reticulocytes, exosomes, protein carriers (e.g., a protein
covalently linked to the circular
polyribonucleotide), or cationic carriers (e.g., a cationic lipopolymer or
transfection reagent).
As used herein, the terms "circular polyribonucleotide," "circular RNA," and
"circRNA" are used
interchangeably and mean a polyribonucleotide molecule that has a structure
having no free ends (i.e., no
free 3' or 5' ends), for example a polyribonucleotide molecule that forms a
circular or end-less structure
through covalent or non-covalent bonds. The circular polyribonucleotide may
be, e.g., a covalently closed
polyribonucleotide.
As used herein, the term "circularization efficiency" is a measurement of
resultant circular
polyribonucleotide versus its non-circular starting material.
The term "diluent" means a vehicle including an inactive solvent in which a
composition described
herein (e.g., a composition including a circular polyribonucleotide) may be
diluted or dissolved. A diluent
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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 "expression sequence" is a nucleic acid sequence that
encodes a
product, e.g., a peptide or polypeptide (e.g., an antifusogenic polypeptide).
An exemplary expression
sequence 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 term "fragment" with respect to a polypeptide or a nucleic
acid sequence,
e.g., an antifusogenic polypeptide or a nucleic acid sequence encoding an
antifusogenic polypeptide,
refers to a continuous, less than a whole portion of a sequence of the
polypeptide or the nucleic acid. A
fragment of a polypeptide or a nucleic acid sequence encoding a polypeptide,
for instance, refers to
continuous, less than a whole fraction (e.g., at least 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%,
or 99% of the entire length) of the sequence such as a sequence disclosed
herein. It is understood that
all the present disclosure contemplates fragments of any antifusogenic
polypeptide disclosed herein.
As used herein, the term "Fc domain" refers to a polypeptide chain that
includes at least a hinge
domain and second and third antibody constant domains (CH2 and CH3) or
functional fragments thereof
(e.g., fragments that that capable of dimerizing and binding to an Fc
receptor). The Fc domain can be
any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD
(e.g., IgG). Additionally, the Fc
domain can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4) (e.g.,
Ig01). An Fc domain
does not include any portion of an immunoglobulin that can act as an antigen-
recognition region, e.g., a
variable domain or a complementarity determining region (CDR). Fc domains in
the conjugates as
described herein can contain one or more changes from a wild-type Fc domain
sequence (e.g., 1-10, 1-8,
1-6, 1-4 amino acid substitutions, additions, or deletions) that alter the
interaction between an Fc domain
and an Fc receptor. Examples of suitable changes are known in the art. Unless
otherwise specified
herein, numbering of amino acid residues in the IgG or Fc domain is according
to the EU numbering
system for antibodies, also called the Kabat EU index, as described, for
example, in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public Health
Service, National Institutes of
Health, Bethesda, MD, 1991
As used herein, the term "GC content" refers to the percentage of guanine (G)
and cytosine (C) in
a nucleic acid sequence_ The formula for calculation of the GC content is
(G+C) / (A+G+C+U) x 100%
(for RNA) or (G+C) / (A+G+C+T) x 100% (for DNA). Likewise, the term "uridine
content" refers to the
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percentage of uridine (U) in a nucleic acid sequence. The formula for
calculation of the uridine content is
U / (A+G+C+U) x 100%. Likewise, the term "thymidine content" refers to the
percentage of thymidine (T)
in a nucleic acid sequence. The formula for calculation of the thymidine
content is T / (A+G+C+T) x
100%.
By "heterologous" is meant to occur in a context other than in the naturally
occurring (native)
context. A "heterologous" polynucleotide sequence indicates that the
polynucleotide sequence is being
used in a way other than what is found in that sequence's native genome. For
example, a "heterologous
promoter" is used to drive transcription of a sequence that is not one that is
natively transcribed by that
promoter; thus, a "heterologous promoter" sequence is often included in an
expression construct by
means of recombinant nucleic acid techniques. The term "heterologous" is also
used to refer to a given
sequence that is placed in a non-naturally occurring relationship to another
sequence; for example, a
heterologous coding or non-coding nucleotide sequence is commonly inserted
into a genome by genomic
transformation techniques, resulting in a genetically modified or recombinant
genome.
As used herein, the term "intron fragment" refers to a portion of an intron,
where a first intron
fragment and a second intron fragment together form an intron, such as a
catalytic intron. An intron
fragment may be a 5' portion of an intron (e.g., a 5' portion of a catalytic
intron) or a 3' portion of an intron
(e.g., a 3' portion of a catalytic intron), such that the 5' intron fragment
and the 3' intron fragment,
together, form a functional intron, such as a functional intron capable of
catalytic self-splicing. The term
intron fragment is meant to refer to an intron split into two portions. The
term intron fragment is not meant
to state, imply, or suggest that the two portion or halves are equal in
length. The term intron fragment is
used synonymously with the term split-intron and may be used instead of the
term "half-intron."
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,
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," "linear polyribonucleotide," and
"linear polyribonucleotide
molecule" are used interchangeably and mean polyribonucleotide molecule having
a 5' and 3' end. One
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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 "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" is 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 polyribonucleotide is a
formulation that includes a circular polyribonucleotide without covalent
modification and is free from a
carrier.
As used herein, the terms "nicked RNA," "nicked linear polyribonucleotide,"
and "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.
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 "a
polyribonucleotide for use in
therapy."
The term "polynucleotide," as used herein, means a molecule including 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. As
used herein, a
polyribonucleotide sequence that recites thymine (T) is understood to
represent uracil (U).
As used herein, the term "polyribonucleotide cargo" herein includes any
sequence including at
least one polyribonucleotide. In embodiments, the polyribonucleotide cargo
includes one or multiple
expression sequences, wherein each expression sequence encodes a polypeptide.
In embodiments, the
polyribonucleotide cargo includes one or multiple noncoding sequences, such as
a polyribonucleotide
having regulatory or catalytic functions. In embodiments, the
polyribonucleotide cargo includes a
combination of expression and noncoding sequences. In embodiments, the
polyribonucleotide cargo
includes one or more polyribonucleotide sequence described herein, such as one
or multiple regulatory
elements, internal ribosomal entry site (IRES) elements, or spacer sequences.
As used interchangeably herein, the terms "polyA" and "polyA sequence" refer
to an untranslated,
contiguous region of a nucleic acid molecule of at least 5 nucleotides in
length and consisting of
adenosine residues. In some embodiments, a polyA sequence is at least 10, at
least 15, at least 20, at
least 30, at least 40, or at least 50 nucleotides in length. In some
embodiments, a polyA sequence is
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located 3' to (e.g., downstream of) an open reason frame (e.g., an open
reading frame encoding a
polypeptide), and the polyA sequence is 3' to a termination element (e.g., a
stop codon) such that the
polyA is not translated. In some embodiments, a polyA sequence is located 3'
to a termination element
and a 3' untranslated region.
As used herein, the elements of a nucleic acid are "operably connected" or
"operably linked" if
they are positioned on the vector such that they can be transcribed to form a
linear RNA that can then be
circularized into a circular RNA using the methods provided herein.
"Polydeoxyribonucleotides," "deoxyribonucleic acids," and "DNA" mean
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
deoxyribo nucleoside 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
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. Embodiments of
polynucleotides include isolated
and purified DNA/RNA molecules, synthetic DNA/RNA molecules, and synthetic
DNA/RNA analogs.
Embodiments of polynucleotides, e.g., polyribonucleotides or
polydeoxyribonucleotides, may
include one or more nucleotide variants, including nonstandard nucleotide(s),
non-natural nucleotide(s),
nucleotide analog(s), 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, 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
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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).
In embodiments, nucleic
acid molecules are 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 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. In embodiments, nucleic acid molecules contain amine -modified
groups, such as amino allyl
1-dUTP (aa-dUTP) and aminohexylacrylamide-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 or
amplification synthesis are
described in Betz K, 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 a multi-
molecular complex such as a dimer, trimer, or tetramer. They can also include
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 (e.g., a viral infection, e.g., HIV, SARS-CoV-2, HCV,
influenza, or RSV), or
alternatively, to reduce the severity or frequency of symptoms in a
subsequently developed disease or
disorder. A therapeutic agent can be administered to a subject who is at
increased risk of developing a
viral infection 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
viral infection.
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, a "spacer" refers to any contiguous nucleotide sequence (e.g.,
of one or more
nucleotides) that provides distance or flexibility between two adjacent
polynucleotide regions.
A "signal sequence" refers to a polypeptide sequence, e.g., between 10 and 45
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.
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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
are referred to as
"substantially identical" or "essentially similar" when they share at least a
certain minimal percentage of
sequence identity when optimally aligned (e.g., when aligned by programs such
as GAP or BESTFIT
using default parameters). 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 are
determined, e.g., using computer programs, such as the COG Wisconsin Package,
Version 10.3,
available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752
USA, or EmbossWin
version 2.1(10 (using the program "needle"). Alternatively, or additionally,
percent identity is determined
by searching against databases, e.g., using algorithms such as FASTA, BLAST,
etc. Sequence identity
refers to the sequence identity over the entire length of the sequence.
As used herein, the term "subject" refers to an organism, such as an animal,
plant, or microbe. In
embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish,
reptile, or amphibian). In
embodiments, the subject is a human. In embodiments, the subject is a non-
human mammal. In
embodiments, the subject is a non-human mammal such as a non-human primate
(e.g., monkeys, apes),
ungulate (e.g., cattle, buffalo, bison, sheep, goat, pig, camel, llama,
alpaca, deer, horses, donkeys),
carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g.,
rabbit). In embodiments, the
subject is a bird, such as a member of the avian taxa Galliformes (e.g.,
chickens, turkeys, pheasants,
quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches,
emus), Columbiformes (e.g.,
pigeons, doves), or Psittaciformes (e.g., parrots). In embodiments, the
subject is an invertebrate such as
an arthropod (e.g., insects, arachnids, crustaceans), a nematode, an annelid,
a helminth, or a mollusc. In
embodiments, the subject is an invertebrate agricultural pest or an
invertebrate that is parasitic on an
invertebrate or vertebrate host. In embodiments, the subject is a plant, such
as an angiosperm plant
(which can be a dicot or a monocot) or a gymnosperm plant (e.g., a conifer, a
cycad, a gnetophyte, a
Ginkgo), a fern, horsetail, clubmoss, or a bryophyte. In embodiments, the
subject is a eukaryotic alga
(unicellular or multicellular). In embodiments, the subject is a plant of
agricultural or horticultural
importance, such as row crop plants, fruit-producing plants and trees,
vegetables, trees, and ornamental
plants including ornamental flowers, shrubs, trees, groundcovers, and turf
grasses.
As used herein, the term "antifusogenic polypeptide" refers to a polypeptide,
such as a
polypeptide of between 10 and 200 amino acids, which inhibits viral fusion-
associated events such as
viral entry or viral fusion. An antifusogenic polypeptide includes, for
example, a polypeptide of Table 1.
An antifusogenic polypeptide includes a polypeptide as well as any
biologically active fragments thereof
(e.g., a fragment of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,
200, 250, 300, 350, 400, 450, or
500 amino acids). An antifusogenic polypeptide includes, for example, a
polypeptide that targets HIV,
SARS-CoV-2, HCV, or RSV. In some embodiments, an antifusogenic polypeptide
includes a polypeptide
having at least 70%, e.g., at least 80%, e.g., at least 85% (e.g., at least
90%, 95%, 97%, 99%, or 100%)
sequence identity to any one of SEQ ID NOs: 1-324. An antifusogenic
polypeptide also refers to a
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polynucleotide (e.g., polyribonucleotide, e.g., circular polyribonucleotide
encoding an antifusogenic
polypeptide (e.g., a polypeptide of Table 1) or a biologically active fragment
thereof.
As used herein, the terms "treat" and "treating" refer to a prophylactic or
therapeutic treatment of
a viral infection, e.g., HIV, SARS-CoV-2, HCV, influenza, or RSV, 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 viral infection,
stabilizing (i.e., not worsening) the state of the viral infection, or
preventing the spread of the viral infection
as compared to the state or the condition of the viral infection 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 "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., a cell-free
translation system like rabbit reticulocyte lysate.
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.
As used herein, a "vector" means a piece of DNA that is synthesized (e.g.,
using PCR), or that is
taken from a virus, plasmid, or cell of a higher organism into which a foreign
DNA fragment can be or has
been inserted for cloning or expression purposes. In some embodiments, a
vector can be stably
maintained in an organism. A vector can include, for example, an origin of
replication, a selectable
marker or reporter gene, such as antibiotic resistance or GFP, or a multiple
cloning site (MCS). The term
includes linear DNA fragments (e.g., PCR products, linearized plasmid
fragments), plasmid vectors, viral
vectors, cosmids, bacterial artificial chromosomes (BACs), yeast artificial
chromosomes (YACs), and the
like. In one embodiment, the vectors provided herein include a multiple
cloning site (MCS). In another
embodiment, the vectors provided herein do not include an MCS.
Brief Description of the Drawings
FIG. 1 is a schematic drawing showing the protein domains or regions of
various Coronavirus S
proteins and sequences of the HR1 and HR2 regions.
FIG. 2 is a schematic drawing showing various antifusogenic polypeptides and
sequences
derived from the HR2 region of SARS CoV-2.
FIG. 3 is a schematic drawing showing exemplary embodiments of multi ORE
antifusogenic
polypeptide constructs.
FIGS. 4A and 4B are graphs showing inhibitory efficacy on fusion using Omicron
and Delta
pseudo viruses. FIG. 4A shows % inhibition of Delta and Omicron using either
HR2 Full length or HR2
Full length with a HiBiT tag. FIG. 4B shows relative expression of the
antifusogenic polypeptide as
compared to a mock control.
FIG. 5 is a graph showing in vivo expression of HR2 full length antifusogenic
polypeptides with
and without HiBiT tag at 6 hours and 24 hours.
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FIGS. 6A and 6B are graphs showing in vitro neutralization of pseudovirus of
the Wuhan and
Omicron strains of SARS CoV-2 using the HR2A polypeptide. FIG. 6A shows
neutralization of the
Wuhan strain of SARS CoV-2 using HR2A. FIG. 6B shows neutralization of the
Omicron strain of SARS
CoV-2 using HR2A.
FIGS. 7A and 7B are graphs showing the inhibition rate (%) of SARS CoV-2
Pseudovirus
Omicron BA4 and BA.5 (FIG. 7A) or SARS CoV-1 Pseudovirus (FIG. 7B) strains.
FIGS. 8A-8D are graphs showing the inhibition rate (%) of SARS CoV-2
Pseudovirus using full
length HR2 (HR2Complete). FIG. 8A shows inhibition of Wuhan strain. FIG. 8B
shows inhibition of
Omicron BA.4 and BA. 5 strain. FIG. 8C shows inhibition of Omicron BA.1
strain. FIG. 80 shows
inhibition of SARS Coy-1 Pseudovirus.
FIG. 9 is a schematic drawing showing construct designs and sequences for
various HIV
antifusogenic polypeptides.
FIGS. 10A and 10B are a graph (FIG. 10A) and a table (FIG. 10B) showing
expression of various
HIV antifusogenic polypeptides from circular RNA.
FIGS. 11A and 11B are graphs showing expression of various HIV antifusogenic
polypeptides
from circular RNA (FIG. 11A) or plasmid DNA (FIG. 11B)
FIG. 12 is a table showing expression of various HIV antifusogenic
polypeptides from circular
RNA.
Detailed Description
The present invention features compositions containing a circular
polyribonucleotide (circular
RNA) encoding an antifusogenic polypeptide and methods of use thereof.
Circular polyribonucleotides
described herein are particularly useful for delivering a polynucleotide cargo
encoding an antifusogenic
polypeptide to a target cell.
The circular polyribonucleotide may include a polyribonucleotide cargo
encoding a polypeptide of
Table 1_ In some embodiments, the polyribonucleotide cargo includes an
expression sequence encoding
a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%)
sequence identity to a
polypeptide of Table 1. In some embodiments, the polyribonucleotide cargo
includes an expression
sequence encoding a polypeptide having at least 85% (e.g., at least 90%, 95%,
97%, 99%, or 100%)
sequence identity to any one of SEQ ID NOs: 1-324.
The circular polyribonucleotide may be produced from a precursor, such as a
linear
deoxyribonucleotide, a circular deoxyribonucleotide, or a circular
polyribonucleotide.
A circular polyribonucleotide may include, for example, a splice junction
joining a 5' oxen
fragment and a 3' exon fragment.
The linear RNA molecules described herein a polyribonucleotide encoding an
antifusogenic
polypeptide. In some embodiments, the linear RNA molecules include, from 5' to
3, (A) a 3' catalytic
intron fragment; (B) a 3' splice site; (C) a 3' exon fragment; (D) a
polyribonucleotide cargo encoding an
antifusogenic polypeptide (e.g., polyribonucleotide cargo encoding an IRES
operably linked to an
expression sequence encoding an antifusogenic polypeptide); (E) a 5' exon
fragment; (F) a 5' splice site;
and (G) a 5' catalytic intron fragment The catalytic intron fragments and
splice sites may then allow the
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linear polyribonucleotide to self-splice, thus forming a circular
polyribonucleotide encoding an
antifusogenic polypeptide.
Also featured are methods of using a circular polyribonucleotide as described
herein. For
example, the circular polyribonucleotide can be formulated as a composition
(e.g., a pharmaceutical
composition) for administration to a subject, e.g., a human subject. The
pharmaceutical composition may
be administered in one or more doses of the composition. The composition may
be administered to the
subject to treat or prevent a viral infection (e.g., HIV, SARS-CoV-2, HCV,
influenza, or RSV).
Polynucleotides
The disclosure features circular polyribonucleotide compositions encoding an
antifusogenic
polypeptide, uses thereof, and methods of making circular polyribonucleotides
encoding an antifusogenic
polypeptide. In some embodiments, a circular polyribonucleotide is produced
from a linear
polyribonucleotide (e.g., by self-splicing compatible ends of the linear
polyribonucleotide). In some
embodiments, a linear polyribonucleotide is transcribed from a
deoxyribonucleotide template (e.g., a
vector, a linearized vector, or a cDNA). Accordingly, the disclosure features
deoxyribonucleotides, linear
polyribonucleotides, and circular polyribonucleotides and compositions thereof
useful in the production of
circular polyribonucleotides encoding an antifusogenic polypeptide.
Template Deoxyribonucleotides
The present invention features a template deoxyribonucleotide for making a
circular RNA as
described herein. In embodiments, the deoxyribonucleotide includes the
following, operably linked in a
5'-to-3' orientation: (A) a 3' catalytic intron fragment; (B) a 3' splice
site; (C) a 3' exon fragment; (D) a
polyribonucleotide cargo encoding an antifusogenic polypeptide; (E) a 5' exon
fragment; (F) a 5' splice
site; and (G) a 5' catalytic intron fragment. In embodiments, the
deoxyribonucleotide includes further
elements, e.g., outside of or between any of elements (A), (B), (C), (D), (E),
(F), or (G). In embodiments,
any of the elements (A), (B), (C), (D), (E), (F), or (G) is separated from
each other by a spacer sequence,
as described herein.
In embodiments, the deoxyribonucleotide is, for example, a circular DNA
vector, a linearized DNA
vector, or a linear DNA (e.g., a cDNA, e.g., produced from a DNA vector).
In some embodiments, the deoxyribonucleotide further includes an RNA
polymerase promoter
operably linked to a sequence encoding a linear RNA described herein. In
embodiments, the RNA
polymerase promoter is heterologous to the sequence encoding the linear RNA.
In some embodiments,
the RNA polymerase promoter is a T7 promoter, a T6 promoter, a T4 promoter, a
T3 promoter, an SP6
virus promoter, or an SP3 promoter.
In some embodiments, the deoxyribonucleotide includes a multiple-cloning site
(MCS).
In some embodiments, the deoxyribonucleotide is used to produce circular RNA
with the size
range of about 100 to about 20,000 nucleotides. In some embodiments, the
circular RNA is at least 100,
500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,100, 1,200, 1,300,
1,400, 1,500, 1,600 1,700,
1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500 or 5,000 nucleotides in
size. In some
embodiments, the circular RNA is no more than 20,000, 15,000 10,000, 9,000,
8,000, 7,000, 6,000, 5,000
or 4,000 nucleotides in size.
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Linear Polyribonucleotides
The present invention also features linear polyribonucleotides encoding an
antifusogenic
polypeptide. The linear polyribonucleotide may be used to create a circular
polyribonucleotide, e.g., by
ligating or splicing (e.g., self-splicing) the linear polyribonucleotide to
produce the circular
polyribonucleotide. In embodiments, the linear polyribonucleotide includes the
following, operably linked
in a 5'-to-3' orientation: (A) a 3' catalytic intron fragment; (B) a 3' splice
site; (C) a 3' exon fragment; (D) a
polyribonucleotide cargo encoding an antifusogenic polypeptide; (E) a 5' exon
fragment; (F) a 5' splice
site; and (G) a 5' catalytic intron fragment. In embodiments, the linear
polyribonucleotide includes further
elements, e.g., outside of or between any of elements (A), (B), (C), (D), (E),
(F), or (G). For example, any
of elements (A), (B), (C), (D), (E), (F), or (G) may be separated by a spacer
sequence, as described
herein.
In certain embodiments, provided herein is a method of generating linear RNA
encoding an
antifusogenic polypeptide by performing transcription in a cell-free system
(e.g., in vitro transcription)
using a deoxyribonucleotide (e.g., a vector, linearized vector, or cDNA)
encoding an antifusogenic
polypeptide provided herein as a template (e.g., a vector, linearized vector,
or cDNA provided herein with
an RNA polymerase promoter positioned upstream of the region that codes for
the linear RNA).
In embodiments, a deoxyribonucleotide template is transcribed to a produce a
linear RNA
containing the components described herein. Upon expression, the linear
polyribonucleotide produces a
splicing-compatible polyribonucleotide, which may be self-spliced in order to
produce a circular
polyribonucleotide.
In some embodiments, the linear polyribonucleotide is from 50 to 20,000, 100
to 20,000, 200 to
20,000, 300 to 20,000 (e.g., 50, 100, 200, 300, 400, 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, 3,000, 3,500, 4,000,
5,000, 6,000, 7,000, 8,000,
9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000,
19,000, or 20,000)
ribonucleotides in length. In embodiments, the linear polyribonucleotide is,
e.g., at least 500, at least
1,000, at least 2,000, at least 3,000, at least 4,000, or at least 5,000
ribonucleotides in length.
Circular Polyribonucleotides
In some embodiments, the invention features a circular polyribonucleotide
including an
expression sequence encoding an antifusogenic polypeptide. In embodiments, the
polyribonucleotide
includes an IRES operably linked to an expression sequence encoding an
antifusogenic polypeptide. The
circular polyribonucleotide may include a splice junction, e.g., joining a 5'
exon fragment and a 3' exon
fragment. The circular polyribonucleotide may include any one or more of the
elements described herein.
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.
In embodiments, the circular polynucleotide further includes a
polyribonucleotide cargo. In
embodiments, the polyribonucleotide cargo includes an expression (or coding)
sequence, a non-coding
sequence, or a combination of an expression (coding) sequence and a non-coding
sequence. In
embodiments, the polyribonucleotide cargo includes an expression (coding)
sequence encoding a
polypeptide. In embodiments, the polyribonucleotide includes an IRES operably
linked to an expression
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sequence encoding a polypeptide. In some embodiments, the IRES is located
upstream of the
expression sequence. In some embodiments, the IRES is located downstream of
the expression
sequence. In some embodiments, the circular polyribonucleotide further
includes a spacer region
between the IRES and the 3' exon fragment or the 5' exon fragment. The spacer
region may be, e.g., at
least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in
length ribonucleotides in length. The
spacer region may be, e.g., from 5 to 500 (e.g., 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 150, 200, 250,
300, 350, 400, 450, or 500) ribonucleotides. In some embodiments, the spacer
region includes a polyA
sequence. In some embodiments, the spacer region includes a polyA-C sequence.
In some
embodiments, the spacer region includes a polyA-G sequence. In some
embodiments, the spacer region
includes a polyA-T sequence. In some embodiments, the spacer region includes a
random sequence. In
some embodiments, the first annealing region and the second annealing region
are joined, thereby
forming a circular polyribonucleotide.
In some embodiments, the circular RNA is produced by a deoxyribonucleotide
template or a
linear RNA described herein. In some embodiments, the circular RNA is produced
by any of the methods
described herein.
In some embodiments, 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 circular polyribonucleotide is between 500
nucleotides and 20,000
nucleotides, between 1,000 and 20,000 nucleotides, between 2,000 and 20,000
nucleotides, or between
5,000 and 20,000 nucleotides. In some embodiments, the circular
polyribonucleotide is between 500
nucleotides and 10,000 nucleotides, between 1,000 and 10,000 nucleotides,
between 2,000 and 10,000
nucleotides, or between 5,000 and 10,000 nucleotides.
As a result of its circularization, the 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, the circular
polyribonucleotide is 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 makes
circular polyribonucleotide
more useful as a cell transforming reagent to produce polypeptides and can be
stored more easily and 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, the circular
polyribonucleotide is less susceptible to
dephosphorylation when the circular polyribonucleotide is incubated with
phosphatase, such as calf
intestine phosphatase.
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The circular polyribonucleotides described herein and compositions or
pharmaceutical
compositions thereof may be used in therapeutic and veterinary methods of
dosing to produce a level of
circular polyribonucleotide, a level of binding to a target, or a level of
protein in a plurality of cells after
providing the plurality with at least two doses of circular
polyribonucleotide. In some embodiments, the
circular polyribonucleotide is non-immunogenic in a mammal, e.g., a human. In
some embodiments, the
circular polyribonucleotide is capable of replicating or replicates in a cell
from an aquaculture animal (fish,
crabs, shrimp, oysters etc.), a mammalian cell, e.g., a cell from a pet or zoo
animal (cats, dogs, lizards,
birds, lions, tigers and bears etc.), a cell from a farm or working animal
(horses, cows, pigs, chickens
etc.), a human cell, cultured cells, primary cells or cell lines, stem cells,
progenitor cells, differentiated
cells, germ cells, cancer cells (e.g., tumorigenic, metastic), non-tumorigenic
cells (normal cells), fetal
cells, embryonic cells, adult cells, mitotic cells, non-mitotic cells, or any
combination thereof. In some
embodiments, the invention includes a cell that includes the circular
polyribonucleotide described herein,
wherein the cell is a cell from an aquaculture animal (fish, crabs, shrimp,
oysters etc.), a mammalian cell,
e.g., a cell from a pet or zoo animal (cats, dogs, lizards, birds, lions,
tigers and bears etc.), a cell from a
farm or working animal (horses, cows, pigs, chickens etc.), a human cell, a
cultured cell, a primary cell or
a cell line, a stem cell, a progenitor cell, a differentiated cell, a germ
cell, a cancer cell (e.g., tumorigenic,
metastatic), a non-tumorigenic cell (normal cells), a fetal cell, an embryonic
cell, an adult cell, a mitotic
cell, a non-mitotic cell, or any combination thereof. In some embodiments, the
cell is modified to include
the circular polyribonucleotide.
In some embodiments, the circular polyribonucleotide includes sequences for
expression
products. In some embodiments, the circular polyribonucleotide includes a
binding site for binding to a
target. In some embodiments, the circular polyribonucleotide is provided to a
plurality of cells via any a
dosing regimen described herein. In some embodiments, the circular
polyribonucleotide as described
herein induces a response or response level in a subject. In some embodiments,
the expression products
encoded by the sequences included in the circular polyribonucleotide are
expressed in one or more of
cells in the plurality of cells.
In some embodiments, the circular polyribonucleotide has a half-life of at
least that of a linear
counterpart, e.g., linear expression sequence, or linear polyribonucleotide.
In some embodiments, the
circular polyribonucleotide has a half-life that is increased over that of a
linear counterpart. In some
embodiments, the half-life is increased by about 5%, 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%,
or more. In some embodiments, the circular polyribonucleotide has a half-life
or persistence in a cell for at
least about 1 hour, e.g., at least 2 hours, 3 hours, 4 hours, 5 hours 6 hours,
12 hours, 24 hours, 1 day, 2
days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2
months, 3 months, 6 months,
or longer. In some embodiments, the circular polyribonucleotide has a half-
life or persistence in a cell for
from about 1 hour to about 60 days, e.g., about 1 hour, 2 hours, 6 hours, 12
hours, 18 hours, 24 hours, 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, 35 days, 40 days, 45 days,
50 days, 55 days, or 60
days. In some embodiments, the circular polyribonucleotide has a half-life or
persistence in a cell while
the cell is dividing. In some embodiments, the circular polyribonucleotide has
a half-life or persistence in
a cell post division. In certain embodiments, the circular polyribonucleotide
has a half-life or persistence
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in a dividing cell for at least about 10 minutes, e.g., at least about 1 hour,
e.g., at least 2 hours, 3 hours, 4
hours, 5 hours 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4 days, 5
days, 6 days, 7 days, 2
weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer. In certain
embodiments, the circular
polyribonucleotide has a half-life or persistence in a dividing cell of from
about 10 minutes to about 60
days, e.g., about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7
hours, 8 hours, 9 hours, 10 hours,
11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18
hours, 24 hours, 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, or 60 days.
