Note: Descriptions are shown in the official language in which they were submitted.
COMPOSITIONS AND METHODS FOR THE PREVENTION AND/OR
TREATMENT OF COVID-19
SEQUENCE LISTING
[0001] The present application is being filed along with a Sequence Listing
in
electronic format. The Sequence Listing file, entitled 20921004CAPRI4.txt, was
created
on April 23, 2021, and is 60,100 bytes in size. The information in electronic
format of
the Sequence Listing is incorporated herein by reference in its entirety.
FIELD OF THE DISCLOSURE
[0002] The present disclosure generally relates to compositions, methods,
formulations, and/or uses of nucleic acid vaccines, specifically nucleic acid
vaccines
(e.g., mRNA vaccines) encoding one or more proteins, peptides, fragments or
variants
thereof of SARS-CoV-2 for the treatment and/or prevention of COVID-19.
BACKGROUND
[0003] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a
new
strain of coronavirus which began infecting mammals in 2019 in China and has
spread to
a pandemic. SARS-CoV-2 infection causes coronavirus disease 2019 (termed
"COVID-
19"), which affects mammals in different ways including individuals who are
asymptomatic to individuals who have a wide range of symptoms that range from
mild
symptoms to severe illness or death.
[0004] Vaccines are an effective way to provide prophylactic protection
against
infective diseases. Currently, there are no vaccines available to prevent
and/or treat
COVID-19. Treatment of COVID-19 has been limited to the management of symptoms
and/or the side effects of the disease. Thus, there remains a strong need for
COVID-19
vaccines including formulations for delivering the vaccines to a range of
different target
cells.
SUMMARY
[0005] The present disclosure provides nucleic acid vaccines and methods of
using
same for the treatment and prevention of COVID-19. The nucleic acid vaccines
may
include polynucleotides which encode at least one structural protein, fragment
or variant
thereof of SARS-CoV-2. The structural protein may be the spike protein, the
membrane
- 1 -
Date Recue/Date Received 2021-04-23
protein, the nucleocapsid phosphoprotein or the envelope protein. Non-limiting
examples
of sequences of these structural proteins are shown in Table 1 (SEQ ID Nos: 1-
5). The
nucleic acid vaccine may encode at least one structural protein with at least
75%, 80%,
85%, 90%, 95%, 99% or 100% of any of the sequences in Table 1 or fragments of
the
sequences in Table 1.
[0006] Provided herein are nucleic acid vaccines for COVID-19 for use in a
method of
vaccinating a subject for COVID-19, wherein the nucleic acid vaccine may
include at
least one polynucleotide encoding at least one structural protein or a
fragment thereof of
SARS-CoV-2.
[0007] Provided herein are methods of inducing an immune response in a
subject by
administering the nucleic acid vaccines described herein in an effective
amount to
produce an immune response. The immune response may be, but is not limited to,
a T
cell response or a B cell response. As a non-limiting example, the immune
response may
be produced by a single administration of the nucleic acid vaccines described
herein. As
another non-limiting example, the immune response may be produced by a booster
administration of the nucleic acid vaccines described herein. The
administering the
pharmaceutical composition may produce a dose-responsive immune response in
the
subject. As a non-limiting example, the dose-responsive immune response may
comprise
induction of one or more of SARS-CoV-2 spike protein specific IgG, IgGl,
IgG2a,
IgG2b, IgM and IgA antibodies in the subject. As another non-limiting example,
the
dose-responsive immune response may comprise induction of one or more of IL-2+
T
cells, IL-4+ T cells, and IFN-gamma+ T cells. In some embodiments, the
administering
the pharmaceutical composition does not induce