In some embodiments, the circular polyribonucleotide modulates a cellular
function, e.g.,
transiently, or long term. In certain embodiments, the cellular function is
stably altered, such as a
modulation that persists for at least about 10 minutes, e.g., at least about 1
hour, e.g., at least 2 hours, 3
hours, 4 hours, 5 hours 6 hours, 12 hours, 24 hours, 1 day, 2 days, 3 days, 4
days, 5 days, 6 days, 7
days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 6 months, or longer. In
certain embodiments, the
cellular function is stably altered, such as a modulation that persists for
from about 1 hour to about 60
days, e.g., from about 1 hour to about 30 days, e.g., for at least about 2
hours, 6 hours, 12 hours, 18
hours, 24 hours, 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, or 60
days.
Elements of Polynucleotides
The polynucleotides (e.g., circular polyribonucleotides) described herein may
include any one or
more of the elements described herein and an expression sequence encoding an
antifusogenic
polypeptide.
Antifusogenic Polypeptides
The disclosure provides circular polyribonucleotides that encode at least one
expression
sequence encoding an antifusogenic polypeptide. In some embodiments, the
antifusogenic polypeptide
inhibits viral entry. In some embodiments, the antifusogenic polypeptide
inhibits viral fusion.
In some embodiments, the antifusogenic polypeptide is a polypeptide or a
variant thereof
including an amino acid sequence selected from a sequence of Table 1. In some
embodiments, the
antifusogenic polypeptide is a polypeptide including a contiguous stretch of
at least 15, 20, 25, 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids of a sequence
in Table 1. In some
embodiments, the antifusogenic polypeptide is a polypeptide including a
contiguous stretch of at least
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the
amino acids of a
sequence in Table 1. In some embodiments, the antifusogenic polypeptide is a
polypeptide including a
sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
99% sequence
identity to a sequence of Table 1. In some embodiments, the antifusogenic
polypeptide is a variant of a
sequence in Table 1 that includes no more than one, two, three, four, five,
six, seven, eight, nine, or ten
mutations (e.g., substitutions, deletions, or insertions). In some
embodiments, the circular
polyribonucleotide includes an expression sequence encoding more than one
antifusogenic polypeptide.
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In some embodiments, the circular polyribonucleotide encoding more than one
fusion protein reduces the
chance for viral escape.
In some embodiments, the antifusogenic polypeptide targets one or more (e.g.,
two, three, four,
or five) viruses. Viruses that may be targeted by the antifusogenic
polypeptide include, but are not limited
to, all strains of viruses listed in Table 1. In some embodiments, the virus
does not infect humans. In
some embodiments, the virus infects humans.
Table 1. Exemplary antifusogenic polypeptides
Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Golden oyster Antiviral protein Tobacco mosaic virus
AACARFIDDFCDTLTPNIYR 1
mushroom, Y3 (TMV), Influenza PRDNGORCYAVNGHRCDF
Pleurotus hernagglutinin (HA), TVFNTNNGGNPIRASTPNC
citrinopileauts Human KTVLRTAANRCPTGGRGKI
immunodeficiency NPNAPFLFAIDPNDGDCST
virus (HIV) NF
pseudovirions
Daruma pond Brevinin-1
Herpes simplex virus-1 FLPVLAGIAAKVVPALFCKIT 2
frog, Rana (HSV-1), Herpes KKC
brevipoda porsa simplex virus-2 (HSV-
2)
European Temporin B CCV, FV3 LLPIVGNLLKSLL
3
common frog,
Rana
temporaria
European Temporin G Influenza A
virus (IAV), FFPVIGRILNGIL 4
common frog, Parainfluenza
Rana respiratory viruses
temporaria (PIV)
North American Ranateurin 3P HIV GLMDTVKNVAKNLAGHMLD
5
frog, Rana KLKCKITGC
pipiens and
Oregon spotted
frog, Rana
pretiosa
Human, Homo Human Pseudotyped viruses
ACYCRIPACIAGERRYGTCI 6
sapiens neutrophil expressing SARS-
YOGRLWAFCC
peptide-1 CoV-2 spike proteins
(HNP-1)
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Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Human, Homo HNP-2 Pseudotyped viruses
CYCRIPACIAGERRYGTCIY 7
sapiens expressing SARS- QGRLWAFCC
CoV-2 spike proteins
Human, Homo HNP-3 Pseudotyped viruses
DCYCRIPACIAGERRYGTCI 8
sapiens expressing SARS- YQGRLWAFCC
CoV-2 spike proteins
Human, Homo HNP-4 Pseudotyped viruses
VCSCRLVFCRRTELRVGNC 9
sapiens expressing SARS- LIGGVSFTYCCTRV
CoV-2 spike proteins
Human, Homo Human BK virus (BKV)
ATCYCRTGRCATRESLSGV 10
sapiens defensin (HD- CEISGRLYRLCCR
5)
Human, Homo HD-6 Respiratory syncytial
AFTCHCRRSCYSTEYSYGT 11
sapiens virus (RSV), human CTVMGINHRFCCL
Ply (HPIV), HIV, HSV,
influenza A virus (IAV),
BKV, human
adenoviruses (HAdV),
human papilloma virus
(H PV)
Rabbit, Rabbit HSV, enveloped
VVCACRRALCLPRERRAGF 12
Oryctolagus neutrophil viruses
CRIRGRIHPLCCRR
cuniculus peptide 1 (NP-
1)
Rabbit, Rabbit RSV, HIV, HSV VVCACRRALCLPLERRAGF
13
Oryctolagus neutrophil CRIRGRIHPLCCRR
cuniculus defensin 2 (NP-
2)
Porcine Protegrin 1 HIV, HSV-1, HSV-2,
RGGRLCYCRRRFCVCVGR 14
neutrophil, Sus (PG-1) Porcine reproductive
scrofa and respiratory
syndrome virus
(PRRSV)
Atlantic Polyphemusin I HIV RRWCFRVCYRGFCYRKCR
15
horseshoe crab,
Limulus
polyphemus
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Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Atlantic Polyphemusin HIV RRWCFRVCYKGFCYRKCR 16
horseshoe crab, II
Limulus
polyphemus
Southeast Asian Tachyplesin I HIV, HSV-1, HSV-2,
KWCFRVCYRGICYRRCR 17
hemocytes, (TP1) Singapore grouper
Tachypleus iridovirus (SGIV)
tridentatus,
Tachypleus
gigas,
Carcinoscorpius
rotundicauda
Rabbit, Rabbit RSK, HIV, HSV GICACRRRFCPNSERFSGY
18
Oryctolagus neutropgil CRVNGARYVRCCSRR
cuniculus defensin 3a
(NP-3a)
Porcine PG-2 Pseudorabies virus RGGRLCYCRRRFCICV
19
neutrophil, Sus (PRV), PRRSV
scrofa
Porcine PG-3 PRRSV RGGGLCYCRRRFCVCVGR 20
neutrophil, Sus
scrofa
Porcine PG-4 PRRSV RGGRLCYCRGWICFCVGR
21
neutrophil, Sus
scrofa
Rat, Rattus Rat NP-1, rat HIV, HSV
VTCYCRRTRCGFRERLSGA 22
novegicus defensin CGYRGRIYRLCCR
Rat, Rattus Rat NP-2 HIV, HSV
VTCYCRSTRCGFRERLSGA 23
novegicus CGYRGRIYRLCCR
Rat, Rattus Rat NP-3 HIV, HSV
CSCRTSSCRFGERLSGACR 24
novegicus LNGRIYRLCC
Rat, Rattus Rat NP-4 HIV, HSV
ACYCRIGACVSGERLTGAC 25
novegicus GLNGRIYRLCCR
Australian green Caerin 1.1 HIV, Murine leukemia
GLLSVLGSVAKHVLPHVVP 26
tree frog, Litoria virus VIAEHL
splendida,
Litoria rothii
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Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Australian green Caerin 1.2 HIV GLLGVLGSVAKHVLPHVVP
27
tree frog, Litoria VIAEHL
caerula
Australian green Caerin 1.3 HIV GLLSVLGSVAQHVLPHVVP
28
tree frog, Litoria VIAEHL
caerula
Australian green Caerin 1.4 HIV GLLSSLSSVAKHVLPHVVPV
29
tree frog, Litoria IAEHL
caerula
Australian green Caerin 1.5 HIV GLLSVLGSVVKHVIPHVVPV
30
tree frog, Litoria IAEHL
caerula
Australian Caerin 1.6 HIV GLFSVLGAVAKHVLPHVVP
31
orange-thighed VIAEK
frog, Litoria
xanthomera
Australian Caerin 1.7 HIV GLFKVLGSVAKHLLPHVAP
32
orange-thighed VIAEK
frog, Litoria
xanthomera
Australian green Caerin 1.9 HIV GLFGVLGSIAKHVLPHVVPV
33
tree frog, Litoria IAEKL-NH2
caerula
Australian green Caerin 1.10 HIV GLLSVLGSVAKHVLPHVVO
34
tree frog, Litoria VIAEKL-NH2
caerula
Australian green Caerin 1.19 HIV GLFKVLGSVAKHLLPHVAPII
35
tree frog, Litoria AEKL-NH2
caerula
Australian green Caerin 1.20 HIV GLFGILGSVAKHVLPHVIPV
36
tree frog, Litoria VAEHL-NH2
caerula
Australian green Caerin 4.1 HIV GLWQKIKSAAGDLASGIVE
37
tree frog, Litoria GIKS
caerula
Mouse, Mus Murine beta- HIV, IAV DQYKCLQHGGFCLRSSCP
38
musculus defensin 1 SNTKLQGTCKPDKPNCCKS
(mBD-1)
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Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Mouse, Mus Mouse RSV GLLRKGGEKIGEKLKKIGQK
39
musculus cathelin-related IKNFFQKLVPQPEQ
antimicrobial
peptide
(mCRAMP)
Human, Homo Human beta HIV
GIINTLQKYYCRVRGGRCA 40
sapiens defensin 3 VLSCLPKEEQIGKCSTRGR
(hBD-3) KCCRRKK
Southern brown Ewingiin 2.1 HIV GLLDMVTGLLGNL-NH2
41
tree frog, Litoria (formerly
ewingii named
Caeridin 7.1)
Australian tree Maculatin 1.1 HIV, PRV
GLFGVLAKVAAHVVPAIAEH 42
frog, Litoria F-NH2
genimaculata
Hybrid striped Piscidin 1H HIV FFHHIFRGIVHVGKTIHRLVT
43
bass, Morone (Pis-1H)
saxatilis,
Morone
chrysops
Hybrid striped Piscidin 1N HIV FFHHIFRGIVHVGKTIHRLVT
44
bass, Morone (Pis-1N)
saxatilis,
Morone
chrysops
Hybrid striped Piscidin 2 (Pis- HIV FFHHIFRGIVHVGKTIHKLVT
45
bass, Morone 2)
saxatilis,
Morone
chrysops
Hybrid striped Piscidin 3 (Pis- HIV FIHHIFRGIVHAGRSIGRFLT
46
bass, Morone 3)
saxatilis,
Morone
chrysops
Blue mussel, Mytilin B Viral hemorrhagic
SCASRCKGHCRARRCGYY 47
Mytilus edulis septicemia virus VSVLYRGRCYCKCLRC
(VHSV) VHSV, IPNV
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Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Mediterranean Mytilin C VHSV, IPNV SCASRCKSRCRARRCRYY
48
mussel, Mytilus VSVRYGWFCYCRCLRC
galloprovincialis
Fungus growing Spinigerin HIV HVDKKVADKVLLLKQLRIMR
49
termite, LLTRL
Pseudocanthote
rmes spiniger
North American Ranateurin 6 HIV FISAIASMLGKFL
50
frog, Rana
catasbeiana
North American Ranateurin 9 HIV FLFPKITSFLSKVL
51
frog, Rana
catasbeiana
Rhesus Rhesus theta HIV GFCRCLCRRGVCRCICTR
52
macaque, defensin-1
Macaca mulatta (RID-1)
Human, Homo Human beta- HIV, HSV DHYNCVSSGGQCLYSACPI
53
sapiens defensin 1 FTKIQGTCYRGKAKCCK
(hBD-1)
Human, Homo hBD-2 HIV, HSV GIGDPVTCLKSGAICHPVFC
54
sapiens PRRYKQIGTCGLPGTKCCK
KP
Streptomyces Siamycin II HIV CLGIGSCNDFAGCGYAIVCF
55
strain AA3891
North American Brevinin-2- HIV GIWDTIKSMGKVFAGKILQN
56
mink frog, Rana related peptide
septentrionalis
Guinea pig, CAP11 IAV GLRKKFRKTRKRIQKLGRKI
57
Cavia porcellus GKTGRKVWKAWREYGQIP
YPCRI
Rhesus RTD-2 HIV GVCRCLCRRGVCRCLCRR
58
macaque,
Macaca mulatta
Rhesus RTD-3 HIV GFCRCICTRGFCRCICTR
59
macaque,
Macaca mulatta
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Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
African clawed Magainin 1 HSV-1, HSV-2 GIGKFLHSAGKFGKAFVGEI
60
frog, Xenopus MKS
laevis
African herb, Kalata B1 HIV
GLPVCGETCVGGTCNTPG 61
Oldenlandia (KB1) CTCSWPVCTRN
affinis
Chicken, Gallus Chicken avian IAV SPIHACRYQRGVCIPGPCR
62
gal/us domestic, beta defensin 6 WPYYRVGSCGSGLKSCCV
Duck, Anas (AvBD-6) RNRWA
platyrhynchos
Chicken, Gallus AvBD-5 IAV GLPQDCERRGGFCSHKSC
63
gal/us domestic PPGIGRIGLCSKEDFCCRSR
WYS
Chinese red Maximin H1 Nipah virus (NiV)
ILGPVISTIGGVLGGLLKNL 64
belly toad,
Bombina
maxima
Sahara frog, Temporin-Sha HSV FLSGIVGMLGKLF
65
Pelophylax
saharicus
Leonia cymosa Cycloviolin A HIV
GVIPCGESCVFIPCISAAIGC 66
SCKNKVCYRN
Leonia cymosa Cycloviolin B HIV
GTACGESCYVLPCFTVGCT 67
CTSSQCFKN
Leonia cymosa Cycloviolin C HIV
GIPCGESCVFIPCLTTVAGC 68
SCKNKVCYRN
Leonia cymosa Cycloviolin D HIV
GFPCGESCVFIPCISAAIGC 69
SCKNKVCYRN
European field Vary peptide E HIV
GLPICGETCVGGTCNTPGC 70
pansy, Viola (Vary E) SCSWPVCTRN
arvensis
African herb, KB8 HIV GSVLNCGETCLLGTCYTTG
71
Oldenlandia CTCNKYRVCTKD
affinis
Palicourea Pal icourein HIV GDPTFCGETCRVIPVCTYS
72
condensata AALGCTCDDRSDGLCKRN
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Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Arrowhead KB2 HIV GLPVCGETCFGGTCNTPG
73
violet, Viola CSCTWPICTRD
betonicifolia
Australian violet, Vhl-1 HIV SISCGESCAMISFCFTEVIG
74
Viola hederacea CSCKNKVCYLN
Tropical tree, Circulin C HIV
GI PCGESCVFIPCITSVAGC 75
Chassalia SCKSKVCYRN
parvifola
Tropical tree, Circulin D HIV
KIPCGESCVWIPCVTSIFNC 76
Chassalia KCKENKVCYHD
parvifola
Tropical tree, Circulin E HIV
KIPCGESCVWIPCLTSVFNC 77
Chassalia KCENKVCYHD
parvifola
Tropical tree, Circulin F HIV
KVCYRAIPCGESCVWIPCIS 78
Chassalia AAIGCSCKN
parvifola
Tilapia, Hepcidin 1-5 Nervous necrosis virus
GIKCRFCCGCCTPGICGVC 79
Oreochromis (TH1-5) (NNV) CRF
mossambicus
Sweet violet, Cycloviolacin HIV Cl PCGESCVWIPCISAAIGC
80
Viola odorata 013 SCKSKVCYRN
Sweet violet, Cycloviolacin HIV GSIPACGESCFKGKCYTPG
81
Viola odorata 014 CSCSKYPLCAKN
Sweet violet, Cycloviolacin HIV GLPTCGETCFGGTCNTPGC
82
Viola odorata 024 TCDPWPVCTHN
Chinese herb, Cycloviolacin HIV GGTIFDCGETCFLGTCYTP
83
Viola yedoensis Y1 GCSCGNYGFCYGTN
Chinese herb, Cycloviolacin HIV GVPCGESCVFIPCITGVIGC
84
Viola yedoensis Y4 SCSSNVCYLN
Chinese herb, Cycloviolacin HIV GI PCAESCVWIPCTVTALVG
85
Viola yedoensis Y5 CSCSDKVCYN
Chinese herb, Cycloviolacin IAV CGESCVFIPCITTVLGCSCSI
86
Viola yedoensis VY1 KVCYKNGSIP
Streptomyces Siamycin I HIV CLGVGSCNDFAGCGYAVV
87
strain AA6532 CFW
Streptomyces NP-06 HIV CLGVGSCNDFAGCGYAIVC
88
strain AA6532 FW
26
CA 03241026 2024- 6- 13
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PCT/US2022/082345
Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Human, Homo Retrocylin-1 HIV GICRCICGRGICRCICGR
89
sapiens (RC1)
Human, Homo RC2 HIV GICRCICGRRICRCICGR
90
sapiens
Human, Homo RC3 HIV RICRCICGRRICRCICGR
91
sapiens
Red sea turtle, Turtle egg Rhabdovirus
QKKCPGRCTLKCGKHERPT 92
Caretta caretta white protein Chandipura virus LPYNCGKYICCVPVKVK
(TEWP)
Mediterranean Myticin C (Myt VHSV
QEAQSVACTSYYCSKFCGS 93
mussel, Mytilus C) AGCSLYGCYLLHPGKICYCL
galloprovincialis HCSR
Australian white- Frenatin 2 Yellow fever virus
GLLGTLGNLLNGLGL 94
lipped tree frog, (YFV)
Litoria
infrafrenata
Coconut, Cocos Coconut HIV EQCREEEDDR 95
nucifera antifungal
peptide
Human, Homo Secretory HIV
SGKSFKAGVCPPKKSAQCL 96
sapiens leukocyte RYKKPECQSDWQCPGKKR
protease CCPDTCGIKCLDPVDTPNP
inhibitor (SLPI) TRRKPGKCPVTYGQCLMLN
PP
Cyanobacterium Antiviral lectin HIV-1 GSGPTYCWNEANNPGGPN
97
, Scytonema scytovirin RCSNNKQCDGARTCSSSG
varium (SVN) FCQGTSRKPDPGPKGPTY
CWDEAKNPGGPNRCSNSK
QCD
Cyanobacterium Cyanovirin-N HIV-1 LGKFSQTCYNSAIQGSVLTS
98
, Nostoc (CVN) TCERTNGGYNTSSIDLNSVI
ellipsosporum ENVDGSLKWQPSNFIETCR
NTQLAGSSELAAECKTRAQ
OF
27
CA 03241026 2024- 6- 13
WO 2023/122789
PCT/US2022/082345
Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Cyanobacterium Microcystis HIV-1 MASYKVNIPAGPLWSNAEA
99
, Microcystis viridis lectin QQVGPKIAAAHOGNFTGO
viridis (MVL) WTTVVESAMSVVEVELQVE
NTGIHEFKTDVLAGPLWSN
Red algae, Griffithsin HIV-1 SLTHRKFGGSGGSPFSGLS
100
Griffithsia sp (GRFT) SIAVRSGSYLDAIIIDGVHHG
GSGGNLSPTFTFGSGEYIS
NMTIRSGDYIDNISFETNMG
RR
Asian common Scylla White spot syndrome
YETLIASVLGKLTGLWHNNS 101
mud crab, Scylla paramamosain virus (WSSV) VDFMGHTCHFRRRPKVRK
paramamosain anti- FKLYHEGKFWCPGWAPFE
lipopolysacchar GRSRTKSRSGSSREAIKDF
ide factor VRK
isoform 1 (Sp-
ALF1)
Asian common Sp-ALF2 WSSV YEALVASILGKLSGLWHSDT
102
mud crab, Scylla VDFMGHTCHIRRRPKFRKF
paramamosain KLYHEGKFWCPGWTHLEG
NSRTKSRSGSARDAIKDFV
YKA
Peking duck, Apl-AvBD-16 Duck hepatitis virus
FFLLFLQGAAGNSVLCRIRG 103
Anas (DHV) GRCHVGSCHFPERHIGRCS
platyrhynchos GFOACCIRTWG
Asian forest Hp1090 Hepatitis C virus (HCV) IFKAIWSGIKSLF
104
scorpion,
Heterometrus
petersii
Red swamp Procambarin WSSV HRPYCGSKGGIGGGHGGG
105
crayfish, SGGFGGGGGFGGGGLGG
Procambarus GKPIGIGGGGGFGGGSGFG
clarkii GGVGLKPNVGGGGGFGGG
GGGFGGGIGLKPNVGGGG
GFGGGIGLKPNVGGGGGF
GGGGGGFGGGGGGFGGG
FGGGKLIGGGIGWRRWWL
CRKQRLRKVNHL
28
CA 03241026 2024- 6- 13
WO 2023/122789
PCT/US2022/082345
Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Scorpion, Hpl 036 HSV-1
ILGKIWEGIKSIF 106
Heterometrus
petersii
Scorpion, Hpl 239 HSV-1
ILSYLWNGIKSIF 107
Heterometrus
petersii
Mouse, Mus mBD-3 IAV KINNPVSCLRKGGRCWNR
108
muscu/us CIGNTRQIGSCGVPFLKCCK
RK
Staphylococcus Micrococcin P1 HCV SCTTCVCTCSCCTT
109
epidermidis (MP1)
strain 115,
Staphylococcus
equorum strain
WS 2733
Turbot, S_ maximus Megalocytivirus (MCV)
QSHISLCRWCCNCCKANK 110
Scophthalmus hepcidin-1 GCGFCCKF
maximus (SmHepl P)
Turbot, SmHep2P MCV GMKCKFCCNCCNLNGCGV
111
Scophthalmus CCRF
maximus
Actinomadura Labyrinthopepti HIV, HSV, RSV, SNASVWECCSTGSWVPFT
112
namibiensis n Al (LabyAl) DENV, Zika virus CC
DSM 6313 (ZIKV), HCV,
Chikungunya virus
(CHIKV), KSHV, CMV
Actinomadura LabyA2 HIV, HSV, RSV,
SDWSLWECCSTGSLFACC 113
namibiensis DENV, ZIKV, HCV,
CHIKV, KSHV, CMV
Spotted scat, SA-hepcidin2 Siniperca chuatsi NPAGCRFCCGCCPNMIGC
114
Scatophagus rhabdovirus (SCRV), GVCCRF
argus Micropterus salmoides
reovirus (MsReV)
Hemolymph, HEdefensin Langat virus (LGTV)
EEESEVAHLRVRRGFGCPL 115
Haemaphysalis NQGACHRHCRSIRRRGGY
longicornis CSGIIKQTCTCYRN
29
CA 03241026 2024- 6- 13
WO 2023/122789
PCT/US2022/082345
Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Scorpion, BmKDfsin4 Hepatitis B virus (HBV)
GFCCPFNQGQCHKHCQS1 116
Mesobuthus RRRGGYCDGFLKTRCVCY
martensii
Karsch
Tiger shrimp, Cyclic shrimp NNV ECKFTVKPYLKRFQVYYKG
117
Penaeus anti- RMWCP
monodon lipopolysacchar
ide factor
(cSALF)
Human, Homo Heparin- IAV
GKRKKKGKGLGKKRDPCL 118
sapiens binding EGF- RKYK
like growth
factor (HB-
EGF)
Severe acute SARSww-1 SARS-CoV
MWKTPTLKYFGGFNFSQIL 119
respriatory
syndrome
coronavirus
(SARS-CoV)
Class I fusion
protein S2
SARS-CoV SARSww-ii SARS-CoV ATAGWTFGAGAALQIPFAM
120
Class I fusion QMAY
protein S2
SARS-CoV SARSww-ill SARS-CoV GYHLMSFPQAAPHGVVFLH
121
Class I fusion VTW
protein S2
SARS-CoV SARSww_iv SARS-CoV GVFVFNGTSWFITQRNFFS
122
Class I fusion
protein S2
SARS-CoV SARSww-vb SARS-CoV AACEVAKNLNESLIDLQELG
123
Class I fusion KYEQYIKW
protein S2
CMV Class III 174-200 CMV
WEIHHINKFAQAYSSYSRVI 124
fusion protein Gb GGTVFA
CMV Class III 233-263 CMV
WHSRGSTWLLYRETANLN 125
fusion protein Gb AMLTITTARSKYPY
CA 03241026 2024- 6- 13
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PCT/US2022/082345
Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
CMV Class III 264-291 CMV HFFATSTGDVVYISPFYNGT
126
fusion protein Gb NRNASYFG
CMV Class III 297-315 CMV FFIFPNYTIVSDFGRPNAA
127
fusion protein Gb
Rift Valley fever RVFY-6 RVFY WNFFDWFSGLMWFGGPLK
128
virus (RFVY)
Class I fusion
protein Gn
RFVY Class I RVFY-6 RVFY WNFFDWFSGLMSWFGGPL
129
fusion protein Gn KTI
RFVY Class I RVFY-6 RVFY SWNFFDWFSGLMSWFGGP
130
fusion protein Gn LK
RFVY Class I RVFY-6 RVFY SGSWNFFDWFSGLMSWFG
131
fusion protein Gn
RFVY Class I RVFY-6 RVFY SGSWNFFDWFSGLMSWFG
132
fusion protein Gn GPLKPL
IAV Class I Residues 84-99 IAV VDDGFLDIWTYNAELL
133
fusion protein of A/H1
Hemagglutinin
(HA)
IAV Class I Residues 84-99 IAV VEDTKIDLWSYNAELL
134
fusion protein of NH3, A/H4,
HA and A/H14
IAV Class I Residues 84-99 IAV MEDGFLDVWTYNAELL
135
fusion protein of A/H5
HA
Pichinde virus Peptide 4.6 Pichinde virus (PICV)
GHTLKWLLELHFNVLHVTR 136
fusion protein (GP1 143-170) HIGARCKT
complex GP1
protein
Pichinde virus Peptide 5.0 PICV HLIASLAQIIGDPKIAWVGK
137
fusion protein (GP1 194-212)
complex GP1
protein
Pichinde virus Peptide 6.0 PICV HYNFLIIQNTTWENHCTYT
138
fusion protein (GP1 233-251)
complex GP1
protein
31
CA 03241026 2024- 6- 13
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Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Pichinde virus Peptide 7.0 PICV
PGGYCLEQWAIIWAGIKCF 139
fusion protein (GP2291-307)
complex GP2
protein
Pichinde virus Peptide 8.0 PICV
LNLFKKTINGLISDSLVIR 140
fusion protein (G P2348-366)
complex GP2
protein
HCV fusion [1197-214 HCV VSGIYHVTNDCSNSSIVY
141
protein El
HCV fusion E2407-424 HCV PSQKIQLVNTNGSWHINR
142
protein E2
HCV fusion E2610-627 HCV DYPYRLWHYPCTVNFTVF
143
protein E2
HCV fusion E2701-718 HCV YLYGIGSAVVSFAIKWEY
144
protein E2
Human, Homo EB HSV, IAV, Vaccinia
RRKKAAVALLPAVLLALLAP 145
sapiens virus
(Fibroblast
growth factor 4
signal
sequence)
Human, Homo FluPep 1 IAV WLVFFVIFYFFR
146
sapiens (FP1), Tkip
Human, Homo FP2 IAV WLVFFVIAYFAR
147
sapiens
Human, Homo FP3 IAV
WLVFFVIFYFFRRRKK 148
sapiens
Human, Homo FP4 IAV
RRKKWLVFFVIFYFFR 149
sapiens
Human, Homo FP7 IAV RRKKIFYFFR
150
sapiens
Human, Homo FPS IAV WLVFFVRRKK
151
sapiens
Human, Homo FP9 IAV FFVIFYRRKK
152
sapiens
32
CA 03241026 2024- 6- 13
WO 2023/122789
PCT/US2022/082345
Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Human, Homo RhoA RSV, HIV ILMCFSIDSPDSLEN
153
sapiens GTPase
RhoA
HSV fusion gB64 HSV TTPKFTVAWDWVPKR
154
protein Gb
HSV fusion gB94 HSV KTTSSIEFARLQFTY
155
protein Gb
HSV fusion gB122 HSV GHRRYFTFGGGYVYF
156
protein Gb
HSV fusion gB131 HSV HEVVPLEVYTRHEIK
157
protein Gb
HCV C5A HCV SWLRDIWDWICEVLSDFK
158
nonstructural
protein NS5A
Human, Homo CL-58 HCV MANAGLQLLGFILAFLGW
159
sapiens Claudin
1
Human, Homo CL-59 HCV AFLGWIGAIVSTALPQWR
160
sapiens Claudin
1
Murine brain P1 West Nile Virus (WNV) DTRACDVIALLCHLNT
161
cDNA phage
display library
Murine brain P9 WNV CDVIALLACHLNT
162
cDNA phage
display library
GB virus C P4-7 HIV WDRGNVTLLCDCPNGPWV
163
(GBV-C) surface WV
glycoprotein E2
GBV-C surface P6-2 HIV LCDCPNGPWVWVPAFCQA
164
glycoprotein E2 VG
Human, Homo P5 SARS-CoV-2 GGGYSKAQKAQAKQAK
165
sapiens QAQKAQKAQAKQAKQ
Human, Homo P5+14 SARS-CoV-2 GGGYSKAOKAQAKOAK
166
sapiens QAQKAOKAQAKQAKQA
QKAQKAQAKQAKO
Human, Homo (P5R)D SARS-CoV-2 GGGYSRAORAQAIROAR
167
sapiens QAQRAQRAQARQARQ
33
CA 03241026 2024- 6- 13
WO 2023/122789
PCT/US2022/082345
Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Human, Homo Lactoferricin
CMV, HIV, HSV, HPV KCFQWQRNMRKVRGPPVS 168
sapiens and CIKRDS
Domestic cow,
Bos taurus
Human, Homo Lactoferrin CMV, HIV, HSV, HPV
MKLVFLVLLFLGALGLCLAG 169
sapiens and RRRRSVQWCAVSQPEATK
Domestic cow, CFQWQRNMRKVRGPPVSC
Bos taurus IKRDSPIQCIQAIAENRADAV
TLDGGFIYEAGLAPYKLRPV
AAEVYGTERQPRTHYYAVA
VVKKGGSFQLNELQGLKSC
HTGLRRTAGWNVPIGTLRP
FLNWTGPPEPIEAAVARFFS
ASCVPGADKGQFPNLCRLC
AGTGENKCAFSSQEPYFSY
SGAFKCLRDGAGDVAFIRE
STV
Human, Homo CAP37 HSV-1, Adenovirus
GRHFCGGALIHARFVMTAA 170
sapiens SCFQ
HIV-1 HIV-1 Tat HIV-1 GRKKRRQRRR
171
HIV C46 HIV WMEWDREINNYTSLIHSLIE 172
ESQNQQEKNEQELLELDK
WASLWNWF
HIV C46mutGlyco HIV WMEWDREINNYASLIHSLIE 173
ESQNQQEKNEQELLELDK
WASLWNWF
HIV C34 HIV WMEWDREINNYTSLIHSLIE 174
ESQNQQEKNEQELL
HIV C36 HIV YTSLIHSLIEESQNQQEKNE 175
QELLELDKWASLWNWF
HIV 1-1249 HIV WQEWEQKITALLEQAQIQQ 176
EKNEYELOKLDKWASLWE
WF
HIV T-649 HIV WMEWDREINNYTSLIHSLIE 177
ESQNQQEKNEQELLEL
HIV SC35EK HIV WEEWDKKIEEYTKKIEELIK 178
KSEEQQKKNEEELKK
34
CA 03241026 2024- 6- 13
WO 2023/122789
PCT/US2022/082345
Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
HIV 1-2635 HIV TTWEAWDRAIAEYAARIEAL 179
IRAAQEQQEKNEAALREL
HIV HIV-2 EHO-C46 HIV WQQWERQVRFLDANITKLL 180
EEAQIQQEKNMYELQELDK
WASLWNWF
HIV DP178 analog HIV TNTIYTLLEESQNQQEKNE 181
derived from QELLELDKWASLWNWF
gp41 peptide
region of
isolate HIV-1
SF2
HIV DP178 analog HIV YTGIIYNLLEESQNQOEKNE 182
derived from QELLELDKW ANLWNWF
gp41 peptide
region of
isolate HIV-1 RF
HIV DP178 analog HIV YTSLIYSLLEKSQ100EKNE 183
derived from QELLELDKWASLWNWF
gp41 peptide
region of
isolate HIV-1
MN
HIV Partial C34 HIV WMEWDREINNYTSLIHSLIE 184
peptide ESONCXDEKNEDELL
(originally
derived from
gp41)
RSV DP178 and/or RSV YTSVITIELSNIKENKCNGAK 185
DP107 analog VKLIKOELDKYK
RSV DP178 and/or RSV TSVITIELSNIKENKCNGAKV 186
DP107 analog KLIKQELDKYKN
RSV DP178 and/or RSV VITIELSNIKENKCNGAKVKL 187
DP107 analog IKOELDKYKNAV
RSV DP178 and/or RSV IALLSTNKAVVSLSNGVSVL 188
DP107 analog TSKVLDLKNYI DK
HPIV DP178 and/or HPIV VEAKQARSDIEKLKEAIRDT 189
DP107 analog NKAVQSVQSSIGNLI
CA 03241026 2024- 6- 13
WO 2023/122789
PCT/US2022/082345
Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
HPIV DP178 and/or HPIV RSDIEKLKEAIRDTNKAVQS
190
DP107 analog VQSSIGNLIVAIKSV
HPIV DP178 and/or HPIV NSVALDPIDISIELNKAKSDL
191
DP107 analog EESKEWIRRSNQKL
HPIV DP178 and/or HPIV ALDPIDISIELNKAKSDLEES
192
DP107 analog KEWIRRSNQKLDSI
HPIV DP178 and/or HPIV LDPIDISIELNKAKSDLEESK
193
DP107 analog EWIRRSNQKLDSIG
HPIV DP178 and/or HPIV DPIDISIELNKAKSDLEESKE
194
DP107 analog WIRRSNQKLDSIGN
HPIV DP178 and/or HPIV PIDISIELNKAKSDLEESKEW
195
DP107 analog IRRSNQKLDSIGNW
HPIV DP178 and/or HPIV IDISIELNKAKSDLEESKEWI
196
DP107 analog RRSNQKLDSIGNWH
MeV DP178 and/or MeV HRIDLGPPISLERLDVGTNL
197
DP107 analog GNAIAKLEAKELLE
MeV DP178 and/or MeV IDLGPPISLERLDVGTNLGN
198
DP107 analog AIAKLEAKELLESS
MeV DP178 and/or MeV LGPPISLERLDVGTNLGNAI
199
DP107 analog AKLEAKELLESSDO
MeV DP178 and/or MeV PISLERLDVGTNLGNAIAKL
200
DP107 analog EAKELLESSDQILR
SIV DP178 and/or SIV WQEWERKVDFLEENITALL
201
DP107 analog EEAQIQQEKNMYELQK
SIV DP178 and/or SIV QEWERKVDFLEENITALLEE
202
DP107 analog AQIQQEKNMYELQKL
SIV DP178 and/or SIV EWERKVDFLEENITALLEEA
203
DP107 analog QIQQEKNMYELQKLN
SIV DP178 and/or SIV WERKVDFLEENITALLEEAQ
204
DP107 analog IQQEKNMYELQKLNS
SIV DP178 and/or SIV ERKVDFLEENITALLEEAQI
205
DP107 analog QQEKNMYELQKLNSW
SIV DP178 and/or SIV RKVDFLEENITALLEEAQIQ
206
DP107 analog QEKNMYELQKLNSWD
SIV DP178 and/or SIV KVDFLEENITALLEEAQIQQ
207
DP107 analog EKNMYELQKLNSWDV
SIV DP178 and/or SIV VDFLEENITALLEEAQIQQE
208
DP107 analog KNMYELQKLNSWDVF
36
CA 03241026 2024- 6- 13
WO 2023/122789
PCT/US2022/082345
Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
SIV DP178 and/or SIV DFLEENITALLEEAQIQQEK
209
DP107 analog NMYELQKLNSWDVFG
SIV DP178 and/or SIV FLEENITALLEEAQIQQEKN
210
DP107 analog MYELQKLNSWDVFGN
HIV DP178 peptide HIV YTSLIHSLIEESQNQQEKNE
211
(AA 638-673 of QELLELDKWASLWNWF
gp41 from the
HIV-1 LAI
isolate)
HIV DP107 peptide HIV NNLLRAIEAQQHLLQLTVW
212
(AA 558-595 of QIKQLQARILAVERYLKDQ
gp41 from the
HIV-1 LAI
isolate)
HIV DP178 analog HIV YTNTIYTLLEESQNQQEKNE
213
derived from QELLELDKWASLWNWF
gp41 peptide
region of HIV-
1 SF2
HIV DP178 analog HIV YTGIIYNLLEESQNQQEKNE
214
derived from QELLELDKWANLWNWF
gp41 peptide
region of HIV-
1 RF
HIV DP178 analog HIV YTSLIYSLLEKSQIQQEKNE
215
derived from QELLELDKWASLWNWF
gp41 peptide
region of HIV-
1 MN
RSV DP178 and/or RSV YTSWITIELSNIKENKCNGA
216
DP107 analog KWKLIKQELDKYK
RSV DP178 and/or RSV TSWITIELSNIKENKONGAK
217
DP107 analog WKLIKQELDKYKN
RSV DP178 and/or RSV WITIELSNIKENKCNGAKWK
218
DP107 analog LIKQELDKYKNAW
RSV DP178 and/or RSV IALLSTNKAWWSLSNGWS
219
DP107 analog WLTSKWLDLKNYI DK
37
CA 03241026 2024- 6- 13
WO 2023/122789
PCT/US2022/082345
Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
HPIV DP178 and/or HPIV VEAKQARSDIEKLKEAIRDT
220
DP107 analog NKAVQSVQSSIGNLI
HPIV DP178 and/or HPIV RSDIEKLKEAIRDTNKAVQS
221
DP107 analog VQSSIGNLIVAIKSV
HPIV DP178 and/or HPIV NSVALDPIDISIELNKAKSDL
222
DP107 analog EESKEWIRRSNOKL
HPIV DP178 and/or HPIV ALDPIDISIELNKAKSDLEES
223
DP107 analog KEWIRRSNQKLDSI
HPIV DP178 and/or HPIV LDPIDISIELNKAKSDLEESK
224
DP107 analog EWIRRSNOKLDSIG
HPIV DP178 and/or HPIV DPIDISIELNKAKSDLEESKE
225
DP107 analog WIRRSNOKLDSIGN
HPIV DP178 and/or HPIV PI
DISIELNKAKSDLEESKEW 226
DP107 analog IRRSNOKLDSIGNW
HPIV DP178 and/or HPIV IDISIELNKAKSDLEESKEWI
227
DP107 analog RRSNQKLDSIGNWE
MeV DP178 and/or MeV HRIDLGPPISLERLDWGTNL
228
DP107 analog GNAIAKLEAKELLE
MeV DP178 and/or MeV IDLGPPISLERLDWGTNLGN
229
DP107 analog AIAKLEAKELLESS
MeV DP178 and/or MeV LGPPISLERLDWGTNLGNAI
230
DP107 analog AKLEAKELLESSDQ
MeV DP178 and/or MeV PISLERLDWGTNLGNAIAKL
231
DP107 analog EAKELLESSDQILR
SIV DP178 and/or SIV WQEWERKVDFLEENITALL
232
DP107 analog EEAQIQQEKNMYELQK
SIV DP178 and/or SIV QEWERKVDFLEENITALLEE
233
DP107 analog AQIQQEKNMYELQKL
SIV DP178 and/or SIV EWERKVDFLEENITALLEEA
234
DP107 analog QIQQEKNMYELQKLN
SIV DP178 and/or SIV WERKVDFLEENITALLEEAQ
235
DP107 analog IQQEKNMYELQKLNS
SIV DP178 and/or SIV ERKVDFLEENITALLEEAQI
236
DP107 analog QQEKNMYELQKLNSW
SIV DP178 and/or SIV RKVDFLEENITALLEEAQIQ
237
DP107 analog QEKNMYELQKLNSWD
SIV DP178 and/or SIV KVDFLEENITALLEEAQIQQ
238
DP107 analog EKNMYELQKLNSWDV
38
CA 03241026 2024- 6- 13
WO 2023/122789
PCT/US2022/082345
Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
SIV DP178 and/or SIV WDFLEENITALLEEAQIQQE
239
DP107 analog KNMYELQKLNSWDVF
SIV DP178 and/or SIV DFLEENITALLEEAQIQQEK
240
DP107 analog NMYELQKLNSWDVFG
SIV DP178 and/or SIV FLEENITALLEEAQIQQEKN
241
DP107 analog MYELQKLNSWDVFGN
HIV Enfuvirtide (T- HIV YTSLIHSLIEESQNQQEKNE
242
1249) QELLELNKWA SLWNWF
HIV HIV CLLLGTEVSEALGGAGLT
243
RSV RSV DEFDASISQVNEKINQSLAFI
244
RKSDELLHNVNAGK
Hendra Virus; Hendra Virus; Human
DITLNNSVALDPIDISIELNKA 245
Human parainfluenza virus
KSDLEESKEWIRRSNQKLD
parainfluenza type 3 SIGN
virus type 3
RSV RSV DPLVFPSDEFDASISQVNEK
246
INQSLAFIRKSDELL
HIV; Influenza A HIV; Influenza A DOYKCLOFIGGFCLRSSCP
247
SNTKLQGTCKPDKPNCCKS
RSV RSV EFDASISQVNEKINQSLAFIR
248
KSDELLHNVNAGKS
RSV RSV FDASISQVNEKINQSLAFIRK
249
SDELLHNVNAGKST
Chikungunya Chikungunya virus;
FLGAILKIGHALAKTVLPMVT 250
virus; Dengue Dengue virus NAFKPKQ
virus
RSV RSV FPSDEFDASISQVNEKINQS
251
LAFIRKSDELLHNVN
RSV RSV FYDPLVFPSDEFDASISQVN
252
EKINQSLAFIRKSDE
Influenza B Influenza B virus;
GADDVVDSSKSFVMENFSS 253
virus:Hepatitis E Hepatitis E virus YHGTKPGYVDSIQKGIQKP
virus KSGTQGNYDDDWKEFYST
DKNYDAAGYSVDNENPLSG
KAGGVVKVTYPGLTKVLAL
KVDNAETIKKELGLSLTEPL
MEQVGT
39
CA 03241026 2024- 6- 13
WO 2023/122789
PCT/US2022/082345
Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
Dengue Dengue virus; Zika GFCCPLDQMOCHNHCQSV
254
virus:Zika virus virus RYRGGYCTNFLKMTCKCY
HIV HIV GIFPKIIGKGIVNGIKSLAKG
255
VGMKVFKAGLNNIGNTGCN
NRDEC
HIV HIV HSLIEESQNQQEKNEQELL
256
ELDKWASLWNWFNITNW
RSV RSV IINFYDPLVFPSDEFDASISQ
257
VNEKINQSLAFIRK
HIV HIV
IKKEIEAIKKEQEAIKKKIEAIE 258
KEISGIVQQQNNLLRAIEAQ
QHLUDLTVWGIKOLOARIL
RSV RSV INFYDPLVFPSDEFDASISQ
259
VNEKINQSLAFIRKS
Hendra Hendra Virus:Nipah
KVDISSOISSMNOSLQQSK 260
Virus:Nipah virus DYIKEAQRLLDTVNPSL
virus
HIV HIV LGTEVSEALGGAGLTGGF
261
Measles Measles virus:Human
LHRIDLGPPISLERLDVGTN 262
virus:Human parainfluenza virus LGNAIAKLEDAKELL
parainfluenza type 3
virus type 3
HIV HIV LNNCLLLGTEVSEALGGA
263
RSV RSV LVFPSDEFDASISQVNEKIN
264
QSLAFIRKSDELLHN
HIV HIV MTWMEWDREINNYTSLIHS
265
LIEESQNQQEKNEQELL
RSV RSV NFYDPLVFPSDEFDASISQV
266
NEKINQSLAFIRKSD
HIV HIV NNLLRAIEAQQHLLQLTVW
267
GIKQLQARILAVERYLKDQ
RSV RSV PLVFPSDEFDASISQVNEKI
268
NQSLAFIRKSDELLH
Hendra Virus; Hendra Virus; Human
PPVYTKDVDISSQISSMNQS 269
Human parainfluenza virus
LQQSKDYIKEAQKILDTVNP
parainfluenza type 3 SL
virus type 3
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Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
RSV RSV PSDEFDASISQVNEKINQSL
270
AFIRKSDELLHNVNA
HIV HIV RFPFHRCGAGPKLTKDLE
271
HIV HIV RSQKEGLHYTCSSHFPYSQ
272
YQFWK
RSV RSV SDEFDASISQVNEKINQSLA
273
FIRKSDELLHNVNAG
HIV HIV SGIVQQQNNLLRAIEAQQHL
274
LQLTVWGIKQLQARIL
Hendra Virus; Hendra Virus; Human
SIELNKAKSDLEESKEWIRR 275
Human parainfluenza virus SNOKLDSI
parainfluenza type 3
virus type 3
HIV HIV TTWEAWDRAIAEYAARIEAL
276
IRAAQEQQEKLEAALREL
Hendra Virus; Hendra Virus; Human
VALDPIDISIELNKAKSDLEE 277
Human parainfluenza virus SKEWIRR
parainfluenza type 3
virus type 3
Hendra Virus; Hendra Virus; Human
VALDPIDISIELNKAKSDLEE 278
Human parainfluenza virus SKEWIRRSNQKLDSD
parainfluenza type 3
virus type 3
Hendra Virus; Hendra Virus; Human
VALDPIDISIELNKAKSDLEE 278
Human parainfluenza virus SKEWIRRSNQKLDSI
parainfluenza type 3
virus type 3
Hendra Virus; Hendra Virus; Human
VANDPIDISIELNKAKSDLEE 280
Human parainfluenza virus SKEWIRRSNQKLDSD
parainfluenza type 3
virus type 3
Hendra Virus; Hendra Virus; Human
VANDPIDISIELNKAKSDLEE 281
Human parainfluenza virus SKEWIRRSNOKLDSI
parainfluenza type 3
virus type 3
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Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
RSV RSV VFPSDEFDASISQVNEKINQ
282
SLAFIRKSDELLHNV
Hendra Virus; Hendra Virus; Human
VYTDKVDISSQISSMNQSLQ 283
Human parainfluenza virus QSKDYIKEAQKILDTV
parainfluenza type 3
virus type 3
HCV HCV WVAVTPTVATRDGKLPTT
284
RSV RSV YDPLVFPSDEFDASISQVNE
285
KINQSLAFIRKSDEL
HIV HIV YTSLIHSLIEESQNQQEKNE
286
QQLLELDKWASLWNWF
HIV-1 gp41 Albuviritide HIV Ac-
287
WEEWDREINNYT(Mpa)LIH
ELIEESQNQQEKNEQELL-
CONH2 (Mpa=3-
maleimidopropionic acid)
CoV-0C43 EK1 CoV SLDQINVTFLDLEYEMKKLE
288
EAIKKLEESYIDLKEL
CoV-0043 0043-HR2P CoV SLDYINVTFLDLQDEMNRLQ
289
EAIKVLNQSYINLKDI
SARS-CoV-2 Wuhan-Hu-1 CoV NVLYENQKLIANQFNSAIGKI
290
HR1 QDSLSSTASALGKLQDVVN
QNAQALNTLVKQLSSNFGAI
SSVLNDILSRLDKVEAEVQI
DRLITGRLQSLQTYVTQQLI
RAAEIRASAN
SARS-CoV-2 Alpha HR1 CoV NVLYENQKLIANQFNSAIGKI
291
QDSLSSTASALGKLQDVVN
QNAQALNTLVKQLSSNFGAI
SSVLNDILARLDKVEAEVQI
DRLITGRLQSLQTYVTQQLI
RAAEIRASAN
SARS-CoV-2 Beta HR1 CoV NVLYENQKLIANQFNSAIGKI
292
QDSLSSTASALGKLQDVVN
QNADALNTLVKQLSSNFGAI
SSVLNDILSRLDKVEAEVQI
DRLITGRLQSLQTYVTQQLI
RAAEIRASAN
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Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
SARS-CoV-2 Gamma HR1 CoV
NVLYENQKLIANQFNSAIGKI 293
QDSLSSTASALGKLQDVVN
QNAQALNTLVKQLSSNFGAI
SSVLNDILSRLDKVEAEVQI
DRLITGRLQSLQTYVTQQLI
RAAEIRASAN
SARS-CoV-2 Delta HR1 CoV NVLYENQKLIANQFNSAIGKI
294
QDSLSSTASALGKLQNVVN
QNAQALNTLVKQLSSNFGAI
SSVLNDILSRLDKVEAEVQI
DRLITGRLQSLQTYVTQQLI
RAAEIRASAN
SARS-CoV-2 Omicron HR1 CoV
NVLYENQKLIANQFNSAIGKI 295
QDSLSSTASALGKLQDVVN
HNAQALNTLVKQLSSKFGAI
SSVLNDIFSRLDKVEAEVQI
DRLITGRLQSLQTYVTQQLI
RAAEIRASAN
SARS-CoV-1 SARS-CoV1 CoV NVLYENQKQIANQFNKAISQ
296
HR1 IQESLTTTSTALGKLQDVVN
QNAQALNTLVKQLSSNFGAI
SSVLNDILSRLDKVEAEVQI
DRLITGRLQSLQTYVTQQLI
RAAEIRASAN
MERS MERS HR1 CoV QVLSENQKLIANKFNQALG
297
AMQTGFTTTNEAFQKVQDA
VNNNAQALSKLASELSNTF
GAISASIGDIIQRLDVLEQDA
01DRLINGRLTTLNAFVAQ0
LVRSESAALSAQ
SARS-CoV-2 Wuhan-Hu-1 CoV VVIGIVNNTVYDPLQPELDS
298
HR2 FKEELDKYFKNHTSPDVDL
GDISGINASVVNIQKEIDRLN
EVAKNLNESLIDLQEL
SARS-CoV-2 Alpha HR2 CoV VVIGIVNNTVYDPLQPELDS
299
FKEELDKYFKNHTSPDVDL
GDISGINASVVNIQKEIDRLN
EVAKNLNESLIDLQEL
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Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
SARS-CoV-2 Beta HR2 CoV VVIGIVNNTVYDPLQPELDS
300
FKEELDKYFKNHTSPDVDL
GDISGINASVVNIQKEIDRLN
EVAKNLNESLIDLQEL
SARS-CoV-2 Gamma HR2 CoV VVIGIVNNTVYDPLQPELDS
301
FKEELDKYFKNHTSPDVDL
GDISGINASFVNIQKEIDRLN
EVAKNLNESLIDLQEL
SARS-CoV-2 Delta HR2 CoV VVIGIVNNTVYDPLQPELDS
302
FKEELDKYFKNHTSPDVDL
GDISGINASVVNIQKEIDRLN
EVAKNLNESLIDLQEL
SARS-CoV-2 Omicron HR2 CoV
VVIGIVNNTVYDPLQPELDS 303
FKEELDKYFKNHTSPDVDL
GDISGINASVVNIQKEIDRLN
EVAKNLNESLIDLQEL
SARS-CoV-1 SARS-CoV1 CoV VVIGIINNTVYDPLOPELDSF
304
HR2 KEELDKYFKNHTSPDVDLG
DISGINASVVNIQKEIDRLNE
VAKNLNESLIDLQEL
MERS MERS HR2 CoV VTYQNISTNLPPPLLGNSTG
305
IDFQDELDEFFKNVSTSIPN
FGSLTQINTTLLDLTYEMLS
LQQVVKALNESYIDLKEL
SARS-CoV-2 HR2A CoV DISGINASVVNIQKEIDRLNE
306
VAKNLNESLIDLQEL
SARS-CoV-2 HR2B CoV VVIGIVNNTVYDPLQPELDS
307
FKEELDKYFKNHTSPD
SARS-CoV-2 HR2C CoV FKNHTSPDVDLGDISGINAS
308
VVNIQKEIDRLNEVAK
SARS-CoV-2 HR2 Full CoV GIVNNTVYDPLOPELDSFKE
309
Length ELDKYFKNHTSPDVDLGDIS
GINASVVNIQKEIDRLNEVA
KNLNESLIDLQEL
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Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
SARS-CoV-2 HR2 Full CoV MGWSCIILFLVATATGVHS
310
Length Fc GIVNNTVYDPLQPELDSFKE
fusion ELDKYFKNHTSPDVDLGDIS
GINASVVNIQKEIDRLNEVA
KNLNESLIDLQELGGGGSG
GGGSGGGGSAESKYGPPC
PPCPAPEAAGGPSVFLEPP
KPKDTLMISRTPEVTCVVVD
VSQEDPEVQFNWYVDGVE
VHNAKTKPREEQFNSTYRV
VSVLTVLHQDWLNGKEYKC
KVSNKGLPSSIEKTISKAKG
QPREPOVYTLPPSOEEMTK
NOVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVL
DSDGSFFLYSRLTVDKSRW
QEGNVFSCSVMHEALHNH
YTQKSLSLSLG
SARS-CoV-2 HR2 Full Coy GIVNNTVYDPLQPELDSFKE
311
Length Fc ELDKYFKNHTSPDVDLGDIS
fusion without GINASVVNIQKEIDRLNEVA
signal peptide KNLNESLIDLQELGGGGSG
GGGSGGGGSAESKYGPPC
PPCPAPEAAGGPSVFLFPP
KPKDTLMISRTPEVTCVVVD
VSQEDPEVQFNWYVDGVE
VHNAKTKPREEQFNSTYRV
VSVLTVLFIQDWLNGKEYKC
KVSNKGLPSSIEKTISKAKG
QPREPQVYTLPPSQEEMTK
NOVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVL
DSDGSFFLYSRLTVDKSRW
QEGNVFSCSVMHEALHNH
YTQKSLSLSLG
HIV HIV CHR HIV TTWMEWDREINNYTSLIHS
312
LIEESQNQQEKNEQELLELD
KWASLWNWF
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Source Peptide Target Amino Acid Sequence
SEQ ID
NO:
HIV 120 HIV YTSLIHSLIEESQNQQEKNE
313
QELLELDKWASLWNWF
HIV T2410 HIV MTWMEWDREINNYTSLIHS
314
LIEESQNQQEKNEQELLEL
HIV 12410_2.0 HIV MTWMEWDREINNYTSLIHS
315
LIEESQNQQEKNEQELLELD
KWASLWNWF
HIV T1144 HIV TTWEAWDRAIAEYAARIEAL
316
LRALQEQQEKNEAALREL
HIV T144_2.0 HIV TTWEAWDRAIAEYAARIEAL
317
LRALQEQQEKNEAALRELD
KWASLWNWF
HIV T1249 HIV WQEWEQKITALLEQAQIQQ
318
EKNEYELQKLDKWASLWE
WF
HIV 11249_2.0 HIV TTWQEWEQKITALLEQAQI
319
QQEKNEYELQKLDKWASL
WEWF
HIV T2635 HIV TTWEAWDRAIAEYAARIEAL
320
IRAAQEQQEKNEAALREL
HIV 12635 2.0 HIV TTWEAWDRAIAEYAARIEAL
321
IRAAQEQQEKNEAALRELD
KWASLWNWF
HIV 12635_3.0 HIV MTWEAWDRAIAEYAARIEA
322
LIRAAQEQQEKNEAALREL
DKWASLWNWF
HIV T290676 HIV TTWEAWDRAIAEYAARIEAL
323
IRASQEQQEKNEAELREL
HIV T290676_2.0 HIV
TTWEAWDRAIAEYAARIEAL 324
IRASQEQQEKNEAELRELD
KWASLWNWF
In some embodiments, the virus is human immunodeficiency virus (HIV) (e.g.,
the antifusogenic
polypeptide inhibits viral entry of HIV). In some embodiments, the HIV is HIV-
1. In some embodiments,
the HIV is a strain of HIV-1 (e.g., HIV-1 HE, HIV-1111B, HIV-1 MN, HIV-1 NDK,
HIV-1 N14-3, HIV-1 IRK, Or HIV-1sF2). In
some embodiments, the HIV is HIV-2. In some embodiments, the HIV is a strain
of HIV-2 (e.g., HIV-2EHo
or HIV-2RoD). In some embodiments, the HIV is an HIV pseudovirion. In some
embodiments, the
antifusogenic polypeptide prevents HIV viral fusion by specifically binding to
HIV glycoprotein 120
(gp120). In some embodiments, the antifusogenic polypeptide prevents binding
of gp120 to the cluster of
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differentiation 4 (CD4) co-receptor. In some embodiments, the antifusogenic
polypeptide reduces the
affinity of gp120 for a co-receptor (e.g., C-C chemokine receptor type 5
(CCR5) and C-X-C chemokine
receptor type 4 (CXCR4)). In some embodiments, the antifusogenic polypeptide
prevents binding of
gp120 to a co-receptor (e.g., CCR5 and CXCR4). In some embodiments, the
antifusogenic polypeptide
prevents HIV viral fusion by specifically binding to HIV glycoprotein 41
(gp41). In some embodiments, the
antifusogenic polypeptide inhibits entry of the HIV viral core into the cell.
In some embodiments, the
polyribonucleotide cargo includes an expression sequence encoding a
polypeptide (e.g., a polypeptide
that inhibits viral entry of HIV) of any one of SEQ ID NOs: 1, 5, 11, 13-18,
22-38, 40-46, 49-56, 58, 59, 61,
66-78, 80-85, 87-91, 95-100, 112, 113, 153, 163, 64, 168, 169, 171-184, 211-
215, 242, 243, 247, 255,
256, 258, 261, 261, 265, 267, 271, 272, 274, 276, 286, 287, or 312-324. In
some embodiments, the
polyribonucleotide cargo includes an expression sequence encoding a
polypeptide having at least 85%
(e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a
polypeptide of any one of SEQ ID
NOs: 1, 5, 11, 13-18, 22-38, 40-46, 49-56, 58, 59, 61, 66-78, 80-85, 87-91, 95-
100, 112, 113, 153, 163,
64, 168, 169, 171-184, 211-215, 242, 243, 247, 255, 256, 258, 261, 261, 265,
267, 271, 272, 274, 276,
286, 287, or 312-324.
In some embodiments, the virus is hepatitis virus (e.g., the antifusogenic
polypeptide inhibits viral
entry of a hepatitis virus). In some embodiments, the hepatitis virus is
hepatitis A virus (HAV). In some
embodiments, the hepatitis virus is hepatitis B virus (HBV). In some
embodiments, the hepatitis virus is
hepatitis C virus (HCV). In some embodiments, the hepatitis virus is hepatitis
D virus (HDV). In some
embodiments, the hepatitis virus is hepatitis E virus (HEV). In some
embodiments, the hepatitis virus is
duck hepatitis virus (DHV). In some embodiments, the polyribonucleotide cargo
includes an expression
sequence encoding a polypeptide (e.g., a polypeptide that inhibits viral entry
of a hepatitis virus such as
HCV) of any one of SEQ ID NOs: 104, 109, 112, 113, 141-145, 158-160, or 284.
In some embodiments,
the polyribonucleotide cargo includes an expression sequence encoding a
polypeptide having at least
85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a
polypeptide of any one of SEQ
ID NOs: 104, 109, 112, 113,141-145, 158-160, or 284.
In some embodiments, the virus is a coronavirus, such as a betacoronavirus
(e.g., the
antifusogenic polypeptide inhibits viral entry of a coronavirus). In some
embodiments, the
betacoronavirus is SARS-CoV type-1 (SARS-CoV-1). In some embodiments, the
betacoronavirus is
SARS-CoV type 2 (SARS-CoV-2). In some embodiments, the betacoronavirus is a
pseudotyped virus
expressing the SARS-CoV-2 spike protein. In some embodiments, the
polyribonucleotide cargo includes
an expression sequence encoding a polypeptide (e.g., a polypeptide that
inhibits viral entry of a
betacoronavirus such as SARS-CoV-1 or SARS-CoV-2) of any one of SEQ ID NOs: 6-
9, 119-123, 165-
167, or 288-311. In some embodiments, the polyribonucleotide cargo includes an
expression sequence
encoding a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%,
or 100%) sequence
identity to a polypeptide of any one of SEQ ID NOs: 6-9, 119-123, 165-167, or
288-311.
In some embodiments, the virus is respiratory syncytial virus (RSV) (e.g., the
antifusogenic
polypeptide inhibits viral entry of RSV). In some embodiments, the RSV is RSV
subtype A (RSVA). In
some embodiments, the RSV is RSV subtype B (RSVB). In some embodiments, the
polyribonucleotide
cargo includes an expression sequence encoding a polypeptide (e.g., a
polypeptide that inhibits viral
entry of RSV) of any one of SEQ ID NOs: 11, 13, 39, 112, 113, 153, 185-188,
216-219, 244, 246, 247-
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249, 251, 252, 257, 259, 264, 266, 268, 270, 273, 282, or 285. In some
embodiments, the
polyribonucleotide cargo includes an expression sequence encoding a
polypeptide having at least 85%
(e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a
polypeptide of any one of SEQ ID
NOs: 11, 13, 39, 112, 113, 153, 185-188, 216-219, 244, 246, 247-249, 251, 252,
257, 259, 264, 266, 268,
270, 273, 282, or 285.
In some embodiments, the virus is influenza (e.g., seasonal influenza,
pandemic influenza,
influenza A, influenza Hi Ni subtype, influenza B, influenza C, influenza D).
In some embodiments, the
influenza is influenza A. In some embodiments, the influenza is influenza B.
In some embodiments, the
influenza is influenza C. In some embodiments, the influenza is influenza D.
In some embodiments, the
polyribonucleotide cargo includes an expression sequence encoding a
polypeptide (e.g., a polypeptide
that inhibits viral entry of influenza) of any one of SEQ ID NOs: 1, 4, 11,
245, 247, 253, 262, 269, 275,
277, 278, 279, 280, 281, or 283. In some embodiments, the polyribonucleotide
cargo includes an
expression sequence encoding a polypeptide having at least 85% (e.g., at least
90%, 95%, 97%, 99%, or
100%) sequence identity to a polypeptide of any one of SEQ ID NOs: 1, 4, 11,
245, 247, 253, 262, 269,
275, 277, 278, 279, 280, 281, or 283.
In some embodiments, the virus is influenza virus (e.g., the antifusogenic
polypeptide inhibits viral
entry of influenza). In some embodiments, the influenza virus is influenza A
virus (IAV). In some
embodiments, the influenza virus is influenza B virus (IBV). In some
embodiments, the influenza virus is
an influenza virus expressing hemagglutinin (HA).
In some embodiments, the virus is herpes simplex virus (HSV) (e.g., the
antifusogenic
polypeptide inhibits viral entry of HSV). In some embodiments, the HSV is HSV-
1. In some
embodiments, the HSV is HSV-2.
In some embodiments, the virus is human papilloma virus (HPV) (e.g., the
antifusogenic
polypeptide inhibits viral entry of HPV). In some embodiments, the HPV is a
high-risk HPV strain (e.g.,
HPV 16, HPV 18, HPV 31, HPV 33, HPV 45, HPV 52, or HPV 58). In some
embodiments, the HPV is a
low-risk HPV strain (e.g., HPV 6, HPV 11, HPV 42, HPV 43, HPV or 44).
In some embodiments, the virus is any virus listed in Table 1.
In some embodiments, the GC content of a nucleic acid sequence encoding an
antifusogenic
polypeptide is at least 51% (e.g., at least 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, or 60%). In some
embodiments, the GC content of a nucleic acid sequence encoding an
antifusogenic polypeptide is at
most 52%, 53%, 54%, 55%, 56%, 57%, 58% or 59%, or 60%. In some embodiments,
the GC content of
a nucleic acid sequence encoding an antifusogenic polypeptide is 51% to 60%,
52% to 60%, 53% to
60%, 54% to 60%, 55% to 60%, 52% to 58%, 53% to 58%.
In some embodiments, the uridine content (for RNA) or the thymidine content
(for DNA) of a
nucleic acid sequence encoding an antifusogenic polypeptide is more than 10%
(e.g., more than 11%,
12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 0r25%). In
some
embodiments, the uridine content (for RNA) or the thymidine content (for DNA)
of a nucleic acid
sequence encoding an antifusogenic polypeptide is at most 30% (e.g., at most
29%, 28%, 27%, 26%,
25%, 24%, 23%, 22%, 21%, or 20%). In some embodiments, the uridine content
(for RNA) or the
thymidine content (for DNA) of a nucleic acid sequence encoding an
antifusogenic polypeptide is 20% to
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28%, 21% to 26%, 10% to 24%, 15% to 24%, 20% to 24%, 21% to 24%, 22% to 24%,
23% to 24%, 10%
to 23%, 15% to 23%, 20% to 23%, 21% to 23%, or 22% to 23%.
The GC content of an expression sequence encoding the antifusogenic
polypeptide refers to the
GC content of the expression sequence that exclusively encodes the
antifusogenic polypeptide with no
other coding regions that encode polypeptides other than the antifusogenic
polypeptide. Likewise, the
uridine content or thymidine of an expression sequence encoding the
antifusogenic polypeptide refers to
the uridine content of the expression sequence that exclusively encodes the
antifusogenic polypeptide
with no other coding regions that encode polypeptides other than the
antifusogenic polypeptide. In some
embodiments, the calculation of the GC content or the uridine (or thymidine)
content of the expression
sequence encoding the antifusogenic polypeptide only takes into account the
continuous nucleic acid
sequence that starts in a 5' to 3' direction from the first nucleoside of the
start codon of the open reading
frame that encodes the antifusogenic polypeptide to the last nucleoside of the
stop codon of the same
open reading frame. In other embodiments, the calculation of the GC content or
the uridine (or thymidine)
content of the expression sequence encoding the antifusogenic polypeptide only
takes into account the
continuous nucleic acid sequence that starts in a 5' to 3' direction from the
first nucleoside of the codon
that encodes the N-terminal end amino acid residue of the antifusogenic
polypeptide to the last
nucleoside of the codon that encodes the C-terminal end amino acid residue of
the antifusogenic
polypeptide.
In some embodiments, the nucleic acid sequence encoding the antifusogenic
polypeptide has a
uridine content of more than 20%. In some embodiments, the uridine content of
a nucleic acid sequence
encoding an antifusogenic polypeptide is more than 10% (e.g., more than 11%,
12%, 13%, 14%, 15%,
16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25%). In some embodiments, the
uridine content
of a nucleic acid sequence encoding an antifusogenic polypeptide is at most
30% (e.g., at most 29%,
28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20%). In some embodiments, the
uridine content of a
nucleic acid sequence encoding an antifusogenic polypeptide is 20% to 28%, 21%
to 26%, 10% to 24%,
15% to 24%, 20% to 24%, 21% to 24%, 22% to 24%, 23% to 24%, 10% to 23%, 15% to
23%, 20% to
23%, 21% to 23%, or 22% to 23%. In some embodiments, the nucleic acid sequence
encoding the
antifusogenic polypeptide has a uridine content of 20% to 28%.
Multiple Antifusogenic Polypeptides
In some embodiments, the circular polyribonucleotide encodes multiple
expression sequences
each encoding an antifusogenic polypeptide (e.g., two or more, such as 2 to
100,2 to 50, 2 to 20, 2 to 10,
5 to 100, 5 to 50, 5 to 20, or 5 to 10 expression sequences).
In some embodiments, the circular polyribonucleotide encodes two or more
(e.g., 2 to 100, 2 to
50, 2 to 20, 2 to 10, 5 to 100, 5 to 50, 5 to 20, or 5 to 10) copies of the
same antifusogenic polypeptide.
In some embodiments, the circular polyribonucleotide encodes two or more
(e.g., 2, 3, 4, 5, 6, 7,
8, 9, or 10) different (e.g., sharing less than 100% sequence identity)
antifusogenic polypeptides. In
some embodiments, the two or more different antifusogenic polypeptides are
each selected from an
antifusogenic polypeptide of Table 1. In some embodiments, the two or more
different antifusogenic
polypeptides each inhibit a different virus_ For example, a circular
polyribonucleotide may encode a first
antifusogenic polypeptide that inhibits influenza and a second an
antifusogenic polypeptide that inhibits
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RSV. A circular polyribonucleotide may include a first antifusogenic
polypeptide that inhibits influenza
and a second antifusogenic polypeptide that inhibits SARS-CoV-2. A circular
polyribonucleotide may
include a first antifusogenic polypeptide that inhibits HIV and a second
antifusogenic polypeptide that
inhibits SARS-CoV-2. A circular polyribonucleotide may include a first
antifusogenic polypeptide that
inhibits HIV and a second antifusogenic polypeptide that inhibits HCV. Where
either the first or second
antifusogenic polypeptide inhibits a plurality of viruses, the first and
second antifusogenic polypeptides
may have different virus specificity.
Wherein a circular polyribonucleotide encodes two or more antifusogenic
polypeptides, the
antifusogenic polypeptides may be encoded in a single open reading frame or
multiple open reading
frames.
In some embodiments, the disclosure provides a circular polyribonucleotide
including an open
reading frame (e.g., an open reading frame operably linked to an IRES) that
includes two or more
expression sequences, where each expression sequence encodes an antifusogenic
polypeptide. In
some embodiments, translation of the open reading frame produces a polypeptide
fusion including the
two or more antifusogenic polypeptides. The antifusogenic polypeptides may be
linked, e.g., by a linker
described herein (e.g., a peptide linker encoded by the open reading frame,
such as a glycine-serine
linker described below with respect to peptide-Fc fusions). In some
embodiments, the antifusogenic
polypeptides may be separated by a cleavage domain (e.g., a stagger sequence),
for example as
described herein.
In some embodiments, the disclosure provides a circular polyribonucleotide
including a first open
reading frame encoding a first antifusogenic polypeptide (e.g., operably
linked to a first IRES) and a
second open reading frame encoding a second antifusogenic polypeptide (e.g.,
operably linked to a
second IRES).
Antifusogenic Polypeptide-Fc Fusions
In some embodiments, the circular polyribonucleotide includes an expression
sequence encoding
a fusion protein including an antifusogenic polypeptide. In some embodiments,
the fusion protein
includes an antifusogenic polypeptide fused to an Fc domain (e.g., a single
chain of an Fc domain) of an
immunoglobulin. In some embodiments, the antifusogenic polypeptide is selected
from Table 1. In some
embodiments, the circular polyribonucleotide includes an expression sequence
encoding more than one
fusion protein including an antifusogenic polypeptide. In some embodiments,
the circular
polyribonucleotide includes an expression sequence encoding more than one
antifusogenic polypeptide.
In some embodiments, the circular polyribonucleotide encoding more than one
fusion protein reduces the
chance for viral escape.
In some embodiments, the Fc domain is an IgG4 Fc domain or a fragment thereof.
In some
embodiments, the Fc domain is an IgG1 Fc domain or a fragment thereof. In some
embodiments, the Fc
domain is an IgG2 Fc domain or a fragment thereof. In some embodiments, the Fc
domain is an IgG2a
Fc domain or a fragment thereof. In some embodiments, the Fc domain is an
IgG2b Fc domain or a
fragment thereof. In some embodiments, the Fc domain is an IgG3 Fc domain or a
fragment thereof.
In some embodiments, the C-terminal amino acid residue of the antifusogenic
polypeptide is
fused to the N-terminal amino acid residue of the Fc domain, optionally via a
peptide linker. In some
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embodiments, the N-terminal amino acid residue of the antifusogenic
polypeptide is fused to the C-
terminal amino acid residue of the Fc domain, optionally via a peptide linker.
In some embodiments, the
peptide linker between the antifusogenic polypeptide and the Fc domain
includes at least two amino acid
residues (e.g., 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, or at least 20 amino acid residues). In some embodiments, the
peptide linker between the
antifusogenic polypeptide and the Fc domain includes 2-200 amino acids residue
(e.g., 2-200, 2-180, 2-
160, 2-140, 2-120, 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-45, 2-40, 2-35, 2-
30, 2-25, 2-20, 2-15, 2-10, 2-9,
2-8, 2-7, 2-6, 2-5, 2-4, 4-200, 5-200, 6-200, 7-200, 8-200, 9-200, 10-200, 15-
200, 20-200, 25-200, 30-200,
35-200, 40-200, 45-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-
200, 140-200, 160-200,
or 180-200 amino acids residues). In some embodiments, the peptide linker
consists of glycine (Gly) and
serine (Ser) residues. In some embodiments, the peptide linker includes the
amino acid sequence of any
one of (GS)., (GGS)., (GGGGS)., (GGSG)., or (SGGG)., wherein x is an integer
from 1 to 50 (e.g., 1-40,
1-30, 1-20, 1-10, or 1-5). In some embodiments, the peptide linker includes
the amino acid sequence of
any one of (GS)x, (GGS)x, (GGGGS)x, (GGSG)x, or (SGGG)x, wherein x is an
integer from 1 to 10 (e.g., 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the peptide linker
includes 6 to 36 amino acids. In
some embodiments, the peptide linker includes 21 to 31 amino acids.
Polyribonucteotide Cargo
A polyribonucleotide cargo described herein includes any sequence including at
least one
polyribonucleotide. In some embodiments, the polyribonucleotide cargo includes
an expression
sequence, a non-coding sequence, or an expression sequence and a non-coding
sequence. In some
embodiments, the polyribonucleotide cargo includes an expression sequence
encoding an antifusogenic
polypeptide. In some embodiments, the polyribonucleotide cargo includes an
IRES operably linked to an
expression sequence encoding an antifusogenic polypeptide. In some
embodiments, the
polyribonucleotide cargo includes an expression sequence that encodes an
antifusogenic polypeptide
that has a biological effect on a subject.
A polyribonucleotide cargo may, for example, include 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 cargo
includes from 1-20,000 nucleotides, 1-10,000 nucleotides, 1-5,000 nucleotides,
100-20,000 nucleotide,
100-10,000 nucleotides, 100-5,000 nucleotides, 500-20,000 nucleotides, 500-
10,000 nucleotides, 500-
5,000 nucleotides, 1,000-20,000 nucleotides, 1,000-10,000 nucleotides, or
1,000-5,000 nucleotides.
In embodiments, the polyribonucleotide cargo includes one or multiple
expression (or coding)
sequences, wherein each expression (or coding) sequence encodes a polypeptide
(e.g., an antifusogenic
polypeptide). In embodiments, the polyribonucleotide cargo includes one or
multiple noncoding
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sequences. In embodiments, the polyribonucleotide cargo consists entirely of
non-coding sequence(s).
In embodiments, the polyribonucleotide cargo includes a combination of
expression (or coding) and
noncoding sequences.
In some embodiments, the 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.
Polypeptide expression sequences
In some embodiments, the polyribonucleotide described herein (e.g., the
polyribonucleotide cargo
of the circular polyribonucleotide) includes one or more expression (or
coding) sequences, wherein each
expression sequence encodes an antifusogenic polypeptide. In some embodiments,
the circular
polyribonucleotide includes two, three, four, five, six, seven, eight, nine,
ten or more expression (or
coding) sequences.
Each encoded polypeptide may be linear or branched. In various embodiments,
the polypeptide
has a length from about 5 to about 40,000 amino acids, about 15 to about
35,000 amino acids, about 20
to about 30,000 amino acids, about 25 to about 25,000 amino acids, about 50 to
about 20,000 amino
acids, about 100 to about 15,000 amino acids, about 200 to about 10,000 amino
acids, about 500 to
about 5,000 amino acids, about 1,000 to about 2,500 amino acids, or any range
therebetween. In some
embodiments, the polypeptide 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, or less may be useful.
Polypeptides included herein may include naturally occurring polypeptides or
non-naturally
occurring polypeptides. In some embodiments, the polypeptide is or includes a
functional fragment or
variant of a reference polypeptide (e.g., a biologically active fragment or
variant of an antifusogenic
polypeptide). For example, the polypeptide may be a functionally active
variant of any of the polypeptides
described herein with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,
79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99%
identity, e.g., over a specified region or over the entire sequence, to a
sequence of a polypeptide
described herein or a naturally occurring polypeptide. In some instances, the
polypeptide may have at
least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or greater)
identity to a protein of
interest.
In embodiments, polypeptides include multiple polypeptides, e.g., multiple
copies of one
polypeptide sequence, or multiple different polypeptide sequences. In
embodiments, multiple
polypeptides are connected by linker amino acids or spacer amino acids.
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In embodiments, the polynucleotide cargo includes a sequence encoding a signal
peptide. Many
signal peptide sequences have been described, for example, the Tat (Twin-
arginine translocation) signal
sequence is typically an N-terminal peptide sequence containing a consensus
SRRxFLK "twin-arginine"
motif, which serves to translocate a folded protein containing such a Tat
signal peptide across a lipid
bilayer. See also, e.g., the Signal Peptide Database publicly available at
www[dot]signalpeptide[dot]cle.
Signal peptides are also useful for directing a protein to specific
organelles; see, e.g., the experimentally
determined and computationally predicted signal peptides disclosed in the Spdb
signal peptide database,
publicly available at proline.bic.nus.edu.sg/spdb.
In some embodiments, the expression (or coding) sequence includes a poly-A
sequence (e.g., at
the 3' end of an expression 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, the expression sequence lacks a poly-A sequence
(e.g., at the 3' end of
an expression sequence).
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, and/or expression efficiency.
Internal Ribosomal Entry Sites
In some embodiments, a circular polyribonucleotide described herein includes
one or more
internal ribosome entry site (IRES) elements. In some embodiments, the IRES is
operably linked to one
or more expression sequences (e.g., each IRES is operably linked to one or
more expression sequences.
In embodiments, the IRES is located between a heterologous promoter and the 5'
end of a coding
sequence.
A suitable IRES element to include in a polyribonucleotide includes an RNA
sequence capable of
engaging a eukaryotic ribosome. In some embodiments, the IRES element is at
least about 5 nt, at least
about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt,
at least about 20 nt, at least about
25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at
least about 100 nt, at least about
200 nt, at least about 250 nt, at least about 350 nt, or at least about 500
nt.
In some embodiments, the IRES element is derived from the DNA of an organism
including, but
not limited to, a virus, a mammal, and a Drosophila. Such viral DNA may be
derived from, but is not
limited to, picomavirus complementary DNA (cDNA), with encephalomyocarditis
virus (EMCV) cDNA and
poliovirus cDNA. In one embodiment, Drosophila DNA from which an IRES element
is derived includes,
but is not limited to, an Antennapedia gene from Drosophila melanogaster.
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In some embodiments, the IRES sequence is an IRES sequence of Taura syndrome
virus,
Triatoma virus, Theiler's encephalomyelitis virus, simian Virus 40, Solenopsis
invicta virus 1,
Rhopalosiphum padi virus, Reticuloendotheliosis virus, fuman poliovirus 1,
Plautia stall intestine virus,
Kashmir bee virus, Human rhinovirus 2 (HRV-2), Homalodisca coagulata virus-1,
Human
Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P
virus, Hepatitis C virus,
Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human
enterovirus 71, Equine rhinitis
virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus (EMCV),
Drosophila C Virus,
Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1,
Black Queen Cell Virus, Aphid
lethal paralysis virus, Avian encephalomyelitis virus (AEV), Acute bee
paralysis virus, Hibiscus chlorotic
ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human
AML1/RUNX1,
Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-I, Human BCL2,
Human BiP,
Human c-IAPI , Human c-myc, Human elF4G, Mouse NDST4L, Human LEF1, Mouse HIFI
alpha, Human
n.myc, Mouse Gtx, Human p27kip1, Human PDGF2/c-sis, Human p53, Human Pim-I,
Mouse Rbm3,
Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA,
Human VEGF-A,
Human XIAP, Salivirus, Cosavirus, Parechovirus, Drosophila hairless, S.
cerevisiae TFIID, S. cerevisiae
YAP1, Human c-src, Human FGF-I, Simian picomavirus, Turnip crinkle virus,
Aichivirus, Crohivirus,
Echovirus 11, an aptamer to elF4G, Coxsackievirus B3 (CVB3) or Coxsackievirus
A (CVB1/2). In yet
another embodiment, the IRES is an IRES sequence of Coxsackievirus B3 (CVB3).
In a further
embodiment, the IRES is an IRES sequence of Encephalomyocarditis virus. In a
further embodiment, the
IRES is an IRES sequence of Theiler's encephalomyelitis virus.
The IRES sequence may have a modified sequence in comparison to the wild-type
IRES
sequence. In some embodiments, when the last nucleotide of the wild-type IRES
is not a cytosine nucleic
acid residue, the last nucleotide of the wild-type IRES sequence may be
modified such that it is a cytosine
residue. For example, the IRES sequence may be a CVB3 IRES sequence wherein
the terminal
adenosine residue is modified to cytosine residue. In some embodiments, the
modified CVB3 IRES may
have the nucleic acid sequence of:
TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGT
ACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACA
GTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAATAGA
CTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACAC
CGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCA
TTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGAC
GCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCG
GCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGTGIGTCGTAACGGGCAACTCTG
CAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAA
TTGAGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCC
TTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACA
GCAAC (SEQ ID NO: 325).
In some embodiments, the IRES sequence is an Enterovirus 71 (EV17) !RES. In
some
embodiments, the terminal guanosine residue of the EV17 IRES sequence is
modified to a cytosine
residue. In some embodiments, the modified EV71 IRES may have the nucleic acid
sequence of:
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ACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCAT
ATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGG
GGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGG
AAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCG
ACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGT
GCCACGTIGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGG
GCTGAAGGATGCCCAGAAGGTACCCCATTGTATOGGATCTGATCTGOGGCCTCGGTGCACATGCTT
TACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTT
GAAAAACACGATGATAATA (SEQ ID NO: 326).
In some embodiments, the polyribonucleotide includes at least one IRES
flanking at least one
(e.g., 2, 3, 4, 5 or more) expression sequence. In some embodiments, the IRES
flanks both sides of at
least one (e.g., 2, 3, 4, 5 or more) expression sequence. In some embodiments,
the 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). For example, a
polyribonucleotide described
herein may include a first IRES operably linked to a first expression sequence
and a second IRES
operably linked to a second expression sequence.
In some embodiments, a polyribonucleotide described herein includes an IRES
(e.g., an IRES
operably linked to a coding region). For example, the polyribonucleotide may
include any IRES as
described in Chen et al. MOL. CELL 81(20):4300-18, 2021; Jopling et al.
ONCOGENE 20:2664-70, 2001;
Baranick et al. PNAS 105(12):4733-38, 2008; Lang et al. MOLECULAR BIOLOGY OF
THE CELL 13(5):1792-
1801, 2002; Dorokhov et al. PNAS 99(8):5301-06, 2002; Wang et al. NUCLEIC
ACIDS RESEARCH
33(7):2248-58, 2005; Petz et al. NUCLEIC ACIDS RESEARCH 35(8):2473-82, 2007;
Chen et al. SCIENCE
268:415-417, 1995; Fan et al. NATURE COMMUNICATION 13(1):3751-3765, 2022, and
International
Publication No. W02021/263124, each of which is hereby incorporated by
reference in their entirety.
Signal Sequences
In some embodiments, an antifusogenic polypeptide expressed from a circular
polyribonucleotide
disclosed herein includes a secreted protein, for example, a protein 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 antifusogenic polypeptide encoded by the circular
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 antifusogenic polypeptide encoded
by the circular
polyribonucleotide does not include a secretion signal.
In some embodiments, the signal sequence is selected from SecSP38
(MWWRLWWLLLLLLLLWPMVWA; SEQ ID NO: 327); SecD4 (MWWLLLLLLLLWPMVWA; SEQ ID NO:
328), gLuc (MGVKVLFALICIAVAEAK; SEQ ID NO: 329); INHC1
(MASRLTLLTLLLLLLAGDRASS; SEQ
ID NO: 330); Epo (MGVHECPAWLWLLLSLLSLPLGLPVLG; SEQ ID NO: 331); and IL-2
(MYRMOLLSCIALSLALVTNS; SEQ ID NO: 332).
In some embodiments, a circular polyribonucleotide encodes multiple copies of
the same
antifusogenic polypeptide (e.g., one, two, three, four, five, six, seven,
eight, nine, ten, or more). In some
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embodiments, at least one copy of the antifusogenic polypeptide includes a
signal sequence and at least
one copy of the antifusogenic polypeptide does not include a signal sequence.
In some embodiments, a
circular polyribonucleotide encodes plurality of an antifusogenic polypeptides
(e.g., a plurality of different
antifusogenic polypeptides or a plurality of an antifusogenic polypeptides
having less than 100%
sequence identity), where at least one of the plurality of an antifusogenic
polypeptides includes a signal
sequence and at least one copy of the plurality of an antifusogenic
polypeptides 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 antifusogenic polypeptide, e.g.,
when expressed
endogenously. In some embodiments, the signal sequence is heterologous to the
antifusogenic
polypeptide, e.g., is not present when the wild-type antifusogenic polypeptide
is expressed endogenously.
A polyribonucleotide sequence encoding an antifusogenic polypeptide 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., an antifusogenic
polypeptide) may include a
signal sequence that directs the antifusogenic polypeptide to the secretory
pathway. In some
embodiments, the signal sequence may direct the antifusogenic polypeptide to
reside in certain
organelles (e.g., the endoplasmic reticulum, Golgi apparatus, or endosomes).
In some embodiments, the
signal sequence directs the antifusogenic polypeptide 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, the secretion signal is a human interleukin-2 (IL-2)
secretion signal. In
some embodiments, the IL-2 secretion signal has an amino acid sequence of at
least 90% sequence
identity to MYRMQLLSCIALSLALVTNS (SEQ ID NO: 332). In some embodiments, the IL-
2 secretion
signal has an amino acid sequence of at least 95% sequence identity to SEQ ID
NO: 332. In some
embodiments, the IL-2 secretion signal has an amino acid sequence of at least
99% sequence identity to
SEQ ID NO: 332. In some embodiments, the IL-2 secretion signal has an amino
acid sequence of 100%
sequence identity to SEQ ID NO: 332.
In some embodiments, the secretion signal is Gaussia luciferase secretion
signal. In some
embodiments, the Gaussia luciferase secretion signal has an amino acid
sequence of at least 90%
sequence identity of MGVKVLFALICIAVAEAK (SEQ ID NO: 329). In some embodiments,
the Gaussia
luciferase secretion signal has an amino acid sequence of at least 95%
sequence identity of SEQ ID NO:
329. In some embodiments, the Gaussia luciferase secretion signal has an amino
acid sequence of at
least 99% sequence identity of SEQ ID NO: 329. In some embodiments, the
Gaussia luciferase secretion
signal has an amino acid sequence of 100% sequence identity of SEQ ID NO: 329.
In some embodiments, the secretion signal is an EPO (e.g_, a human EPO)
secretion signal. In
some embodiments, the EPO secretion signal has an amino acid sequence of at
least 90% sequence
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identity of MGVHECPAWLWLLLSLLSLPLGLPVLGA (SEQ ID NO: 333). In some
embodiments, the EPO
secretion signal has an amino acid sequence of at least 95% sequence identity
of SEQ ID NO: 333. In
some embodiments, the EPO secretion signal has an amino acid sequence of at
least 99% sequence
identity of SEQ ID NO: 333. In some embodiments, the EPO secretion signal has
an amino acid
sequence of 100% sequence identity of SEQ ID NO: 333.
In some embodiments, the secretion signal is a wildtype SARS-CoV-2 secretion
signal. In some
embodiments, the wildtype SARS-CoV-2 secretion signal has an amino acid
sequence of at least 90%
sequence identity of MFVFLVLLPLVSS (SEQ ID NO: 334). In some embodiments, the
wildtype SARS-
CoV-2 secretion signal has an amino acid sequence of at least 95% sequence
identity of SEQ ID NO:
334. In some embodiments, the wildtype SARS-CoV-2 secretion signal has an
amino acid sequence of
at least 99% sequence identity of SEQ ID NO: 334. In some embodiments, the
wildtype SARS-CoV-2
secretion signal has an amino acid sequence of 100% sequence identity of SEQ
ID NO: 334.
In some embodiments, an antifusogenic polypeptide encoded by a
polyribonucleotide includes
either a secretion signal sequence, a transmembrane insertion signal sequence,
or does not include a
signal sequence.
Regulatory Elements
In some embodiments, the polyribonucleotide described herein (e.g., the
polyribonucleotide cargo
of the polyribonucleotide) includes one or more regulatory elements. In some
embodiments, the
polyribonucleotide includes a regulatory element, e.g., a sequence that
modifies expression of an
expression sequence within the 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 linked
operatively 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 exists. In
addition, one regulatory element
can increase an amount of products expressed for multiple expression sequences
attached in tandem_
Hence, one regulatory element can enhance the expression of one or more
expression sequences.
Multiple regulatory elements are well-known to persons of ordinary skill in
the art.
In some embodiments, the regulatory element is a translation modulator. A
translation modulator
can modulate translation of the expression sequence in the polyribonucleotide.
A translation modulator
can be a translation enhancer or suppressor. In some embodiments, the
polyribonucleotide includes at
least one translation modulator adjacent to at least one expression sequence.
In some embodiments, the
polyribonucleotide includes a translation modulator adjacent each expression
sequence. In some
embodiments, the translation modulator is present 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, the regulatory element is a microRNA (miRNA) or a miRNA
binding site.
Further examples of regulatory elements are described, e.g., in paragraphs
[0154] ¨[0161] of
International Patent Publication No. W02019/118919, which is hereby
incorporated by reference in its
entirety.
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Cleavage Domains
A circular 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: 335). 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, the circular polyribonucleotide includes at least one
stagger element
adjacent to an expression sequence. In some embodiments, the circular
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., peptide(s) and or polypeptide(s). In some embodiments, the
stagger element is a portion
of the one or more expression sequences. In some embodiments, the circular
polyribonucleotide
includes one or more expression sequences, and each of the one or more
expression sequences is
separated from a succeeding expression sequence by a stagger element on the
circular
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.
In some embodiments, the circular polyribonucleotide includes a stagger
element. To avoid
production of a continuous expression product, e.g., peptide or polypeptide,
while maintaining rolling
circle translation, a stagger element may be included to induce ribosomal
pausing 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 polyribonucleotide. The stagger element may include, but is not
limited to a 2A-like, or
CHYSEL (SEQ ID NO: 336) (cis-acting hydrolase element) sequence. In some
embodiments, the
stagger element encodes a sequence with a C-terminal consensus sequence that
is X1X2X3EX5NPGP
(SEQ ID NO: 337), 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. 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
(SEQ ID NO: 338), where x= any amino acid. Some nonlimiting examples of
stagger elements includes
GDVESNPGP (SEQ ID NO: 339), GDIEEN POP (SEQ ID NO: 340), VEPNPGP (SEQ ID NO:
341),
IETNPGP (SEQ ID NO: 342), GDIESNPGP (SEQ ID NO: 343), GDVELNPGP (SEQ ID NO:
344),
GDIETNPGP (SEQ ID NO: 345), GDVENPGP (SEQ ID NO: 346), GDVEENPGP (SEQ ID NO:
347),
GDVEQNPGP (SEQ ID NO: 348), IESNPGP (SEQ ID NO: 349), GDIELNPGP (SEQ ID NO:
350),
HDIETNPGP (SEQ ID NO: 351), HDVETNPGP (SEQ ID NO: 352), HDVEMNPGP (SEQ ID NO:
353),
GDMESNPGP (SEQ ID NO: 354), GDVETNPGP (SEQ ID NO: 355), GDIEQNPGP (SEQ ID NO:
356),
and DSEFNPGP (SEQ ID NO: 357).
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In some embodiments, the 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 polyribonucleotide includes at least one stagger element to
cleave the expression product. In
some embodiments, the circular polyribonucleotide includes a stagger element
adjacent to at least one
expression sequence. In some embodiments, the circular polyribonucleotide
includes a stagger element
after each expression sequence. In some embodiments, the circular
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 intemucleoside 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, the stagger element is present in the circular
polyribonucleotide in other
forms. For example, in some exemplary circular polyribonucleotides, a stagger
element includes a
termination element of a first expression sequence in the circular
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 polyribonucleotide.
In some cases, the first
expression sequence and the expression sequence succeeding the first
expression sequence are two
separate expression sequences in the circular 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
polyribonucleotide including the first stagger element upstream of the first
translation initiation sequence
of the succeeding sequence in the circular polyribonucleotide is continuously
translated, while a
corresponding circular 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 polyribonucleotide, and the first expression sequence
and its succeeding
expression sequence are the same expression sequence. In some exemplary
circular
polyribonucleotides, a stagger element includes a first termination element of
a first expression sequence
in the circular polyribonucleotide, and a nucleotide spacer sequence that
separates the termination
element from a downstream translation initiation sequence. In some such
examples, the first stagger
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element is upstream of (5' to) a first translation initiation sequence of the
first expression sequence in the
circular 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
polyribonucleotide including
the first stagger element upstream of the first translation initiation
sequence of the first expression
sequence in the circular polyribonucleotide is continuously translated, while
a corresponding circular
polyribonucleotide including a stagger element upstream of a second
translation initiation sequence of a
second expression sequence in the corresponding circular 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
polyribonucleotide than a distance between the first stagger element and the
first translation initiation in
the circular 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 polyribonucleotide includes more than one expression
sequence.
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, a plurality of an antifusogenic polypeptides encoded by a
circular
ribonucleotide may be separated by an IRES between each antifusogenic
polypeptide (e.g., each
antifusogenic polypeptide is operably linked to a separate IRES). For example,
a circular
polyribonucleotide may include a first IRES operably linked to a first
expression sequence and a second
IRES operably linked to a second expression sequence. The IRES may be the same
IRES between all
antifusogenic polypeptides. The IRES may be different between different
antifusogenic polypeptides.
In some embodiments, the plurality of an antifusogenic polypeptides may be
separated by a 2A
self-cleaving peptide. For example, a circular polyribonucleotide may encode
an IRES operably linked to
an open reading frame encoding a first antifusogenic polypeptide, a 2A, and a
second antifusogenic
polypeptide.
In some embodiments, the plurality of an antifusogenic polypeptides may be
separated by a
protease cleavage site (e.g., a furin cleavage site). For example, a circular
polyribonucleotide may
encode an IRES operably linked to an open reading frame encoding a first
antifusogenic polypeptide, a
protease cleavage site (e.g., a furin cleavage site), and a second
antifusogenic polypeptide.
In some embodiments, the plurality of an antifusogenic polypeptides may be
separated by a 2A
self-cleaving peptide and a protease cleavage site (e.g., a furin cleavage
site). For example, a circular
polyribonucleotide may encode an IRES operably linked to an open reading frame
encoding a first
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antifusogenic polypeptide, a 2A, a protease cleavage site (e.g., a furin
cleavage site), and a second
antifusogenic polypeptide. A circular polyribonucleotide may also encode an
IRES operably linked to an
open reading frame encoding a first antifusogenic polypeptide, a protease
cleavage site (e.g., a furin
cleavage site), a 2A, and a second antifusogenic polypeptide. A tandem 2A and
furin cleavage site may
be referred to as a furin-2A (which includes furin-2A or 2A-furin, arranged in
either orientation).
Furthermore, the plurality of an antifusogenic polypeptides encoded by the
circular ribonucleotide
may be separated by both IRES and 2A sequences. For example, an IRES may be
between one
antifusogenic polypeptide and a second antifusogenic polypeptide while a 2A
peptide may be between
the second antifusogenic polypeptide and the third antifusogenic polypeptide.
The selection of a
particular IRES or 2A self-cleaving peptide may be used to control the
expression level of an
antifusogenic polypeptide 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.
In some embodiments, a circular 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 embodiments,
cleavage sequence is included in an expression sequence. In some embodiments,
the circular
polyribonucleotide includes between 2 and 10 cleavage sequences. In some
embodiments, the circular
polyribonucleotide includes between 2 and 5 cleavage sequences. In some
embodiments, the multiple
cleavage sequences are between multiple expression sequences; for example, a
circular
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
polyribonucleotide includes a cleavage sequence, such as in an immolating
circRNA or cleavable
circRNA or self-cleaving circRNA. In some embodiments, the circular
polyribonucleotide includes two or
more cleavage sequences, leading to separation of the circular
polyribonucleotide into multiple products,
e.g., miRNAs, linear RNAs, smaller circular 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 antifusogenic
polypeptides, e.g., where the two or
more antifusogenic polypeptides are encoded by a single open-reading frame
(ORF). For example, two
or more antifusogenic polypeptides 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
antifusogenic polypeptides each
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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 antifusogenic
polypeptides is cleaved upon
expression, such that the two or more antifusogenic polypeptides 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 (e.g.,
furin), 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 polyribonucleotide described herein is an
immolating circular
polyribonucleotide, a cleavable circular polyribonucleotide, or a self-
cleaving circular polyribonucleotide.
A circular polyribonucleotide can deliver cellular components including, for
example, RNA, IncRNA,
lincRNA, miRNA, tRNA, rRNA, snoRNA, ncRNA, siRNA, or shRNA. In some
embodiments, a circular
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.
Translation Initiation Sequences
In some embodiments, the polyribonucleotide described herein (e.g., the
polyribonucleotide cargo
of the polyribonucleotide) includes at least one translation initiation
sequence. In some embodiments, the
polyribonucleotide includes a translation initiation sequence operably linked
to an expression sequence.
In some embodiments, the polyribonucleotide encodes a polypeptide and may
include 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
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 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 polyribonucleotide. In some embodiments, the translation initiation
sequence is within a substantially
single stranded region of the 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.
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The 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, the polyribonucleotide may initiate at a codon which is
not the first start
codon, e.g., AUG. Translation of the polyribonucleotide may initiate at an
alternative translation initiation
sequence, such as, but not limited to, ACG, AGO, AAG, CTG/CUG, GTG/GUG,
ATA/AUA, ATT/AUU,
TTG/UUG. In some embodiments, translation begins at an alternative translation
initiation sequence
under selective conditions, e.g., stress induced conditions. As a non-limiting
example, the translation of
the polyribonucleotide may begin at alternative translation initiation
sequence, such as ACG. As another
non-limiting example, the polyribonucleotide translation may begin at
alternative translation initiation
sequence, CTG/CUG. As another non-limiting example, the polyribonucleotide
translation may begin at
alternative translation initiation sequence, GTG/GUG. As another non-limiting
example, the
polyribonucleotide may begin translation at a repeat-associated non-AUG (RAN)
sequence, such as an
alternative translation initiation sequence that includes short stretches of
repetitive RNA e.g., COG,
GGGGCC, CAG, CTG.
Termination Elements
In some embodiments, the polyribonucleotide described herein (e.g., the
polyribonucleotide cargo
of the polyribonucleotide) includes least one termination element. In some
embodiments, the
polyribonucleotide includes a termination element operably linked to an
expression sequence. In some
embodiments, the polynucleotide lacks a termination element.
In some embodiments, the polyribonucleotide includes one or more expression
sequences, and
each expression sequence may or may not have a termination element. In some
embodiments, the
polyribonucleotide includes one or more expression sequences, and the
expression sequences lack a
termination element, such that the polyribonucleotide is continuously
translated. Exclusion of a
termination element may result in rolling circle translation or continuous
expression of expression product.
In some embodiments, the circular polyribonucleotide includes one or more
expression
sequences, and each expression sequence may or may not have a termination
element. In some
embodiments, the circular polyribonucleotide includes one or more expression
sequences, and the
expression sequences lack a termination element, such that the circular
polyribonucleotide is
continuously translated. Exclusion of a termination element may result in
rolling circle translation or
continuous expression of expression product, e.g., peptides or polypeptides,
due to lack of ribosome
stalling or fall-off. In such an embodiment, rolling circle translation
expresses a continuous expression
product through each expression sequence. In some other embodiments, a
termination element of an
expression sequence can be part of a stagger element. In some embodiments, one
or more expression
sequences in the circular polyribonucleotide includes a termination element.
However, rolling circle
translation or expression of a succeeding (e.g., second, third, fourth, fifth,
etc.) expression sequence in
the circular polyribonucleotide is performed. In such instances, the
expression product may fall off the
ribosome when the ribosome encounters the termination element, e.g., a stop
codon, and terminates
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translation. In some embodiments, translation is terminated while the
ribosome, e.g., at least one subunit
of the ribosome, remains in contact with the circular polyribonucleotide.
In some embodiments, the circular polyribonucleotide includes a termination
element at the end
of one or more expression sequences. In some embodiments, one or more
expression sequences
includes two or more termination elements in succession. In such embodiments,
translation is terminated
and rolling circle translation is terminated. In some embodiments, the
ribosome completely disengages
with the circular polyribonucleotide. In some such embodiments, production of
a succeeding (e.g.,
second, third, fourth, fifth, etc.) expression sequence in the circular
polyribonucleotide may require the
ribosome to reengage with the circular polyribonucleotide prior to initiation
of translation. Generally,
termination elements include an in-frame nucleotide triplet that signals
termination of translation, e.g.,
UAA, UGA, UAG. In some embodiments, one or more termination elements in the
circular
polyribonucleotide are frame-shifted termination elements, such as but not
limited to, off-frame or -1 and
4 1 shifted reading frames (e.g., hidden stop) that may terminate translation.
Frame-shifted termination
elements include nucleotide triples, TAA, TAG, and TGA that appear in the
second and third reading
frames of an expression sequence. Frame-shifted termination elements may be
important in preventing
misreads of mRNA, which is often detrimental to the cell. In some embodiments,
the termination element
is a stop codon.
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.
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.
In some embodiments, a circular polyribonucleotide includes a polyA sequence.
Exemplary
polyA 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 polyA 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
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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 polyA 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
polyA 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
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.
Protein-Binding Sequences
In some embodiments, a circular polyribonucleotide includes one or more
protein binding sites
that allow a protein, e.g., a ribosome, to bind to an internal site in the RNA
sequence. By engineering
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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 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: 358), where R is a purine (adenine or guanine) three bases upstream of the
start codon (AUG),
which is followed by another G. 5' UTRs also have been known to form secondary
structures which are
involved in elongation factor binding.
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, E1F4A3, E1F4G2, 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, MS11, MSI2, NONO, NONO-, NOP58, NPM1, NUDT21, PCBP2, POLR2A,
PRPF8,
PTBP1, RBFOX2, RBM10, RBM22, RBM27, RBM47, RNPS1, SAFB2, SBDS, SF3A3, SF3B4,
SIRT7,
SLBP, SLIM, 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.
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Spacer Sequences
In some embodiments, the polyribonucleotide described herein includes one or
more spacer
sequences. A spacer refers to any contiguous nucleotide sequence (e.g., of one
or more nucleotides)
that provides distance or flexibility between two adjacent polynucleotide
regions. Spacers may be
present in between any of the nucleic acid elements described herein. Spacer
may also be present within
a nucleic acid element described herein.
For example, wherein a nucleic acid includes any two or more of the following
elements: (A) a 3'
catalytic intron fragment; (B) a 3' splice site; (C) a 3' exon fragment; (D) a
polyribonucleotide cargo; (E) a
5' exon fragment; (F) a 5' splice site; and (G) a 5' catalytic intron
fragment; a spacer region may be
present between any one or more of the elements. Any of elements (A), (B),
(C), (D), (E), (F), or (G) may
be separated by a spacer sequence, as described herein. For example, there may
be a spacer between
(A) and (B), between (B) and (C), between (C) and (D), between (D) and (E),
between (E) and (F), or
between (F) and (G).
In some embodiments, the polyribonucleotide further includes a first spacer
region between the 5'
exon fragment of (C) and the polyribonucleotide cargo of (D). The spacer may
be, e.g., at least 5 (e.g., at
least 10, at least 15, at least 20) ribonucleotides in length. In some
embodiments, the polyribonucleotide
further includes a second spacer region between the polyribonucleotide cargo
of (D) and the 5' exon
fragment of (E).
A spacer sequences may be used to separate an IRES from adjacent structural
elements to
martini the structure and function of the IRES or the adjacent element. A
spacer can be specifically
engineered depending on the !RES. In some embodiments, an RNA folding computer
software, such as
RNAFold, can be utilized to guide designs of the various elements of the
vector, including the spacers.
The spacer may be, e.g., at least 5 (e.g., at least 10, at least 15, at least
20) ribonucleotides in
length. In some embodiments, each spacer region is at least 5 (e.g., at least
10, at least 15, at least 20)
ribonucleotides in length. Each spacer region may be, e.g., from 5 to 500
(e.g., 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500) ribonucleotides in
length. The first spacer region,
the second spacer region, or the first spacer region and the second spacer
region may include a polyA
sequence. The first spacer region, the second spacer region, or the first
spacer region and the second
spacer region may include a polyA-C sequence. In some embodiments, the first
spacer region, the
second spacer region, or the first spacer region and the second spacer region
includes a polyA-G
sequence. In some embodiments, the first spacer region, the second spacer
region, or the first spacer
region and the second spacer region includes a polyA-T sequence. In some
embodiments, the first
spacer region, the second spacer region, or the first spacer region and the
second spacer region includes
a random sequence.
Spacers may also be present within a nucleic acid region described herein. For
example, a
polynucleotide cargo region may include one or multiple spacers. Spacers may
separate regions within
the polynucleotide cargo.
In some embodiments, the spacer sequence can be, for example, at least 10
nucleotides in
length, at least 15 nucleotides in length, or at least 30 nucleotides in
length. In some embodiments, the
spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 25 or 30 nucleotides in
length. In some embodiments, the spacer sequence is no more than 100, 90, 80,
70, 60, 50, 45, 40, 35
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or 30 nucleotides in length. In some embodiments the spacer sequence is from
20 to 50 nucleotides in
length. In certain embodiments, the spacer sequence is 10,11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49 or
50 nucleotides in length.
The spacer sequences can be polyA sequences, polyA-C sequences, polyC
sequences, or poly-
U sequences.
In some embodiments, the spacer sequences can be polyA-T, polyA-C, polyA-G, or
a random
sequence.
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.
In some embodiments, the polyribonucleotide includes a 5' spacer sequence
(e.g., between the
5' annealing region and the polyribonucleotide cargo). In some embodiments,
the 5' spacer sequence is
at least 10 nucleotides in length_ In another embodiment, the 5' spacer
sequence is at least 15
nucleotides in length. In a further embodiment, the 5' spacer sequence is at
least 30 nucleotides in
length. In some embodiments, the 5' spacer sequence is at least 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the 5' spacer
sequence is no more
than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some
embodiments the 5' spacer
sequence is between 20 and 50 nucleotides in length. In certain embodiments,
the 5' spacer sequence is
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In
one embodiment, the 5'
spacer sequence is a polyA sequence. In another embodiment, the 5' spacer
sequence is a polyA-C
sequence. In some embodiments, the 5' spacer sequence includes a polyA-G
sequence. In some
embodiments, the 5' spacer sequence includes a polyA-T sequence. In some
embodiments, the 5'
spacer sequence includes a random sequence.
In some embodiments, the polyribonucleotide includes a 3' spacer sequence
(e.g., between the
3' annealing region and the polyribonucleotide cargo). In some embodiments,
the 3' spacer sequence is
at least 10 nucleotides in length. In another embodiment, the 3' spacer
sequence is at least 15
nucleotides in length. In a further embodiment, the 3' spacer sequence is at
least 30 nucleotides in
length. In some embodiments, the 3' spacer sequence is at least 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 25 or 30 nucleotides in length. In some embodiments, the 3' spacer
sequence is no more
than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides in length. In some
embodiments the 3' spacer
sequence is from 20 to 50 nucleotides in length. In certain embodiments, the
3' spacer sequence is 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38,
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In one
embodiment, the 3' spacer
sequence is a polyA sequence. In another embodiment, the 5' spacer sequence is
a polyA-C sequence.
In some embodiments, the 5 spacer sequence includes a polyA-G sequence. In
some embodiments, the
5' spacer sequence includes a polyA-T sequence. In some embodiments, the 5'
spacer sequence
includes a random sequence.
In one embodiment, the polyribonucleotide includes a 5' spacer sequence, but
not a 3' spacer
sequence. In another embodiment, the polyribonucleotide includes a 3' spacer
sequence, but not a 5'
spacer sequence. In another embodiment, the polyribonucleotide includes
neither a 5' spacer sequence,
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nor a 3' spacer sequence. In another embodiment, the polyribonucleotide does
not include an IRES
sequence. In a further embodiment, the polyribonucleotide does not include an
IRES sequence, a 5'
spacer sequence or a 3' spacer sequence.
In some embodiments, the spacer sequence includes at least 3 ribonucleotides,
at least 4
ribonucleotides, at least 5 ribonucleotides, at least about 8 ribonucleotides,
at least about 10
ribonucleotides, at least about 12 ribonucleotides, at least about 15
ribonucleotides, at least about 20
ribonucleotides, at least about 25 ribonucleotides, at least about 30
ribonucleotides, at least about 40
ribonucleotides, at least about 50 ribonucleotides, at least about 60
ribonucleotides, at least about 70
ribonucleotides, at least about 80 ribonucleotides, at least about 90
ribonucleotides, at least about 100
ribonucleotides, at least about 120 ribonucleotides, at least about 150
ribonucleotides, at least about 200
ribonucleotides, at least about 250 ribonucleotides, at least about 300
ribonucleotides, at least about 400
ribonucleotides, at least about 500 ribonucleotides, at least about 600
ribonucleotides, at least about 700
ribonucleotides, at least about 800 ribonucleotides, at least about 900
ribonucleotides, or at least about
100 ribonucleotides.
Modifications
A polyribonucleotide (e.g., circular polyribonucleotide) as described herein
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, polyA
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
polyribonucleotide 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 polyribonucleotide may include any useful modification, such as to the
sugar, the nucleobase,
or the internucleoside linkage (e.g., to a linking phosphate Ito a
phosphodiester linkage / to the
phosphodiester backbone). One or more atoms of a pyrirnidine 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 polyribonucleotide includes at least one
N(6)methyladenosine (m6A)
modification to increase translation efficiency. In some embodiments, the m6A
modification can reduce
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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.,
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 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_
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The modified nucleotides, which may be incorporated into the
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.
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-(1-
thiophosphate)-adenosine, 5'-0-(1-thiophosphate)-cytidine (a- thio-cytidine),
5'-0-(1-thiophosphate)-
guanosine, 5'-0-(1-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,1-(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-1-(tetrahydrofuran-2- yl)pyrimidine-2,4(IH,3H)-dione), troxacitabine,
tezacitabine, 2'- deoxy-2'-
methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include
fludarabine phosphate,
N4-behenoy1-1-beta-D-arabinofuranosylcytosine, N4-octadecy1-1-beta-D-
arabinofuranosylcytosine, N4-
palmitoy1-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-
4055 (cytarabine 5'-
elaidic acid ester).
A polyribonucleotide may or may not be uniformly modified along the entire
length of the
molecule. For example, one or more or all types of nucleotides (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
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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 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 intern
ucleoside 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`)/0 to 80%, from 1% to 90%, from 1% to 95%,
from 10% to 20%, from
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%).
Methods of Circularization
The disclosure provides methods for producing circular polyribonucleotides
encoding an
antifusogenic polypeptide (e.g., a polypeptide of Table 1), including, e.g.,
recombinant technology or
chemical synthesis. For example, 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). 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 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.
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
circular polyribonucleotide may be
in a mixture with linear polyribonucleotides. In some embodiments, the linear
polyribonucleotides have
the same nucleic acid sequence as the circular polyribonucleotides.
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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
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'-/3'-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 SplintRO ligase, can be
used for splint ligation, RNA
ligase II, T4 RNA ligase, or T4 DNA ligase. 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
concatemerized 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
concatemerized 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.
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In some embodiments, the linear polyribonucleotide for circularization is
synthesized using IVT
and an RNA polymerase, where the nucleotide mixture used for IVT may contain
an excess of guanosine
monophosphate relative to guanosine triphosphate to preferentially produce RNA
with a 5'
monophosphate; the purified IVT product may be circularized using a splint
DNA.
In some embodiments, a linear polyribonucleotide for circularization is
cyclized or
concatemerized 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
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
concatemerized. 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
concatemerized 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 invention 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). In some
embodiments, the 5'
end of at least a portion of the linear polyribonucleotides includes a
monophosphate moiety. In some
embodiments, the population of polyribonucleotides including circular and
linear polyribonucleotides is
contacted with RppH prior to digesting at least a portion of the linear
polyribonucleotides with a 5'
exonuclease and/or a 3' exonuclease. 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,
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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%.
In some embodiments, the 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 back splice
events such as sequences in
the 200 bp preceding (upstream of) or following (downstream from) a back
splice site with flanking exons.
In some embodiments, the linear 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 Muscle blind and Quaking (QKI) splicing factors).
In some embodiments, the linear polyribonucleotide may include canonical
splice sites that flank
head-to-tail junctions of the circular polyribonucleotide.
In some embodiments, the linear 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, the linear polyribonucleotide may include a multimeric
repeating RNA
sequence that harbors a HPR element. The HPR includes a 2',3'-cyclic phosphate
and 5'-OH termini.
The HPR element self-processes the 5'- and 3'-ends of the linear linear
polyribonucleotide, thereby
ligating the ends together.
In some embodiments, the linear polyribonucleotide may include a sequence that
mediates self-ligation.
In one embodiment, the linear polyribonucleotide may include a HDV sequence,
e.g., HDV replication
domain conserved sequence,
GGCUCAUCUCGACAAGAGGCGGCAG UCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUG
CUGGACUCGCCGCCCAAGUUCGAGCAUGAGCC (Beeharry et al 2004) (SEQ ID NO: 359) or
GGCUAGAGGCGGCAGUCCUCAGUACUCUUACUCUUUUCUGUAAAGAGGAGACUGCUGGACUCGC
CGCCCGAGCC (SEQ ID NO: 360), to self-ligate. In one embodiment, the linear
polyribonucleotide may
include loop E sequence (e.g., in PSTVd) to self-ligate. In another
embodiment, the linear
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. Nonlimiting examples of
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group I intron self-splicing sequences may include self-splicing permuted
intron-exon sequences derived
from 14 bacteriophage gene td, and the intervening sequence (IVS) rRNA of
Tetrahymena.
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 linear polyribonucleotide. In some embodiments, the linear
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 linear polyribonucleotide includes
at least one repetitive
nucleic acid sequence that hybridizes to a complementary repetitive nucleic
acid sequence in another
segment of the linear polyribonucleotide, with the hybridized segment forming
an internal double strand.
In some embodiments, the linear 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 linear polyribonucleotide, with the
hybridized segment forming an
internal double strand. In some embodiments, the linear polyribonucleotide
includes 2 repetitive nucleic
acid sequences that hybridize to a complementary repetitive nucleic acid
sequence in another segment of
the linear 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 linear 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 polyribonucleotide or complement, a
complementary strand of the
circular polyribonucleotide, or the circular polyribonucleotide.
Circularization of the linear 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.
In some embodiments, linear polyribonucleotides 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
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circular polyribonucleotide. In some embodiments, the linear
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 linear polyribonucleotide includes
at least one repetitive
nucleic acid sequence that hybridizes to a complementary repetitive nucleic
acid sequence in another
segment of the linear 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 linear 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. 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.
Methods of making the 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); Muller and Appel, from
RNA Biol, 2017, 14(8):1018-1027; and Egli & Herdewijn, Chemistry and Biology
of Artificial Nucleic Acids,
(First Edition), Wiley-VCH (2012). Other methods of making circular
polyribonucleotides are described,
for example, in International Publication No. W02022/247943, US Patent No.
US11000547, International
Publication No. 2018/191722, International Publication No. W02019/236673,
International Publication
No. W02020/023595, International Publication No. W02022/204460, International
Publication No.
W02022/204464, and International Publication No. W02022/204466.
Various methods of synthesizing circular polyribonucleotides are also
described elsewhere (see,
e.g., US Patent No. US6210931, US Patent No. US5773244, US Patent No.
US5766903, US Patent No.
US5712128, US Patent No. US5426180, US Publication No. US20100137407,
International Publication
No. W01992001813, International Publication No. W02010084371, and Petkovic et
al., Nucleic Acids
Res. 43:2454-65 (2015); the contents of each of which are herein incorporated
by reference in their
entirety).
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.
Methods of Production
Methods of production in a cell-free system
The disclosure also provides methods of producing a circular RNA. For example,
a
deoxyribonucleotide template may be transcribed in a cell-free system (e.g.,
by in vitro transcription) to a
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produce a linear RNA. The linear polyribonucleotide produces a splicing-
compatible polyribonucleotide,
which may be self-spliced to produce a circular polyribonucleotide.
In some embodiments, the disclosure provides a method of producing a circular
polyribonucleotide (e.g., in a cell-free system) by providing a linear
polyribonucleotide; and self-splicing
linear polyribonucleotide under conditions suitable for splicing of the 3 and
5' splice sites of the linear
polyribonucleotide; thereby producing a circular polyribonucleotide.
In some embodiments, the disclosure provides a method of producing a circular
polyribonucleotide by providing a deoxyribonucleotide encoding the linear
polyribonucleotide; transcribing
the deoxyribonucleotide in a cell-free system to produce the linear
polyribonucleotide; optionally purifying
the splicing-compatible linear polyribonucleotide; and self-splicing the
linear polyribonucleotide under
conditions suitable for splicing of the 3' and 5' splice sites of the linear
polyribonucleotide, thereby
producing a circular polyribonucleotide.
In some embodiments, the disclosure provides a method of producing a circular
polyribonucleotide by providing a deoxyribonucleotide encoding a linear
polyribonucleotide; transcribing
the deoxyribonucleotide in a cell-free system to produce the linear
polyribonucleotide, wherein the
transcribing occurs in a solution under conditions suitable for splicing of
the 3' and 5' splice sites of the
linear polyribonucleotide, thereby producing a circular polyribonucleotide. In
some embodiments, the
linear polyribonucleotide comprises a 5' split-intron and a 3' split-intron
(e.g., a self-splicing construct for
producing a circular polyribonucleotide). In some embodiments, the linear
polyribonucleotide comprises a
5' annealing region and a 3' annealing region.
Suitable conditions for in vitro transcriptions and or self-splicing may
include any conditions (e.g.,
a solution or a buffer, such as an aqueous buffer or solution) that mimic
physiological conditions in one or
more respects. In some embodiments, suitable conditions include between 0.1-
100mM Mg2+ ions or a
salt thereof (e.g., 1-100mM, 1-50mM, 1-20mM, 5- 50mM, 5-20 mM, or 5-15mM). In
some embodiments,
suitable conditions include between 1-1000mM K+ ions or a salt thereof such as
KCI (e.g., 1-1000mM, 1-
500mM, 1-200mM, 50- 500mM, 100-500mM, or 100-300mM) In some embodiments,
suitable conditions
include between 1-1000mM Cl- ions or a salt thereof such as KCI (e.g., 1-
1000mM, 1-500mM, 1-200mM,
50- 500mM, 100-500mM, or 100-300mM). In some embodiments, suitable conditions
include between
0.1-100mM Mn2+ ions or a salt thereof such as MnCl2 (e.g., 0.1-100mM, 0.1-
50mM, 0.1-20mM, 0.1-
10mM, 0.1-5mM, 0.1-2mM, 0.5- 50mM, 0.5-20 mM, 0.5-15mM, 0.5-5mM, 0.5-2mM, or
0.1-10mM). In
some embodiments, suitable conditions include dithiothreitol (DTT) (e.g., 1-
1000 pM, 1-500 pM, 1-200pM,
50- 500pM, 100-500pM, 100-300pM, 0.1-100mM, 0.1-50mM, 0.1-20mM, 0.1-10mM, 0.1-
5mM, 0.1-2mM,
0.5- 50mM, 0.5-20 mM, 0.5-15mM, 0.5-5mM, 0.5-2mM, or 0.1-10mM). In some
embodiments, suitable
conditions include between 0.1mM and 100mM ribonucleoside triphosphate (NTP)
(e.g., 0.1-100 mM,
0.1-50mM, 0.1-10mM, 1- 100mM, 1-50mM, or 1-10mM). In some embodiments,
suitable conditions
include a pH of 4 to 10 (e.g., pH of 5 to 9, pH of 6 to 9, or pH of 6.5 to
8.5). In some embodiments,
suitable conditions include a temperature of 4 C to 50 C (e.g., 10 C to 40 C,
15 C to 40 C, 20 C to
C, or 30 C to 40 C),
In some embodiments the linear polyribonucleotide is produced from a
deoxyribonucleic acid,
40 e.g., a deoxyribonucleic acid described herein, such as a DNA vector, a
linearized DNA vector, or a
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cDNA. In some embodiments, the linear polyribonucleotide is transcribed from
the deoxyribonucleic acid
by transcription in a cell-free system (e.g., in vitro transcription).
Methods of production in a cell
The disclosure also provides methods of producing a circular RNA in a cell,
e.g., a prokaryotic
cell or a eukaryotic cell. In some embodiments, an exogenous
polyribonucleotide is provided to a cell
(e.g., a linear polyribonucleotide described herein or a DNA molecule encoding
for the transcription of a
linear polyribonucleotide described here). The linear polyribonucleotides may
be transcribed in the cell
from an exogenous DNA molecule provided to the cell. The linear
polyribonucleotide may be transcribed
in the cell from an exogenous recombinant DNA molecule transiently provided to
the cell. In some
embodiments, the exogenous DNA molecule does not integrate into the cell's
genome. In some
embodiments, the linear polyribonucleotide is transcribed in the cell from a
recombinant DNA molecule
that is incorporated into the cell's genome.
In some embodiments, the cell is a prokaryotic cell. In some embodiments, the
prokaryotic cell
including the polyribonucleotides described herein may be a bacterial cell or
an archaeal cell. For
example, the prokaryotic cell including the polyribonucleotides described
herein may be E coli, halophilic
archaea (e.g., Haloferax volcaniii), Sphingomonas, cyanobacteria (e.g.,
Synechococcus elongatus,
Spirulina (Arthrospira) spp., and Synechocystis spp.), Streptomyces,
actinonnycetes (e.g., Nonomuraea,
Kitasatospora, or Thermobifida), Bacillus spp. (e.g., Bacillus subtilis,
Bacillus anthracis, Bacillus cereus),
betaproteobacteria (e.g., Burkholderia), alphaproteobacterial (e.g.,
Agrobacterium), Pseudomonas (e.g.,
Pseudomonas putida), and enterobacteria. The prokaryotic cells may be grown in
a culture medium. The
prokaryotic cells may be contained in a bioreactor.
In some embodiments, the cell is a eukaryotic cell. In some embodiments, the
eukaryotic cell
including the polyribonucleotides described herein is a unicellular eukaryotic
cell. In some embodiments,
the unicellular eukaryotic is a unicellular fungal cell such as a yeast cell
(e.g., Saccharomyces cerevisiae
and other Saccharomyces spp., Brettanomyces spp., Schizosaccharomyces spp.,
Torulaspora spp, and
Pichia sop.). In some embodiments, the unicellular eukaryotic cell is a
unicellular animal cell. A
unicellular animal cell may be a cell isolated from a multicellular animal and
grown in culture, or the
daughter cells thereof. In some embodiments, the unicellular animal cell may
be dedifferentiated. In
some embodiments, the unicellular eukaryotic cell is a unicellular plant cell.
A unicellular plant cell may
be a cell isolated from a multicellular plant and grown in culture, or the
daughter cells thereof. In some
embodiments, the unicellular plant cell may be dedifferentiated. In some
embodiments, the unicellular
plant cell is from a plant callus. In embodiments, the unicellular cell is a
plant cell protoplast. In some
embodiments, the unicellular eukaryotic cell is a unicellular eukaryotic algal
cell, such as a unicellular
green alga, a diatom, a euglenid, or a dinoflagellate. Non-limiting examples
of unicellular eukaryotic
algae of interest include Dunaliella sauna, Chlorella vulgaris, Chlorella
zofingiensis, Haematococcus
pluvialis, Neochloris oleoabundans and other Neochloris spp., Protosiphon
botryoides, Botryococcus
braunii, Cryptococcus spp., Chlamydomonas reinhardtii and other Chlamydomonas
spp. In some
embodiments, the unicellular eukaryotic cell is a protist cell. In some
embodiments, the unicellular
eukaryotic cell is a protozoan cell.
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In some embodiments, the eukaryotic cell is a cell of a multicellular
eukaryote. For example, the
multicellular eukaryote may be selected from the group consisting of a
vertebrate animal, an invertebrate
animal, a multicellular fungus, a multicellular alga, and a multicellular
plant. In some embodiments, the
eukaryotic organism is a human. In some embodiments, the eukaryotic organism
is a non-human
vertebrate animal. In some embodiments, the eukaryotic organism is an
invertebrate animal. In some
embodiments, the eukaryotic organism is a multicellular fungus. In some
embodiments, the eukaryotic
organism is a multicellular plant. In embodiments, the eukaryotic cell is a
cell of a human or a cell of a
non-human mammal such as a non-human primate (e.g., monkeys, apes), ungulate
(e.g., bovids
including cattle, buffalo, bison, sheep, goat, and musk ox; pig; camelids
including camel, llama, and
alpaca; deer, antelope; and equids including horse and donkey), carnivore
(e.g., dog, cat), rodent (e.g.,
rat, mouse, guinea pig, hamster, squirrel), or lagomorph (e.g., rabbit, hare).
In embodiments, the
eukaryotic cell is a cell of a bird, such as a member of the avian taxa
Galliformes (e.g., chickens, turkeys,
pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g.,
ostriches, emus),
Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). In
embodiments, the eukaryotic
cell is a cell of an arthropod (e.g., insects, arachnids, crustaceans), a
nematode, an annelid, a helminth,
or a mollusc. In embodiments, the eukaryotic cell is a cell of a multicellular
plant, such as an angiosperm
plant (which can be a dicot or a monocot) or a gymnosperm plant (e.g., a
conifer, a cycad, a gnetophyte,
a Ginkgo), a fern, horsetail, clubmoss, or a bryophyte. In embodiments, the
eukaryotic cell is a cell of a
eukaryotic multicellular alga.
The eukaryotic cells may be grown in a culture medium. The eukaryotic cells
may be contained
in a bioreactor.
Methods of purification
One or more purification steps may be included in the methods described
herein. For example,
in some embodiments, the linear polyribonucleotide is substantively enriched
or pure (e.g., purified) prior
to self-splicing the linear polyribonucleotide. In other embodiments, the
linear polyribonucleotide is not
purified prior to self-splicing the linear polyribonucleotide. In some
embodiments, the resulting circular
RNA is purified.
Purification may include separating or enriching the desired reaction product
from one or more
undesired components, such as any unreacted stating material, byproducts,
enzymes, or other reaction
components. For example, purification of linear polyribonucleotide following
transcription in a cell-free
system (e.g., in vitro transcription) may include separation or enrichment
from the DNA template prior to
self-splicing the linear polyribonucleotide. Purification of the circular RNA
product following splicing may
be used to separate or enrich the circular RNA from its corresponding linear
RNA. Methods of purification
of RNA are known to those of skill in the art and include enzymatic
purification or by chromatography.
In some embodiments, the methods of purification result in a circular
polyribonucleotide that has
less than 50% (e.g., less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%)
linear polyribonucleotides.
Bioreactors
In some embodiments, any method of producing a circular polyribonucleotide
described herein
may be performed in a bioreactor. A bioreactor refers to any vessel in which a
chemical or biological
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process is carried out which involves organisms or biochemically active
substances derived from such
organisms. Bioreactors may be compatible with the cell-free methods for
production of circular RNA
described herein. A vessel for a bioreactor may include a culture flask, a
dish, or a bag that may be
single use (disposable), autoclavable, or sterilizable. A bioreactor may be
made of glass, or it may be
polymer-based, or it may be made of other materials.
Examples of bioreactors include, without limitation, stirred tank (e.g., well
mixed) bioreactors and
tubular (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred
tanks, spin filter stirred tanks,
vibromixers, fluidized bed reactors, and membrane bioreactors. The mode of
operating the bioreactor
may be a batch or continuous processes. A bioreactor is continuous when the
reagent and product
streams are continuously being fed and withdrawn from the system. A batch
bioreactor may have a
continuous recirculating flow, but no continuous feeding of reagents or
product harvest.
Some methods of the present disclosure are directed to large-scale production
of circular
polyribonucleotides. For large-scale production methods, the method may be
performed in a volume of 1
liter (L) to 50 L, or more (e.g., 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40
L, 45 L, 50 L, or more). In some
embodiments, the method may be performed in a volume of 5 L to 10 L, 5 L to 15
L, 5 L to 20 L, 5 L to 25
L, 5 L to 30 L, 5 L to 35 L, 5 L to 40 L, 5 L to 45 L, 10 L to 15 L, 10 L to
20 L, 10 L to 25 L, 20 L to 30 L, 10
L to 35 L, 10 L to 40 L, 10 L to 45 L, 10 L to 50 L, 15 L to 20 L, 15 L to 25
L, 15 L to 30 L, 15 L to 35 L, 15
L to 40 L, 15 L to 45 L, or 15 to 50 L.
In some embodiments, a bioreactor may produce at least lg of circular RNA. In
some
embodiments, a bioreactor may produce 1-200g of circular RNA (e.g., 1-10g, 1-
20g, 1-50g, 10-50g, 10-
100g, 50-100g, of 50-200g of circular RNA). In some embodiments, the amount
produced is measured
per liter (e.g., 1-200g per liter), per batch or reaction (e.g., 1-200g per
batch or reaction), or per unit time
(e.g., 1-200g per hour or per day).
In some embodiments, more than one bioreactor may be utilized in series to
increase the
production capacity (e.g., one, two, three, four, five, six, seven, eight, or
nine bioreactors may be used in
series).
Methods of Use
In some embodiments, a circular polyribonucleotide encoding an antifusogenic
polypeptide (e.g.,
a polypeptide of Table 1) is used for the treatment or prevention of a viral
infection (e.g., HIV, SARS-CoV-
2, HCV, influenza, or RSV).
In some embodiments, a circular polynucleotide encoding an antifusogenic
polypeptide (e.g., a
polypeptide of Table 1) is used for reducing viral entry.
In some embodiments, a circular polynucleotide encoding an antifusogenic
polypeptide (e.g., a
polypeptide of Table 1) may be administered to a subject to reduce the risk of
a viral infection (e.g., HIV,
SARS-CoV-2, HCV, influenza, or RSV).
For example, a circular polyribonucleotide as described herein may be
administered to a subject
(e.g., in a pharmaceutical composition). In some embodiments, the subject is a
vertebrate animal (e.g.,
mammal, bird, fish, reptile, or amphibian). In some embodiments, the subject
is a human. In some
embodiments, the subject is a non-human mammal. In embodiments, the subject is
a non-human
mammal is such as a non-human primate (e.g., monkeys, apes), ungulate (e.g.,
cattle, buffalo, sheep,
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goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog,
cat), rodent (e.g., rat,
mouse), or lagomorph (e.g., rabbit). In embodiments, the subject is a bird,
such as a member of the
avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail),
Anseriformes (e.g., ducks, geese),
Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves),
or Psittaciformes (e.g.,
parrots). In embodiments, the subject is an invertebrate such as an arthropod
(e.g., insects, arachnids,
crustaceans), a nematode, an annelid, a helminth, or a mollusk.
In some embodiments, the disclosure provides a method of modifying a subject
by providing to
the subject a composition or formulation described herein. In some
embodiments, the composition or
formulation is or includes a nucleic acid molecule (e.g., a DNA molecule or an
RNA molecule described
herein), and the polynucleotide is provided to a eukaryotic subject. In some
embodiments, the
composition or formulation is or includes or a eukaryotic or prokaryotic cell
including a nucleic acid
described herein.
In some embodiments, the disclosure provides a method of treating a viral
infection in a subject in
need thereof by providing to the subject a composition or formulation
described herein. In some
embodiments, the composition or formulation is or includes a nucleic acid
molecule (e.g., a DNA molecule
or an RNA molecule described herein), and the polynucleotide is provided to a
eukaryotic subject. In
some embodiments, the composition or formulation is or includes a eukaryotic
or prokaryotic cell
including a nucleic acid described herein. In some embodiments, the
polyribonucleotide is provided in an
amount and for a duration sufficient to treat a viral infection in a subject,
e.g., in need thereof.
In some embodiments, the method may be used to treat or prevent HIV. For
example, in some
embodiments, the circular polyribonucleotide encodes an antifusogenic
polypeptide that targets HIV, and
the composition may be used to treat or prevent HIV.
In some embodiments, the method may be used to treat or prevent SARS-CoV-2.
For example,
in some embodiments, the circular polyribonucleotide encodes an antifusogenic
polypeptide that targets
SARS-CoV-2, and the composition may be used to treat or prevent SARS-CoV-2.
In some embodiments, the method may be used to treat or prevent HCV. For
example, in some
embodiments, the circular polyribonucleotide encodes an antifusogenic
polypeptide that targets HCV, and
the composition may be used to treat or prevent HCV.
In some embodiments, the method may be used to treat or prevent RSV. For
example, in some
embodiments, the circular polyribonucleotide encodes an antifusogenic
polypeptide that targets RSV, and
the composition may be used to treat or prevent RSV.
Methods of Dosing
A method of dosing to produce a level of circular polyribonucleotide encoding
an antifusogenic
polypeptide (e.g., a polypeptide of Table 1) or express a level of an
antifusogenic polypeptide (e.g., a
polypeptide of Table 1) in a cell after providing the cell with at least two
doses or compositions of circular
polyribonucleotide is disclosed herein. A method of dosing to produce a level
of circular
polyribonucleotide or express a level of an antifusogenic polypeptide (e.g., a
polypeptide of Table 1, e.g.,
a polypeptide of any one of Tables 2-4) in a subject (e.g., a mammal, e.g., a
human) after providing (e.g.,
administering to) the subject with at least two doses or compositions of
circular polyribonucleotide is
disclosed herein. The composition includes a circular polyribonucleotide
encoding an antifusogenic
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polypeptide as described herein. A method of dosing can include administering
two or more doses of a
composition of circular polyribonucleotides, e.g., over short time period or
over an extended period. In
some embodiments, the composition containing a circular polyribonucleotide
further includes a
pharmaceutically acceptable carrier or excipient. The circular
polyribonucleotide encodes an
antifusogenic polypeptide, which can be expressed in a cell, e.g., following
administration.
The methods described herein may include administering a first dose of the
pharmaceutical
composition in an amount sufficient to produce a serum concentration of at
least 500 ng/mL (e.g., at least
600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL, 1,100 ng/mL, 1,200
ng/mL, 1,300 ng/mL,
1,400 ng/mL, 1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800 ng/mL, 1,900 ng/mL,
2,000 ng/mL, 2,100
ng/mL, 2,200 ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL, 2,600 ng/mL, 2,700
ng/mL, 2,800 ng/mL,
2,900 ng/mL, 3,000 ng/mL, or more) of an antifusogenic polypeptide in the
subject.
In some embodiments, the method may further include administering a second
dose of the
pharmaceutical composition. The method may further include administering a
third, fourth, fifth, sixth,
seventh, eighth, ninth, tenth, or more doses of the pharmaceutical
composition. In some embodiments, a
subsequent dose helps maintain a serum concentration of at least 500 ng/mL
(e.g., at least 600 ng/mL,
700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL, 1,100 ng/mL, 1,200 ng/mL, 1,300
ng/mL, 1,400 ng/mL,
1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800 ng/mL, 1,900 ng/mL, 2,000 ng/mL,
2,100 ng/mL, 2,200
ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL, 2,600 ng/mL, 2,700 ng/mL, 2,800
ng/mL, 2,900 ng/mL,
3,000 ng/mL, or more) of an antifusogenic polypeptide in the subject. In some
embodiments, a
subsequent dose is administered before the serum concentration drops below 500
ng/mL of an
antifusogenic polypeptide in the subject.
In some embodiments, multiple doses are provided to produce a level of the
composition or
express a level of the antifusogenic polypeptide in a cell, tissue or subject.
In some embodiments,
multiple doses are provided to produce or maintain a level of the composition,
or to produce or maintain a
level of the antifusogenic polypeptide, in a cell, tissue or subject for a
period of time, for instance, for at
least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120,150 days, or at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 12, 15, 18,
21, or 24 months, or at least 1, 2, 3, 4, or 5 years.
In some embodiments, the second dose is administered at least one hour (e.g.,
at least two
hours, three hours, four hours, five hours, six hours, seven hours, eight
hours, nine hours, 10 hours, 11
hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours,
19 hours, 20 hours, 21
hours, 22 hours, 23 hours, one day, two days, three days, four days, five
days, six days, one week, two
weeks, three weeks, one month, two months, three months, four months, five
months, six months, seven
months, eight months, nine months, ten months, eleven months, one year, or
longer) after the first dose
of the pharmaceutical composition.
In some embodiments, the second dose is administered from 1 hour to 1 year
(e.g., from 1 hour
to 1 day, e.g., one hour, two hours, three hours, four hours, five hours, six
hours, seven hours, eight
hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours,
16 hours, 17 hours, 18
hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or one day, e.g.,
from one day to one week,
e.g., two days, three days, four days, five days, six days, or one week, e.g.,
from one week to one month,
e.g., two weeks, three weeks, or one month, e.g., from one month to one year,
e.g., one month, two
months, three months, four months, five months, six months, seven months,
eight months, nine months,
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ten months, eleven months, or one year) after the first dose of the
pharmaceutical composition. In some
embodiments, the second dose is administered from 1 days to 180 days (e.g.,
from 1 day to 90 days,
from 1 day to 45 days, from one day to 30 days, from 1 day to 14 days, from 1
day to 7 days, from 2 days
to 45 days, from 2 days to 30 days, from 2 days to 14 days, from 2 days to 7
days, from 3 days to 90
days, from 3 days to 45 days, from 3 days to 30 days, from 3 days to 14 days,
from 3 days to 7 days,
from 4 days to 90 days, from 4 days to 45 days, from 4 days to 30 days, from 4
days to 14 days, from 4
days to 7 days, from 5 days to 90 days, from 5 days to 45 days, from 5 days to
30 days, from 5 days to 14
days, from 5 days to 7 days, from 6 days to 90 days, from 6 days to 45 days,
from 6 days to 30 days,
from 6 days to 14 days, from 6 days to 7 days, from 7 days to 90 days, from 7
days to 45 days, from 7
days to 30 days, from 7 days to 14 days, from 14 days to 90 days, from 14 days
to 45 days, from 14 days
to 30 days, from 21 days to 90 days, from 21 days to 60 days, from 21 days to
45 days, from 21 days to
30 days, from 30 days to 90 days, from 30 days to 60 days, from 30 days to 45
days, from 45 to 180
days, from 45 to 120 days, form 45 to 100 days, from 45 to 90 days, from 45 to
60 days, from 60 to 180
days, from 60 to 120 days, from 60 to 100 days, from 60 to 90 days, from 90 to
100 days, from 90 to 120
days, or from 90 to 180 days) after the first dose of the pharmaceutical
composition.
In some embodiments, the third dose is administered at least one hour (e.g.,
at least two hours,
three hours, four hours, five hours, six hours, seven hours, eight hours, nine
hours, 10 hours, 11 hours,
12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19
hours, 20 hours, 21 hours, 22
hours, 23 hours, one day, two days, three days, four days, five days, six
days, one week, two weeks,
three weeks, one month, two months, three months, four months, five months,
six months, seven months,
eight months, nine months, ten months, eleven months, one year, or longer)
after the second dose of the
pharmaceutical composition.
In some embodiments, the third dose is administered from 1 hour to 1 year
(e.g., from 1 hour to 1
day, e.g., one hour, two hours, three hours, four hours, five hours, six
hours, seven hours, eight hours,
nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16
hours, 17 hours, 18 hours, 19
hours, 20 hours, 21 hours, 22 hours, 23 hours, or one day, e.g., from one day
to one week, e.g., two
days, three days, four days, five days, six days, or one week, e.g., from one
week to one month, e.g., two
weeks, three weeks, or one month, e.g., from one month to one year, e.g., one
month, two months, three
months, four months, five months, six months, seven months, eight months, nine
months, ten months,
eleven months, or one year) after the second dose of the pharmaceutical
composition. In some
embodiments, the third dose is administered from 1 days to 180 days (e.g.,
from 1 day to 90 days, from 1
day to 45 days, from one day to 30 days, from 1 day to 14 days, from 1 day to
7 days, from 2 days to 45
days, from 2 days to 30 days, from 2 days to 14 days, from 2 days to 7 days,
from 3 days to 90 days,
from 3 days to 45 days, from 3 days to 30 days, from 3 days to 14 days, from 3
days to 7 days, from 4
days to 90 days, from 4 days to 45 days, from 4 days to 30 days, from 4 days
to 14 days, from 4 days to 7
days, from 5 days to 90 days, from 5 days to 45 days, from 5 days to 30 days,
from 5 days to 14 days,
from 5 days to 7 days, from 6 days to 90 days, from 6 days to 45 days, from 6
days to 30 days, from 6
days to 14 days, from 6 days to 7 days, from 7 days to 90 days, from 7 days to
45 days, from 7 days to 30
days, from 7 days to 14 days, from 14 days to 90 days, from 14 days to 45
days, from 14 days to 30 days,
from 21 days to 90 days, from 21 days to 60 days, from 21 days to 45 days,
from 21 days to 30 days,
from 30 days to 90 days, from 30 days to 60 days, from 30 days to 45 days,
from 45 to 180 days, from 45
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to 120 days, form 45 to 100 days, from 45 to 90 days, from 45 to 60 days, from
60 to 180 days, from 60 to
120 days, from 60 to 100 days, from 60 to 90 days, from 90 to 100 days, from
90 to 120 days, or from 90
to 180 days) after the second dose of the pharmaceutical composition.
In some embodiments, the second dose is administered before a serum
concentration of an
antifusogenic polypeptide is less than about 500 ng/mL in serum of the
subject.
In some embodiments, the method maintains a serum concentration of at least
500 ng/mL (e.g.,
at least 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1,000 ng/mL, 1,100 ng/mL,
1,200 ng/mL, 1,300
ng/mL, 1,400 ng/mL, 1,500 ng/mL, 1,600 ng/mL, 1,700 ng/mL, 1,800 ng/mL, 1,900
ng/mL, 2,000 ng/mL,
2,100 ng/mL, 2,200 ng/mL, 2,300 ng/mL, 2,400 ng/mL, 2,500 ng/mL, 2,600 ng/mL,
2,700 ng/mL, 2,800
ng/mL, 2,900 ng/mL, 3,000 ng/mL, or more) of an antifusogenic polypeptide in
the subject, e.g., for at
least one hour (e.g., at least two hours, three hours, four hours, five hours,
six hours, seven hours, eight
hours, nine hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours,
16 hours, 17 hours, 18
hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, one day, two days,
three days, four days, five
days, six days, one week, two weeks, three weeks, one month, two months, three
months, four months,
five months, six months, seven months, eight months, nine months, ten months,
eleven months, one
year, or longer).
A method of administering multiple doses of a composition of a nucleic acid
molecule described
herein (e.g., a circular polyribonucleotide) includes providing two or more
compositions over a period of
time, to a cell, tissue or subject (e.g., a mammal). According to certain
embodiments, multiple doses of a
composition of a nucleic acid molecule described herein may be administered to
a subject over a defined
time course. The methods according to this aspect of the invention include
sequentially administering to
a subject multiple doses of a composition of a nucleic acid molecule described
herein (e.g., a circular
polyribonucleotide, a linear polyribonucleotide, a circular
polydeoxyribonucleotide, a linear
polydeoxyribonucleotide) (e.g., in a pharmaceutical or veterinary
composition). As used herein,
"sequentially administering" means that each dose of composition of a nucleic
acid molecule described
herein is administered to the subject at a different point in time, e.g., on
different days separated by a
predetermined interval (e.g., hours, days, weeks or months). In some
embodiments, the present invention
provides methods which include sequentially administering to the subject a
single initial dose of a
composition of a nucleic acid molecule described herein, followed by one or
more secondary doses of the
composition, and optionally followed by one or more tertiary doses of the
composition.
The terms "initial dose," "secondary doses," and "tertiary doses," refer to
the temporal sequence
of administration of a composition of a nucleic acid molecule described
herein. Thus, the "initial dose" is
the dose which is administered at the beginning of the treatment regimen; the
"secondary doses" are the
doses which are administered after the initial dose; and the "tertiary doses"
are the doses which are
administered after the secondary doses. The initial, secondary, and tertiary
doses may all contain the
same amount of a composition of a nucleic acid molecule described herein, and
in certain embodiments,
may differ from one another in terms of frequency of administration. In
certain embodiments, the amount
of a composition of a nucleic acid molecule described herein contained in the
initial, secondary and/or
tertiary doses varies from one another (e.g., adjusted up or down as
appropriate) during the course of
treatment In certain embodiments, one or more (e.g., 2, 3, 4, or 5) doses are
administered at the
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beginning of the treatment regimen as "loading doses" followed by subsequent
doses that are
administered on a less frequent basis (e.g., "maintenance doses").
In certain embodiments, each secondary and/or tertiary dose is administered
after the
immediately preceding dose. The phrase "the immediately preceding dose," as
used herein, means, in a
sequence of multiple administrations, the dose of the composition of a nucleic
acid molecule described
herein which is administered to a subject prior to the administration of the
very next dose in the sequence
with no intervening doses. In certain embodiments, each secondary and/or
tertiary dose is administered
every day, every 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the
immediately preceding dose.
In certain embodiments, each secondary and/or tertiary dose is administered
every 0.5 weeks, 1 week, 2
weeks, 3 weeks, or 4 weeks after the immediately preceding dose.
The methods according to this aspect of the invention may include
administering to a subject any
number of secondary and/or tertiary doses of a composition of a nucleic acid
molecule described herein.
For example, in certain embodiments, only a single secondary dose is
administered to the subject In
other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary
doses are administered to
the subject. Likewise, in certain embodiments, only a single tertiary dose is
administered to the subject. In
other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary
doses are administered to the
subject.
In certain embodiments, the frequency at which the secondary and/or tertiary
doses are
administered to a subject can vary over the course of the treatment regimen.
The frequency of
administration may also be adjusted during the course of treatment.
In some embodiments, the method includes providing (e.g., administering) at
least a first
composition and a second composition to the cells, tissue, or subject (e.g., a
mammal, e.g., a human). In
some embodiments, the method further includes providing (e.g., administering)
a third composition, fourth
composition, fifth composition, sixth composition, seventh composition, eighth
composition, ninth
composition, tenth composition, or more. In some embodiments, additional
compositions are provided for
the duration of the life of the cell. In some embodiments, additional
compositions are provided (e.g.,
administered) while the cell, tissue or subject obtains a benefit from the
composition.
In some embodiments, a first composition in a multiple dosing regimen includes
a first amount of
the nucleic acid molecule (e.g., circular polyribonucleotide) disclosed
herein. In some embodiments, a
second composition in a multiple dosing regimen includes a second amount of
the nucleic acid molecule
(e.g., circular polyribonucleotide) disclosed herein. In some embodiments, a
third composition, a fourth
composition, a fifth composition, a sixth composition, a seventh composition,
an eighth composition, a
ninth composition, a tenth composition, or more in a multiple dosing regimen
includes a third, fourth, fifth,
sixth, seventh, eighth, ninth, tenth or more amount of the nucleic acid
molecule (e.g., circular
polyribonucleotide) disclosed herein. In some embodiments, the second amount
of the nucleic acid
molecule (e.g., circular polyribonucleotide) is the same as the first amount
of the nucleic acid molecule
(e.g., circular polyribonucleotide). In some embodiments, the third amount of
the nucleic acid molecule
(e.g., circular polyribonucleotide) is the same as the first amount of the
nucleic acid molecule (e.g.,
circular polyribonucleotide). In some embodiments, the fourth, fifth, sixth,
seventh, eighth, ninth, tenth, or
more amount of the nucleic acid molecule (e.g., circular polyribonucleotide)
is the same as the first
amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In
some embodiments, the second
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amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is
less than the first amount of the
nucleic acid molecule (e.g., circular polyribonucleotide). In some
embodiments, the third amount of the
nucleic acid molecule (e.g., circular polyribonucleotide) is less than the
first amount of the nucleic acid
molecule (e.g., circular polyribonucleotide). In some embodiments, the fourth,
fifth, sixth, seventh, eighth,
ninth, tenth, or more amount of the nucleic acid molecule (e.g., circular
polyribonucleotide) is less than
the first amount of the nucleic acid molecule (e.g., circular
polyribonucleotide). In some embodiments, the
second amount of the nucleic acid molecule (e.g., circular polyribonucleotide)
is greater than the first
amount of the nucleic acid molecule (e.g., circular polyribonucleotide). In
some embodiments, the third
amount of the nucleic acid molecule (e.g., circular polyribonucleotide) is
greater than the first amount of
the nucleic acid molecule (e.g., circular polyribonucleotide). In some
embodiments, the fourth, fifth, sixth,
seventh, eighth, ninth, tenth, or more amount of the nucleic acid molecule
(e.g., circular
polyribonucleotide) is greater than the first amount of the nucleic acid
molecule (e.g., circular
polyribonucleotide). In some embodiments, an amount of the nucleic acid
molecule (e.g., circular
polyribonucleotide) of the second composition varies by no more than 1%, 5%,
10%, 15%, 20%, or 25%
of an amount of the nucleic acid molecule (e.g., circular polyribonucleotide)
of the first composition. In
some embodiments, an amount of the nucleic acid molecule (e.g., circular
polyribonucleotide) of the
second composition is no more than 1%, 5%, 10%, 15%, 20%, or 25% less than an
amount of the nucleic
acid molecule (e.g., circular polyribonucleotide) of the first composition. In
some embodiments, an
amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of a
second composition is from
0.1-fold to 1000-fold higher than an amount of the nucleic acid molecule
(e.g., circular polyribonucleotide)
of a first composition. In some embodiments, an amount of the nucleic acid
molecule (e.g., circular
polyribonucleotide) of a second composition is 0.1-fold, 1-fold, 5-fold, 10-
fold, 100-fold, or 1000-fold
higher than an amount of the nucleic acid molecule (e.g., circular
polyribonucleotide) of a first
composition. In some embodiments, an amount of the nucleic acid molecule
(e.g., circular
polyribonucleotide) of a subsequent composition (e.g., a composition
administered after a first
composition) is 0.1-fold, 1-fold, 5-fold, 10-fold, 100-fold, or 1000-fold
higher than an amount of the nucleic
acid molecule (e.g., circular polyribonucleotide) of a first composition. In
some embodiments, an amount
of the nucleic acid molecule (e.g., circular polyribonucleotide) of a second
composition is from 0.1-fold to
1000-fold lower than an amount of the nucleic acid molecule (e.g., circular
polyribonucleotide) of a first
composition. In some embodiments, an amount of the nucleic acid molecule
(e.g., circular
polyribonucleotide) of a second composition is 0.1-fold, 1-fold, 5-fold, 10-
fold, 100-fold, or 1000-fold lower
than an amount of the nucleic acid molecule (e.g., circular
polyribonucleotide) of a first composition. In
some embodiments, an amount of the nucleic acid molecule (e.g., circular
polyribonucleotide) of a
subsequent composition (e.g., a composition administered after a first
composition) is 0.1-fold, 1-fold, 5-
fold, 10-fold, 100-fold, or 1000-fold lower than an amount of the nucleic acid
molecule (e.g., circular
polyribonucleotide) of a first composition. In some embodiments, an amount of
the nucleic acid molecule
(e.g., circular polyribonucleotide) of a subsequent composition (e.g., after a
first composition of an amount
of nucleic acid molecule (e.g., circular polyribonucleotide)) is from 0.1-fold
to 1000-fold higher or lower
than an amount of the nucleic acid molecule (e.g., circular
polyribonucleotide) of a first composition. In
some embodiments, an amount of the nucleic acid molecule (e.g., circular
polyribonucleotide) of a
subsequent composition (e.g., after a first composition of an amount of
nucleic acid molecule (e.g.,
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circular polyribonucleotide)) is 0.1-fold, 1-fold, 5-fold, 10-fold, 100-fold,
or 1000-fold higher or lower than
an amount of the nucleic acid molecule (e.g., circular polyribonucleotide) of
a first composition. For
example, a first composition includes 1-fold nucleic acid molecule (e.g.,
circular polyribonucleotide), a
second composition includes 5-fold nucleic acid molecule (e.g., circular
polyribonucleotide) compared to
the first composition, and a third composition includes 0.2-fold nucleic acid
molecule (e.g., circular
polyribonucleotide) compared to the first composition. In some embodiments,
the second composition
includes at least 5-fold nucleic acid molecule (e.g., circular
polyribonucleotide) compared to an amount of
nucleic acid molecule (e.g., circular polyribonucleotide) of a first
composition.
In some embodiments, the first composition includes a higher amount of the
nucleic acid
molecule (e.g., circular polyribonucleotide) than the second composition. In
some embodiments, the first
composition includes a higher amount of the nucleic acid molecules (e.g.,
circular polyribonucleotides)
than the third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth
composition.
In some embodiments, the plurality (e.g., two or more) of compositions of a
nucleic acid molecule
(e.g., circular polyribonucleotide) encoding an antifusogenic polypeptide,
which are administered in a
multiple dosing regimen as described herein, are the same compositions. In
some embodiments, the
plurality (e.g., two or more) of compositions of a nucleic acid molecule
(e.g., circular polyribonucleotide)
encoding an antifusogenic polypeptide, which are administered in a multiple
dosing regimen as described
herein, are different compositions. In some embodiments, the same compositions
include the nucleic acid
molecules (e.g., circular polyribonucleotides) encoding the same antifusogenic
polypeptide. In some
embodiments, the different compositions include the nucleic acid molecules
(e.g., circular
polyribonucleotides) encoding different antifusogenic polypeptides, or a
combination thereof.
In some embodiments, in a multiple dosing regimen, the method of administering
the nucleic acid
molecule (e.g., circular polyribonucleotide) provided herein includes
administering to a subject in need
thereof the nucleic acid molecule for multiple times (multiple doses), e.g.,
at least 3, 4, 5, 6, 7, 8, 9, 10,
12, 15, 20, 30, 40, 50, 60, 100, 150, 200, or 500 times, with an interval of
from 1 day to 56 days, such as
about 49 days, 42 days, 35 days, 28 days, 21 days, 14 days, or 7 days. In some
embodiments, in a
multiple dosing regimen, the method provided herein includes administering to
a subject in need thereof
the nucleic acid molecule for at least 3 times, with an interval of about 7
days. In some embodiments, in
a subject that receives administration of multiple doses of the nucleic acid
molecule (e.g., at least 3, 4, 5,
6, 7, 8, or 9 doses) provided herein, a level of the antifusogenic polypeptide
(e.g., a plasma antifusogenic
polypeptide) is maintained at a level with variation of less than 50%, 40%,
30%, 20%, or 10% for a period
of longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, or
20 weeks after the last dose. In
some embodiments, in a subject that receives administration of multiple doses
of the nucleic acid
molecule (e.g., at least 3, 4, 5, 6, 7, 8, or 9 doses) provided herein, a
level of the antifusogenic
polypeptide (e.g., a plasma antifusogenic polypeptide level) is maintained at
a first level for a period of
longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, or
20 weeks after the second, third,
fourth, fifth, sixth, seventh, eight, or the last dose, wherein the first
level is higher than a level of the
antifusogenic polypeptide measured shortly after the first dose (e.g.,
measured about 12, 24, 36, or 48
hours after the first dose). In some embodiments, in a subject that receives
administration of multiple
doses of the nucleic acid molecule (e.g., at least 3 doses) provided herein
with an interval of about 7
days, a level of the antifusogenic polypeptide (e.g., a plasma antifusogenic
polypeptide level) is
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maintained at a first level for a period of longer than 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20
weeks after the second, third, fourth, fifth, sixth, seventh, eight, or the
last dose, wherein the first level is
higher than a level of the antifusogenic polypeptide measured shortly after
the first dose (e.g., measured
about 12, 24, 36, or 48 hours after the first dose).
Methods of Delivery
A circular polyribonucleotide encoding an antifusogenic polypeptide (e.g., a
polypeptide of Table
1) 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.
In some embodiments, circular 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 polyribonucleotide
or partial or complete
encapsulation of the circular polyribonucleotide.
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 and
the circular polyribonucleotide is not partially or completely encapsulated.
In some embodiments, a
circular 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 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 phytoglycogen octenyl succinate,
phytoglycogen beta-
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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)propy1]-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-dimethy1-1-propanaminiurn
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-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
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 (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), 24[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 formulation includes a cell-penetrating agent. In
some embodiments,
the formulation is a topical formulation and includes a cell-penetrating
agent. 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 limited
to, rnonohydric 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 includes ethanol. The cell-penetrating agents can include
any cell-penetrating agent in
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any amount or in any formulation as described in WO 2020/180751 or WO
2020/180752, which are
hereby incorporated by reference in their entirety.
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 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. 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. 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 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.).
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
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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, intraderrnally 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 of the present disclosure a circular polyribonucleotide
described herein
may be formulated in composition, e.g., a composition for delivery to a cell,
a plant, an invertebrate
animal, a non-human vertebrate animal, or a human subject, e.g., an
agricultural, veterinary, or
pharmaceutical composition. In some embodiments, the circular
polyribonucleotide is formulated in a
pharmaceutical composition. In some embodiments, a composition includes a
circular polyribonucleotide
and a diluent, a carrier, an adjuvant, or a combination thereof_ In a
particular embodiment, a composition
includes a circular polyribonucleotide described herein and a carrier or a
diluent free of any carrier. In
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some embodiments, a composition including a circular polyribonucleotide with a
diluent free of any carrier
is used for naked delivery of the circular polyribonucleotide to a subject.
Pharmaceutical compositions may optionally include one or more additional
active substances,
e.g., therapeutically and/or prophylactically active substances.
Pharmaceutical compositions may
optionally include an inactive substance that serves as a vehicle or medium
for the compositions
described herein (e.g., compositions including circular polyribonucleotides,
such as any one of the
inactive ingredients approved by the United States Food and Drug
Administration (FDA) and listed in the
Inactive Ingredient Database). Pharmaceutical compositions of the present
invention may be sterile
and/or pyrogen-free. General considerations in the formulation and/or
manufacture of pharmaceutical
agents may be found, for example, in Remington: The Science and Practice of
Pharmacy 21st ed.,
Lippincott Williams & Wilkins, 2005 (incorporated herein by reference). Non-
limiting examples of an
inactive substance 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.
Although the descriptions of pharmaceutical compositions provided herein are
principally directed
to pharmaceutical compositions which are suitable for administration to
humans, it will be understood by
the skilled artisan that such compositions are generally suitable for
administration to any other animal,
e.g., to non-human animals, e.g., non-human mammals. Modification of
pharmaceutical compositions
suitable for administration to humans to render the compositions suitable for
administration to various
animals is well understood, and the ordinarily skilled veterinary
pharmacologist can design and/or perform
such modification with merely ordinary, if any, experimentation. Subjects to
which administration of the
pharmaceutical compositions is contemplated include, but are not limited to,
humans and/or other
primates; mammals, including commercially relevant mammals such as cattle,
pigs, horses, sheep, cats,
dogs, mice, and/or rats; and/or birds, including commercially relevant birds
such as poultry, chickens,
ducks, geese, and/or turkeys.
Formulations of the pharmaceutical compositions described herein may be
prepared by any
method known or hereafter developed in the art of pharmacology. In general,
such preparatory methods
include the step of bringing the active ingredient into association with an
excipient and/or one or more
other accessory ingredients, and then, if necessary and/or desirable,
dividing, shaping and/or packaging
the product.
In some embodiments, the reference criterion for the amount of circular
polyribonucleotide
molecules present in the preparation is at least 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), 99.1% (w/w), 99.2% (w/w), 99.3% (w/w),
99.4% (w/w), 99.5%
(w/w), 99.6% (w/w), 99.7% (w/w), 99.8% (w/w), 99.9% (w/w), or 100%
(w/w)molecules of the total
ribonucleotide molecules in the pharmaceutical preparation.
In some embodiments, the reference criterion for the amount of linear
polyribonucleotide
molecules present in the preparation is the presence 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, 60 ng/ml,
70 ng/ml, 80 ng/ml, 90 ng/ml,
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100 ng/ml, 200 ng/ml, 300 ng/ml, 400 ng/ml, 500 ng/ml, 600 ng/ml, 114/ ml, 10
p.g/ml, 50 ug/ml, 100
pg/ml, 200 g/ml, 300 g/ml, 400 g/ml, 500 p.g/ml, 600 u.g/ml, 700 p.g/ml, 800
p.g/ml, 900 p.g/ml, 1 mg/ml,
1.5 mg/ml, or 2 mg/ml of linear polyribonucleotide molecules.
In some embodiments, the reference criterion for the amount of linear
polyribonucleotide
molecules present in the preparation is no more than 0.5% (w/w), 1% (w/w), 2%
(w/w), 5% (w/w), 10%
(w/w), 15% (w/w), 20% (w/w), 25% (w/w), 30% (w/w), 40% (w/w), 50% (w/w) linear
polyribonucleotide
molecules of the total ribonucleotide molecules in the pharmaceutical
preparation.
In some embodiments, the reference criterion for the amount of nicked
polyribonucleotide
molecules present in the preparation is no more than 0.5% (w/w), 1% (w/w), 2%
(w/w), 5% (w/w), 10%
(w/w), or 15% (w/w) nicked polyribonucleotide molecules of the total
ribonucleotide molecules in the
pharmaceutical preparation.
In some embodiments, the reference criterion for the amount of combined nicked
and linear
polyribonucleotide molecules present in the preparation is no more than 0.5%
(w/w), 1% (w/w), 2% (w/w),
5% (w/w), 10% (w/w), 15% (w/w), 20% (w/w), 25% (w/w), 30% (w/w), 40% (w/w),
50% (w/w) combined
nicked and linear polyribonucleotide molecules of the total ribonucleotide
molecules in the pharmaceutical
preparation. In some embodiments, a pharmaceutical preparation is an
intermediate pharmaceutical
preparation of a final circular polyribonucleotide drug product. In some
embodiments, a pharmaceutical
preparation is a drug substance or active pharmaceutical ingredient (API). In
some embodiments, a
pharmaceutical preparation is a drug product for administration to a subject.
In some embodiments, a preparation of circular polyribonucleotides is (before,
during or after the
reduction of linear RNA) further processed to substantially remove DNA,
protein contamination (e.g., cell
protein such as a host cell protein or protein process impurities), endotoxin,
mononucleotide molecules,
and/or a process-related impurity.
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
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).
Preservatives
A composition or pharmaceutical composition provided herein can include
material for a single
administration, or can include 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.
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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
includes 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 include 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 include those of the inorganic ions, such as, for
example, sodium, potassium,
calcium, magnesium ions, and the like. Such salts can include 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 include one or
more buffers,
such as a Trig 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.
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 include 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 (E0), 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 phosphatidylcho line (lecithin); nonylphenol
ethoxylates, such as the TergitolTm NP
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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, a composition of the disclosure includes a circular
polyribonucleotide and
a diluent In some embodiments, a 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
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
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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, arninoglycoside-polyamine,
dideoxy-diamino-b-cyclodextrin,
sperrnine, 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)imidazoliniurn chloride (DOTIM),
2,3-dioleyloxy-N-
[2(sperminecarboxamido)ethy1]-N,N-dimethy1-1-propanaminiurn trifluoroacetate
(DOSPA), 3B-[N¨ (N\N'-
Dimethylaminoethane)-carbannoyl]Cholesterol Hydrochloride (DC-Cholesterol
NCI),
diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N-
dirnethylammonium bromide (DDAB),
N-(1,2-dimyristyloxyprop-3-yI)-N,N-dimethyl-N- hydroxyethyl ammonium bromide
(DMRIE), N,N-dioleyl-
N,N-dimethylammonium chloride (DODAC), human serum albumin (HSA), low-density
lipoprotein ([DL),
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,
formamide, and 2-propanol. Examples of a buffer include 2-(N-
morpholino)ethanesulfonic acid (MES),
Bis-Tris, 2-[(2-arnino-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, a composition of the disclosure includes a circular
polyribonucleotide and
a carrier. In some embodiments, a composition of the disclosure includes a
linear polyribonucleotide and
a carrier.
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In certain embodiments, a composition includes a circular polyribonucleotide
as described herein
in a vesicle or other membrane-based carrier. In certain embodiments, a
composition includes a linear
polyribonucleotide as described herein in a vesicle or other membrane-based
carrier.
In other embodiments, a composition includes the circular polyribonucleotide
in or via a cell,
vesicle or other membrane-based carrier. In other embodiments, a composition
includes the linear
polyribonucleotide in or via a cell, vesicle or other membrane-based carrier.
In one embodiment, a
composition includes the circular polyribonucleotide in liposomes or other
similar vesicles. In one
embodiment, a 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, a composition of the disclosure includes a circular
polyribonucleotide
and lipid nanoparticles, for example lipid nanoparticles described herein. In
certain embodiments, a
composition of the disclosure includes a linear polyribonucleotide and lipid
nanoparticles. Lipid
nanoparticles are another example of a carrier that provides a biocompatible
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,
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and prevent leakage of water-soluble drugs. For a review, see, e.g., Li et al.
2017, Nanomaterials 7, 122;
doi:10.3390/nan07060122.
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)propyIN,N,N-
trimethylammonium chloride (DOTMA),1-[2-(oleoyloxy)ethy1]-2-oley1-3-(2-
hydroxyethyl)imidazolinium
chloride (DOTIM), 2,3-dioleyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-
1-propanaminium
trifluoroacetate (DOSPA), 3B[N-(N\N'-Dimethylaminoethane)-
carbamoyl]Cholesterol Hydrochloride (DC-
Cholesterol NCI), diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N-
dimethylammoniunn
bromide (DDAB), N-(1,2-dimyristyloxyprop-3-y1)-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;
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
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
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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., US7115583; 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, include 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 include one or more of the lipids in Table 4 of
W02019217941. Lipid
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 1-(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-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,
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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 includes 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 includes 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,
N
(i)
In some embodiments an LNP including Formula (i) is used to deliver a
polyribonucleotide (e.g., a
circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
(ii)
In some embodiments an LNP including Formula (ii) is used to deliver a
polyribonucleotide (e.g.,
a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
0
(iii)
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In some embodiments an LNP including Formula (iii) is used to deliver a
polyribonucleotide (e.g.,
a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
6
(iv)
N
t I
0
(v)
In some embodiments an LNP including 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 including Formula (vi) is used to deliver a
polyribonucleotide (e.g.,
a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
0
0
H 0 N
0 0
(vii)
0
0
HO N
===
0 0 (viii)
In some embodiments an LNP including Formula (viii) is used to deliver a
polyribonucleotide
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
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t.,
(ix)
In some embodiments an LNP including Formula (ix) is used to deliver a
polyribonucleotide (e.g.,
a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
9 R'
14
Y Zt 0
$0.
(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 C1-3 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 C2-12 alkylene, Y2 is
selected from
0
s
\ 0
(in either orientation), (in either orientation), (in either
orientation),
n is 0 to 3, R4 is C1-15 alkyl, Z1 is C1-6 alkylene or a direct bond,
0
v2is
\ 0
(in either orientation) or absent, provided that if Z1 is a direct bond, Z2 is
absent;
R5 is 05-9 alkyl or C6-10 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 C2 alkyls, X1
is 0, X2 is linear C3 alkylene,
X3 is C(=0), Y1 is linear Ce alkylene, (Y2 )n-R4 is
R4
;
, 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 including Formula (xii) is used to deliver a
polyribonucleotide (e.g.,
a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
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0
cr.."
0
(xi)
In some embodiments an LNP including Formula (xi) is used to deliver a
polyribonucleotide (e.g.,
a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
0
oti
OF=42
where R= (xii)
0
r-NN ¨"Ns_
HO I
H
-if) 21
C101:123
Oil
H
"10 23 (Xiii)
is
40"
(XiV)
In some embodiments an LNP includes a compound of Formula (xiii) and a
compound of Formula
(xiv).
O
5
OH H
N
HO
OH
(xv)
In some embodiments an LNP including Formula (xv) is used to deliver a
polyribonucleotide (e.g.,
a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells.
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PElgoo:Cor6
HOy)
CI3H27 (xvi)
In some embodiments an LNP including a formulation of Formula (xvi) is used to
deliver a
polyribonucleotide (e.g., a circular polyribonucleotide, a linear
polyribonucleotide) composition described
herein to cells.
,
(xvii)
s
=
?
a
x. amino szructur:'! where x=
(xviii)(a)
T Iõ =
\
es,
(xviii)(b)
z
0
t
N
0
it
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:
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HN
1 0
N N
(xx)(a)
0
ft
603 1-12N' 'NH2 + =
(XX)(b).
In somo ombodimonts an LNP including Formula (xxi) is usod to dolivor a
polyribonucicotido
(e.g., a circular polyribonucleotide, a linear polyribonucleotide) composition
described herein to cells. In
some embodiments the [NP of Formula (xxi) is an [NP described by W02021113777
(e.g., a lipid of
Formula (1) such as a lipid of Table 1 of W02021113777).
R1- L14 L3 - R3
_
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, alkenylcarbonylami no, 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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7
---t-4 e ,---N ,,---1:1
N= õ,, 0,.% "N.5
k's
1. ,... v...
,........, ...,.
.,,
Ns
.N- .i:i
?-. Nõ,,,,,,, .,=_
,..,,,,r,õ<õ,
Srsk...,, i vidtok.,
,-,v1/4,4,- , =-..,<Att, ,
= .
es-14:i ,4;......N ,..........N
t,. ...).õ.,.. .4 .%,,,..... .... ti ,,v ......
kq µk
,),, N N. ' N -- '''''' 't,r----
'(' = ' N
N ' ,
i I.,, .., ' 1 ii¨N
1
N,s, ' ,..1
N
,
A¨N ,
' -====":::..., , N
:t".. ==fr ==:, i\fµ". =''' AAA",
= N
N N.:-"=-=......-- ' (.1 Si k,-k. -1, k,,,,,..-:;;P===-=
N' ====
"....,
....1., / ,... . \ ....--
. an
µ..... !I
;:a.......14
=
, d
In some embodiments an LNP including 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 [NP of Formula (xxii) is an [NP described by W02021113777
(e.g., a lipid of
Formula (2) such as a lipid of Table 2 of W02021113777).
Ri
0-_------7 -
--
R2 y 0 R3
0 (xxii)
wherein
each n is independently an integer from 1-15;
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Ri and R2 are each independently selected from a group consisting of:
7
N.--
õ......-...N...,t.0
1
N',..."`eN.,-,'" Nk......", r=-=,'N....X s\,,,....,.0
9
sr.õ......,õ,X;
9 Q
'..,,,..."...,-,=,..,õ...-' N'x)3L-1"NNA'"'",")
, 7
9
0
k , N. ,---,----,..."--,-..-0
, ,
9
0
.
,------,---,-----,--N-0-A------NN,--wyoris,
X
o
o Y
8 0
, ,
_,----,---,--,---so 0 f ....0e
,
Tke f
X.--,---
1 08
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R3 is selected from a group consisting of:
'
N
4,....... N
r-----'-'\ r.--;--
z--\
N
\
r---\
õ\...0,_...õ..¨....õ,....õ,c,--.
Nc.- N--,.-----..õ.--- N---µ,
t ,
NI 11---.
,n0,. r.-----\N
''. N----'/' , and
,.
, - .
In some embodiments an LNP including 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 [NP of Formula (xxiii) is an [NP 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)-Ls.õ...,, N ..õ....so.õõ.õ.õX ¨ Ri
142 (xxiii)
wherein
X is selected from -0-, -S-, or -0C(0)-*, wherein * indicates the attachment
point to Ri;
Fli is selected from a group consisting of:
1
A.... ,.."N%>=,,,,,,,-" ;.4õ....---= ;$<=,-"A'N.,,,.."'µ''''---
-"'
:
:
,and
=
,,
1 09
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and R2 is selected from a group consisting of:
.,...z.:
if 's1 .4-14
r---=N `i:.'1) ---N -14 fr----
.N r---N
b"
..%k) -I
kr.N...,, \N.:-.= N ..,-
,,...,
I .1,,,, ),,, Li 1
..,
1",,,,,...-K Lwi
..-,.....^., kArl.n., :"Ad-s., = .."..k.-v ~",
kr-NL,. ( )1--,./
1" i-s.,µõ,
L (-1,
..N-... N
i
kl. ' x, N
N_
i .1 i.N.,
...--"--...
N.=\
34 ...,
t-,...õ,,,,=-='-'-.. -,t, 'IQ: -..
,
e
"or"' '-=--tsi- , 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 [NP that includes an
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
US2012/0027803 (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-
yl)ethyl)azanediy1)bis(dodecan-2-ol) (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-IH-
cyclopenta[a]phenanthren-3-y13-(1H-irnidazol-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 includes 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
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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 include a second cationic
lipid having a different
effective pKa (e.g., greater than the first effective pKa), than the first
cationic lipid. A lipid nanoparticle
may include 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 including
a cationic lipid. In some
embodiments, the nucleic acid may be encapsulated in an LNP, e.g., an LNP
including a cationic lipid. In
some embodiments, the lipid nanoparticle may include a targeting moiety, e.g.,
coated with a targeting
agent. In embodiments, the LNP formulation is biodegradable. In some
embodiments, a lipid
nanoparticle including 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; 1, 11 or III of US20160151284; 1, IA,
11, or IIA of
US20170210967; 1-c of US20150140070; A of US2013/0178541; 1 of US2013/0303587
or
US2013/0123338; 1 of US2015/0141678; II, Ill, IV, or V of US2015/0239926; 1 of
US2017/0119904; 1 or 11
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; 1 of US2008/042973; 1, II, III, or IV of
US2012/01287670; 1 or 11
of US2014/0200257; 1, II, or III of US2015/0203446; 1 or III of
US2015/0005363; 1, IA, IB, IC, ID, II, IIA, IIB,
IIC, IID, or III-XXIV of US2014/0308304; of US2013/0338210; 1, II, Ill, or IV
of W02009/132131; A of
US2012/01011478; 1 or XXXV of US2012/0027796; XIV or XVII of US2012/0058144;
of
US2013/0323269; 1 of US2011/0117125; 1, II, or III of US2011/0256175; 1, 11,
Ill, IV, V, VI, VII, VIII, IX, X,
XI, XII of US2012/0202871; 1, II, Ill, IV, V, VI, VII, VIII, X, XII, XIII,
XIV, XV, or XVI of US2011/0076335; 1
or Hof US2006/008378;lof US2013/0123338; 1 or X-A-Y-Z of US2015/0064242; XVI,
XVII, or XVIII of
US2013/0022649; 1, 11, or III of US2013/0116307; 1, II, or III of
US2013/0116307; 1 or 11 of
US2010/0062967; I-X of US2013/0189351; 1 of US2014/0039032; V of
US2018/0028664; 1 of
US2016/0317458; 1 of 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-
P4C2 of US9,708,628; 1 of W02020/106946; 1 of 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,3 IZ)-
heptatriaconta- 6,9,28,3 I-
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
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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
(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-phosphatidylethanolarnine
(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 (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin
(SM), dimyristoyl
phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG),
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.nan01ett.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 nnyristate,
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 US2018/0028664, 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 include, 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
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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 include any phospholipids.
In some aspects, the lipid nanoparticle can further include a component, such
as a sterol, to
provide membrane integrity. One exemplary sterol that can be used in the lipid
nanoparticle is cholesterol
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
include 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 include 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
as 1-(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 US5,885,613,
US6,287,591, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058,
US2011/0117125, US2010/0130588, US2016/0376224, US2017/0119904,
US2018/0028664, and
W02017/099823, 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 111,111-a-1, Ill-a-2,
111-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-disterylglycannide, PEG-cholesterol (1-[8'-(Cholest-
5-en-3[beta]-
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oxy)carboxamido-3',6'-dioxaoctanyl] carbamoyHomegal-methyl-poly(ethylene
glycol), PEG- DMB (3,4-
Ditetradecoxylbenzyl- [omega]-methyl-poly(ethylene glycol) ether), and 1,2-
dimyristoyl-sn-glycero-3-
phosphoethanolarnine-Nimethoxy(polyethylene glycol)-2000]. In some
embodiments, the PEG-lipid
includes PEG-DMG, 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-
[methoxy(polyethylene
glycol)-2000]. In some embodiments, the PEG-lipid includes a structure
selected from:
0
0
0
N
0
0
1.1
H
O
and
0
4
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
10 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.
15 In some embodiments, the PEG or the conjugated lipid can include 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 include 30-70% ionizable lipid by mole or by total weight of the
composition, 0-60% cholesterol by
20 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 includes 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
50-75% ionizable lipid by
25 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%
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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 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 including 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, 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
includes 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
includes ionizable lipid, cholesterol
and a PEG-ylated lipid in a molar ratio of 60:38.5: 1.5.
In some embodiments, the lipid particle includes 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 includes 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
including 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 include biodegradable, ionizable lipids. In some
embodiments,
the LNPs include (9Z,12Z)-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-
9,12-dienoate) or another ionizable lipid. See, e.g., lipids of W02019/067992,
WO/2017/173054,
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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
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 an 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
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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
embodiments, the encapsulation efficiency may be at least 90%. In some
embodiments, the
encapsulation efficiency may be at least 95%.
A LNP may optionally include 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-
gRNA 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 U88158601
and US8168775, both
incorporated by reference, which include formulations used in patisiran, sold
under the name
ONPATTRO.
In embodiments, a polyribonucleotide (e.g., a circular polyribonucleotide, a
linear
polyribonucleotide) encoding at least a portion (e.g., an antigenic portion)
of a protein or polypeptide
described herein is formulated in an LNP, wherein: (a) the LNPs comprise a
cationic lipid, a neutral lipid,
a cholesterol, and a PEG lipid, (b) the LNPs have a mean particle size of
between 80 nm and 160 nm,
and (c) the polyribonucleotide. In embodiments, the polyribonucelotide (e.g.,
circular polyribonucleotide,
linear polyribonucleotide) formulated in an LNP is a vaccine.
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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).
In some embodiments, a dose of a polyribonucleotide (e.g., a circular
polyribonucleotide, a linear
polyribonucleotide) antigenic composition described herein is between 30-200
mcg, e.g., 30 mcg, 50 mcg,
75 mcg, 100 mcg, 150 mcg, or 200 mcg.
Kits
In some aspects, the disclosure provides a kit. In some embodiments, the kit
includes (a) a
circular polyribonucleotide encoding an antifusogenic polypeptide (e.g., a
polypeptide of Table 1) or a
pharmaceutical composition described herein, and optionally (b) informational
material. The informational
material may be descriptive, instructional, marketing or other material that
relates to the methods
described herein and/or the use of the pharmaceutical composition or circular
polyribonucleotide for the
methods described herein_ The pharmaceutical composition or circular
polyribonucleotide may include
material for a single administration (e.g., single dosage form), or may
include material for multiple
administrations (e.g., a "multidose" kit).
The informational material of the kits is not limited in its form. In one
embodiment, the
informational material may include information about production of the
pharmaceutical composition, the
pharmaceutical drug substance, or the pharmaceutical drug product, molecular
weight of the
pharmaceutical composition, the pharmaceutical drug substance, or the
pharmaceutical drug product,
concentration, date of expiration, batch or production site information, and
so forth. In one embodiment,
the informational material relates to methods for administering a dosage form
of the pharmaceutical
composition. In one embodiment, the informational material relates to methods
for administering a
dosage form of the circular polyribonucleotide.
In addition to a dosage form of the pharmaceutical composition and circular
polyribonucleotide
described herein, the kit may include other ingredients, such as a solvent or
buffer, a stabilizer, a
preservative, a flavoring agent (e.g., a bitter antagonist or a sweetener), a
fragrance, a dye or coloring
agent, for example, to tint or color one or more components in the kit, or
other cosmetic ingredient, and/or
a second agent for treating a condition or disorder described herein.
Alternatively, the other ingredients
may be included in the kit, but in different compositions or containers than a
pharmaceutical composition
or circular polyribonucleotide described herein. In such embodiments, the kit
may include instructions for
admixing a pharmaceutical composition or nucleic acid molecule (e.g., a
circular polyribonucleotide)
described herein and the other ingredients, or for using a pharmaceutical
composition or nucleic acid
molecule (e.g., a circular polyribonucleotide) described herein together with
the other ingredients.
In some embodiments, the components of the kit are stored under inert
conditions (e.g., under
Nitrogen or another inert gas such as Argon). In some embodiments, the
components of the kit are
stored under anhydrous conditions (e.g., with a desiccant). In some
embodiments, the components are
stored in a light blocking container such as an amber vial.
A dosage form of a pharmaceutical composition or nucleic acid molecule (e.g.,
a circular
polyribonucleotide) described herein may be provided in any form, e.g.,
liquid, dried or lyophilized form. It
is preferred that a pharmaceutical composition or nucleic acid molecule (e.g.,
a circular
polyribonucleotide) described herein be substantially pure and/or sterile.
When a pharmaceutical
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composition or nucleic acid molecule (e.g., a circular polyribonucleotide)
described herein is provided in a
liquid solution, the liquid solution preferably is an aqueous solution, with a
sterile aqueous solution being
preferred. When a pharmaceutical composition or nucleic acid molecule (e.g., a
circular
polyribonucleotide) described herein is provided as a dried form,
reconstitution generally is by the addition
of a suitable solvent. The solvent, e.g., sterile water or buffer, can
optionally be provided in the kit.
The kit may include one or more containers for the composition containing a
dosage form
described herein. In some embodiments, the kit contains separate containers,
dividers or compartments
for the composition and informational material. For example, the
pharmaceutical composition or circular
polyribonucleotide may be contained in a bottle, vial, or syringe, and the
informational material may be
contained in a plastic sleeve or packet. In other embodiments, the separate
elements of the kit are
contained within a single, undivided container. For example, the dosage form
of a pharmaceutical
composition or nucleic acid molecule (e.g., a circular polyribonucleotide)
described herein is contained in
a bottle, vial or syringe that has attached thereto the informational material
in the form of a label. In some
embodiments, the kit includes a plurality (e.g., a pack) of individual
containers, each containing one or
more unit dosage forms of a pharmaceutical composition or circular
polyribonucleotide described herein.
For example, the kit includes a plurality of syringes, ampules, foil packets,
or blister packs, each
containing a single unit dose of a dosage form described herein.
The containers of the kits can be airtight, waterproof (e.g., impermeable to
changes in moisture or
evaporation), and/or light-tight.
The kit optionally includes a device suitable for use of the dosage form,
e.g., a syringe, pipette,
forceps, measured spoon, swab (e.g., a cotton swab or wooden swab), or any
such device.
The kits of the invention may include dosage forms of varying strengths to
provide a subject with
doses suitable for one or more of the initiation phase regimens, induction
phase regimens, or
maintenance phase regimens described herein. Alternatively, the kit may
include a scored tablet to allow
the user to administered divided doses, as needed.
Examples
The following examples are put forth so as 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,
and 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: Expression of antifusogenic polypeptides from RNA in mammalian
cells
This example demonstrates expression of one or more open reading frames (ORFs)
encoding
one or more 0C43-HR2P and EK1 peptides in Huh-7 cells.
In this Example, circular RNAs encoding one 0043-HR2P peptide (SEQ ID NO:
289), one EK1
peptide (SEQ ID NO: 288), multiple 0C43-HR2P peptides, multiple EK1 peptides,
and a combination of
0C43-HR2P peptides, peptide analogs, and EK1 peptides are designed. Circular
RNAs are designed to
include an IRES, a secretion signal, a furin site, one or more 0043-HR2P
peptides, analogs, and/or EK1
sequences, and a spacer element The circular RNAs are transfected into Huh-7
cells using
Lipofectamine MessengerMax (Invitrogen LMRNA001) according to the
manufacturer's instructions.
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In one study, peptide expression is monitored in vitro over a time course.
Example 2: Inhibition of MERS-CoV S protein-mediated cell¨cell fusion
To determine inhibition of MERS-CoV fusion with the 293T target cell, a MERS-
CoV S protein-
mediated cell¨cell fusion assay is used. This example uses 293T cells that are
transfected with the
plasmid pAAV-IRES-EGFP encoding the EG FP (293T/EGFP) or pAAV-IRES-MERS-EGFP
encoding the
MERS-CoV S protein and EGFP (293T/MERS/EGFP) and cultured in DMEM containing
10% FBS at
37 C for 48 h. Huh-7 cells (5 x 104) expressing the MERS-CoV receptor DPP4,
prepared according to
Example 1, are incubated in 96-well plates at 37 C for 5 h, followed by the
addition of 1 x
104 293T/EGFP or 293T/MERS/EGFP cells, respectively.
After co-culture at 37 C for 4 h, the 293T/MERS/EGFP cells (293T/EGFP cells
are used as the
negative control) fused or unfused with Huh-7 cells are counted under an
inverted fluorescence
microscope (Nikon Eclipse Ti-S). The fused cell is seen as one that is 2-fold
or more larger than the
unfused cell, and the differences of intensity of fluorescence in the fused
cell is compared to that of the
unfused cell. The percent inhibition of cell¨cell fusion can be calculated
using the following formula:
(1 ¨(E¨N)I(P¨N)) x 100. 'E' represents the % cell¨cell fusion in the
experimental group. P' represents
the % cell¨cell fusion in the positive control group, to which no circRNA was
added. 'N' is the % cell¨cell
fusion in negative control group, in which 293T/MERS/EGFP cells are replaced
by 293T/EGFP cells. The
concentration for 50% inhibition (IC50) can be calculated using the CalcuSyn
software. Co-culture can
continue at 37 C for 48 h and measurements may be taken, for example of
syncytium formation. In-cell
S protein [LISA can be adapted to measure antiviral activities two days
following viral challenge.
Example 3: Inhibition of pseudotyped SARS-CoV-2 and MERS-CoV infection
SARS or MERS pseudovirus bearing SARS-CoV-2 or MERS-CoV S protein,
respectively, and a
defective HIV-1 genome that expresses luciferase as reporter are prepared by
co-transfecting 293T cells
with the plasmid pNL4-3_luc_RE (encoding Env-defective, luciferase-expressing
HIV-1) and pcDNA3.1-
MERS-CoV-S plasmid. To detect the inhibitory activity of the expressed
peptides on infection by SARS or
MERS pseudovirus, ACE2-transfected 293T (293T/ACE2) cells and Huh-7 cells (1
04 per well in 96-well
plates) that have and have not been transfected with circRNAs of the present
invention are respectively
infected with SARS or MERS-CoV pseudovirus. Following infection, the culture
is re-fed with fresh
medium 12 h post-infection and incubated for an additional 72 h. Cells are
washed with PBS and lysed
using lysis reagent included in a luciferase kit (Promega). Aliquots of cell
lysates are transferred to 96-
well Costar flat-bottom luminometer plates (Corning Costar), followed by the
addition of luciferase
substrate (Promega). Relative light units are determined immediately in the
Ultra 384 luminometer (Tecan
US).
Example 4: Expression of SARS-CoV-2 antifusogenic polypeptides
This example demonstrates expression of SARS-CoV-2 antifusogenic polypeptides
from circular
RNAs.
Several SARS-CoV-2 antifusogenic polypeptides were designed (FIGS. 2 and 3)
based on the
HR2 region show in FIG. 1. Circular RNAs were designed to include an IRES and
a nucleotide sequence
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encoding a SARS-CoV-2 antifusogenic polypeptide. In this example, DNA
constructs were designed to
include a spacer element and a polynucleotide cargo. The constructs were
designed to include a
combination of an I RES and an ORF as the polynucleotide cargo. The ORF was
designed to include an
IL-2 secretion signal sequence, a nucleotide sequence encoding a SARS-CoV-2
antifusogenic
polypeptide, and a nucleotide sequence encoding a HiBiT tag with a GGGGS
peptide linker. The IRES
was EMCV.
Amino acid and nucleic acid sequences for all constructs used are shown below.
N-terminal IL-2 secretion signal shown in uppercase (20 AA or 60 nucleotides)
Bold = Furin
Bold and italics = H iBiT tag with G4S peptide linker
HR2 Full Length
ATGTATAGAATGCAGCTGCTGTCTTGTATTGCTCTTTCTCTGGCTCTTGTGACTAATTCTagactgag gag
aggtattgttaataatactgtttacgatcctcttcagcctgaacttgattcttttaaagaagaactggataaatatttt
aagaatcatacttctcctgacgttga
tctgggtgatatttctggtattaacgcttctgttgttaatattcagaaagaaattgatagactgaacgaagttgctaag
aatctgaacgaatctcttattgatc
ttcaggaacttggaggaggaggaagcgtcagcggctggcggctgttcaagaagatcagc (SEQ ID NO: 378)
MYRMQLLSCIALSLALVTNSRLRRGIVNNTVYDPLQPELDSFKE ELDKYFKNHTSPDVDLGDISGINASVV
NIQKEIDRLNEVAKNLNESLIDUDELGGGGSVSGWRLFKKIS (SEQ ID NO: 379)
HR2A
ATGTATAGAATGCAGCTTCTTTCTTGTATTGCTCTTTCTCTTGCTCTGGTTACTAATTCTagactgag gag
agatatttctggtattaacgcttctgttgttaatattcagaaagaaattgatagacttaacgaagttgctaaaaatctg
aacgaatctctgattgatctgcag
gaactgggaggaggaggaagcgtcageggctggeggctgitcaagaagatcagc (SEQ ID NO: 380)
MYRMQLLSCIALSLALVTNSRLRRDISG INASVVNIQKEIDRLN EVAKNLNESLIDLQELGGGGSVSGWRL
FKKIS (SEQ ID NO: 381)
HR2C
ATGTATAGAATGCAGCTTCTGTCTTGTATTGCTCTGTCTCTTGCTCTTGTTACTAATTCTagactgag gag
atttaaaaatcatacttctcctgacgttgatctgggtgatatttctggtattaacgcttctgttgttaatattcagaaa
gaaattgatagactgaacgaagttg
ctaaaggaggaggaggaagegtcagcggctggcggctgttcaagaagatcagc (SEQ ID NO: 382)
MYRMQLLSCIALSLALVTNSRLRRFKNHTSPDVDLG DISG INASVVNIQKEIDRLN EVAKGGGGSVSGWR
LFKKIS (SEQ ID NO: 383)
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HR2B
ATGTATAGAATGCAGCTTCTGTCTTGTATTGCTCTGTCTCTTGCTCTGGTTACTAATTCTaggctgagaag
agttgttattggtattgttaataatactgtttacgatcctcttcagcctgaacttgattcttttaaggaagaactggat
aagtattttaaaaatcacacttctcct
gatggaggaggaggaagcgtcagcggctggcggctgttcaagaagatcagc (SEQ ID NO: 384)
MYRMQLLSCIALSLALVTNSRLRRVVIG IVNNTVYDPLQPELDSFKEELDKYFKNHTSPDGGGGSVSGWR
LFKK1S (SEQ ID NO: 385)
EK1
ATGTATAGAATGCAGCTTCTTTCTTGTATTGCTCTGTCTCTGGCTCTTGTTACTAATTCTagactgaggag
atctcttgatcagattaacgttactittctggatctggaatacgaaatgaaaaagctggaagaagctattaaaaagctt
gaagaatcttatattgatctga
aagaactgggaggaggaggaagcgtcagcggctggcggctgttcaagaagatcagc (SEQ ID NO: 386)
MYRMOLLSCIALSLALVINSRLRRSLDQINVTFLDLEYEMKKLEEAIKKLEESYIDLKELGGGGSVSGWRL
FKKIS (SEQ ID NO: 387)
Circular RNAs were generated by self-splicing using a method described herein.
Unmodified
linear RNA was synthesized by in vitro transcription using T7 RNA polymerase
from a DNA template
including the motifs listed above in the presence of 7.5 mM of NTP. Template
DNA was removed by
treating with DNase. Synthesized linear RNA was purified with an RNA clean up
kit (New England
Biolabs, T2050). Self-splicing occurred during transcription. Circular RNAs
encoding an antifusogenic
peptide were purified by urea polyacrylamide gel electrophoresis (Urea-PAGE)
or by reversed phase
column chromatography.
To measure the expression of the SARS-CoV-2 antifusogenic polypeptides, 0.4
pmol of circular
RNA was delivered to HEK293 cells using lipofectamine. Expression was measured
after 48 hours.
Various polypeptides were expressed in HEK293 cells. Total expression (ng/ml
and nM) is shown in
Table 2.
Table 2: HR2 construct expression
Expression (ng/mL) Expression (nM)
HR2 Full Length 69.2 8.4
HR2A 5 1.3
HR2C 1.4 0.4
HR2B 0 0
EK1 0.1 0
Example 5: Inhibition of pseudotyped SARS-CoV-2 infection
This example demonstrates inhibition of pseudotyped SARS-CoV2- infection by
antifusogenic
polypeptides expressed from circular RNAs.
Circular RNAs were designed to include an internal ribosome entry site (IRES)
and a nucleotide
sequence encoding an anitfusogenic polypeptide of SARS-CoV-2. In this example,
DNA constructs were
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designed to include a spacer element and a combination of an EMCV IRES and an
ORF as the
polynucleotide cargo. The ORF was designed to include an IL-2 secretion signal
sequence and a
nucleotide sequence encoding an HR2 full length antifusogenic polypeptide, and
a nucleotide sequence
encoding a HiBiT peptide tag. An ORF was also designed to include an IL-2
secretion signal sequence
and a nucleotide sequence encoding an HR2 full length antifusogenic
polypeptide without a nucleotide
sequence encoding a HiBiT peptide tag.
Circular RNAs were generated by self-splicing using a method described herein.
Unmodified
linear RNA was synthesized by in vitro transcription using T7 RNA polymerase
from a DNA template
including the motifs listed above in the presence of 7.5 mM of NTP. Template
DNA was removed by
treating with DNase. Synthesized linear RNA was purified with an RNA clean up
kit (New England
Biolabs, T2050). Self-splicing occurred during transcription. Circular RNAs
encoding the HR2 full length
antifusogenic polypeptide were purified by urea polyacrylamide gel
electrophoresis (Urea-PAGE) or by
reversed phase column chromatography.
To detect the inhibitory activity of the expressed polypeptides on infection
by SARS pseudovirus,
ACE2-transfected 293T (293T/ACE2) cells (104 per well in 96-well plates) that
have and have not been
transfected with circular RNAs were respectively infected with SARS-CoV-2
pseudovirus. Transfection
reagent alone (with no circular RNA) was used as a control ("Mock"). Following
infection, the culture was
re-fed with fresh medium 12 hours post-infection and incubated for an
additional 72 hours. Cells were
washed with PBS and lysed using lysis reagent included in a luciferase kit
(Promega). Aliquots of cell
lysates were transferred to 96-well Costar flat-bottom luminometer plates
(Corning Costar), followed by
the addition of luciferase substrate (Promega). Relative light units were
determined immediately in the
Ultra 384 luminometer (Tecan US).
Efficacy of fusion inhibition in vitro using Omicron and Delta pseudoviruses
was determined using
circular polyribonucleotides encoding antifusogenic polypeptides described
herein. HR2 full length
antifusogenic polypeptide expressed in cells (FIG. 4A), and HR2 full length
and HR2 full length
conjugated to a HiBiT tag antifusogenic polypeptides were shown to inhibit
fusion (FIG. 4A) of Delta and
Omicron strains. There was no change in cell viability (measured by cellTiter
glo) (FIG. 4B) suggesting
that the decrease in luciferase signaling (FIG. 4A) was due to inhibition of
viral fusion.
To measure the expression of the polypeptide in vivo, 120 pmol of circular
RNAs formulated in
lipid nanoparticles were delivered to mice via intravenous injection.
Expression was measured by Nano-
GloO HiBiT Extracellular Detection System (#N3030, Promega) 10% Serum. The
antifusogenic
polypeptide was highly expressed at 6 hours and significantly decreased by 24
hours as shown in Table 3
and FIG. 5.
Table 3: HR2 expression
6 hours (ng/mL) 24 hours (ng/mL)
HR2 Full Length HiBiT 24.8 0.2
No HiBiT 0.02 0.01
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Example 6: Inhibition of pseudotyped SARS-CoV-2 infection
This example demonstrates pseudotyped SARS-CoV2- infection using antifusogenic
polypeptides.
Antifusogenic polypeptides of SARS-CoV-2 were created based on the HR2, HR2A,
HR2B, and
HR2C regions of SARS-CoV-2 Spike polypeptide and the EK1 polypeptide (FIG. 2).
A functional assay was performed to measured pseudoviral neutralization by EK-
1 and HR2A
polypeptides in vitro. HR2A polypeptides showed efficacy against Wuhan and
Omicron strains (FIGS. 6A
and 6B).
Further experiments with polypeptides were performed using HR2A-C and and HR2
full length
polypeptides. For a negative control, IPB19, a HIV peptide, was used, and for
a positive control, ACE-Fc,
an antibody that binds the receptor directly, was used. Polypeptides (starting
dilution 10 pM) were
prepared using 4-fold serial dilution (8 dilutions) and HEK293 ACE2 cells. All
polypeptides were shown to
inhibit Omicron and WT strains, with IC50 values shown in Table 4.
Table 4: IC50 Values
IC50 ( M) IC50 (nM) IC50
(ng/ml)
Omicron Wildtype Omicron Wildtype Omicron Wildtype
HR2 Full Length 0.02625 0.1742 26.3 174.2 215.3
1428.4
HR2A 37.7 614.2 158.1
2579.6
EK-1 64.8 329.3 272.2
1383.1
HR2B Neg. Neg. Neg.
Neg.
HR2C Neg. Neg. Neg.
Neg.
IPB19 1046.0 Neg. 4393.2
Neg.
ACE-Fc 4.3 8.4
Full length HR2 Fc fusions were also tested and shown to maintain inhibitory
activity against
Omicron and Wuhan strains.
HR2 full length polypeptide ("HR2Complete") was shown to successfully inhibit
the fusion of
Omicron BA.4/BA.5, SARS CoV-1, and Wuhan strains (FIGS. 7A, 7B, and 8A-8D).
Example 7: Expression of HIV antifusogenic polypeptides
This example demonstrates expression of HIV antifusogenic polypeptides from
circular RNAs.
Several HIV antifusogenic polypeptides were designed (FIG. 9). Circular RNAs
were designed to
include an IRES and a nucleotide sequence encoding an HIV antifusogenic
polypeptide. In this example,
DNA constructs were designed to include a spacer element and a polynucleotide
cargo (FIG. 9). The
constructs were designed to include a combination of an IRES and an ORF as the
polynucleotide cargo.
The ORF was designed to include an IL-2 secretion signal sequence (SEQ ID NO:
332), a nucleotide
sequence encoding an HIV antifusogenic polypeptide, and a nucleotide sequence
encoding a HiBiT tag
(having sequence VSGWRLFKKIS (SEQ ID NO: 362) with a GGS or GGGGS peptide
linker. The IRES
was either EMCV or a modified CVB3.
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Circular RNAs were generated by self-splicing using a method described herein.
Unmodified
linear RNA was synthesized by in vitro transcription using 17 RNA polymerase
from a DNA template
including the motifs listed above in the presence of 7.5 mM of NIP. Template
DNA was removed by
treating with DNase. Synthesized linear RNA was purified with an RNA clean up
kit (New England
Biolabs, T2050). Self-splicing occurred during transcription. Circular RNAs
encoding an antifusogenic
peptide were purified by urea polyacrylamide gel electrophoresis (Urea-PAGE)
or by reversed phase
column chromatography.
To measure the expression of the HIV antifusogenic polypeptides, 0.4 pmol of
circular RNA was
delivered to HEK293 cells using lipofectamine. Expression was measured after
48 hours. As shown in
FIGS. 10A and 10B, the polypeptides were expressed in HEK293 cells. As shown
in FIGS. 11A and
11B, expression was comparable between circular RNA and DNA plasmid. Total
expression (ng/mL and
nM) is shown in FIG. 12 for the various polypeptides.
HIV polypeptide and nucleic acid sequences
T20
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 313)
tatacttctctgatccactctctgatcgaggaatctcagaaccagcaggagaagaacgaacaggaactgctggaactgg
ataagtgggcttctctgt
ggaactggttc (with EMCV IRES) (SEQ ID NO: 363)
OR
tacaccagcctgatccacagcctgatcgaggaaagccagaaccagcaagagaagaacgagcaggagctgctggagctgg
acaagtgggcca
gcctgtggaactggttc (with modified CVB3 IRES) (SEQ ID NO: 364)
T1249
WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO: 318)
tggcaggagtgggaacagaagatcactgctctgctggaacaggctcagattcagcaggaaaagaacgaatacgaactgc
agaagctggataa
gtgggcttctctgtgggagtggttc (with EMCV IRES) (SEQ ID NO: 365)
OR
tggcaggagtgggagcagaagatcaccgccctgctggagcaggcccagatccagcaagagaagaacgagtacgagctgc
agaagctggac
aagtgggccagcctgtgggagtggttc (with modified CVB3 IRES) (SEQ ID NO: 366)
T1144
TTWEAWDRAIAEYAARIEALLRALQEQQEKNEAALR EL (SEQ ID NO: 316)
actacttgggaagcttgggatagagctatcgctgaatacgctgctagaattgaagctctgctgagagctctgcaggaac
agcaggaaaagaacga
agctgctctgagagaactg (with EMCV IRES) (SEQ ID NO: 367)
OR
accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgctgcgggccctgcaggagc
agcaagagaaga
acgaggccgccctgcgggagctg (with modified CVB3 IRES) (SEQ ID NO: 368)
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ET -9 -4Z0Z 9ZOI-17Z0 VD
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(
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blobubbbobpeoboobbebou
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6e666poeooe
(036 :ON CII 01S) 731=11VV1N>IDOCAOVVE111VAII=IVVAAVIVEICIMVAMII cc
ge9Z
(171
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bppeope 08
(173C :ON CII 03S) JAANM1SVAANCI-13E113V3N>130030SVE111V3ILIVVA3VIVEICIMV3M11
O--9L906
(Ea =ON CII CAS) (S3E11 8EIA0 PeWIDow Lpm) 6136e6b6o6lo6e60066e6oe
ebeebebeeobeobebbembeoobbboolebl000bbeboiebbolobooboelbe633631e33bbbo3e66613366
e666133e33e g3
(En :ON al OAS) -131=11aVaN>ROCAOSVHFIVAIEIVVAAVIVHCIMVAM11
9L906
(Lc :ON CII OAS) (SDEll CCIAO ID9WIDow t_mm) 311661Be66616133Beopb66i6eepe66i3
6ee6e3bl36eboe16eEoeP6ee6ebee3beo31e6e3336Ee36u661o613o3600eo1e6ee6eo6e66616ebb
e3661o3e33e 03
(61-E :ON CII OS) JMD/VCISVMNCI-INO1AADN>,90010V0D-11VIINOAMDOMII
07-617Z
:ON CII OAS) (Sal=11 88A0 PoIl!PoLu LI1I/A)
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ebeebebeeobeooeebepobeeebbebolebloobeoeomebloobuooeoeueeoeemebebbbooebbblbebble
bbloouble
(9 1-6 :ON CII C)3S) JAANM1SVM>ICI-0-MCAN>13C)C)NC)S3317SHI-
ISIANNI3EICIMMAIMIIAI
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bblooeble
(171-e :ON CII OAS) 137130ANNAOONOSAAFISHFISIANNIALICIMAIAIMIIAI
01-173-1
(698 g
:ON CII ODS) (SAEll 88A0 Paq!PoLu qum)
0116610ee661613o6eo3666i6eeoe66lo6e666o6p3363366e6oe
ebeebebeeabeobebbeoblooabbboblabpaabbebolebbalaboaboeMeboobalepobbbooebbbiapbbe
bbbpaeaae
(L :ON CII OHS) JMNM-ISVMNCI1AEFIVVANNA00a01V171-11VAIHVVAAVIVHCIMVAMII
0-Z-1712. L I.
Sta80/ZZOZSIVI3c1 68LZZINZ0Z OAA
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accacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagc
agcaagagaaga
acgaggccgcactgcgggagctggacaagtgggccagcctgtggaactggttc (with modified CVB3
IRES) (SEQ ID NO:
376)
2635_3.0
MTWEAWDRAIAEYAARIEALIRAAQEQQEKNEAALRELDKWASLWNWF (SEQ ID NO: 322)
atgacctgggaggcctgggaccgggccatcgccgagtacgccgctcggatcgaggccctgatccgggccgcccaggagc
agcaagagaaga
acgaggccgcactgcgggagctggacaagtgggccagcctgtggaactggttc (SEQ ID NO: 377)
Other Embodiments
While the invention has been described in connection with specific embodiments
thereof, it will be
understood that it is capable of further modifications and this application is
intended to cover any
variations, uses, or adaptations of the invention following, in general, the
principles of the invention and
including such departures from the invention that come within known or
customary practice within the art
to which the invention pertains and may be applied to the essential features
hereinbefore set forth, and
follows in the scope of the claims. Other embodiments are within the claims.
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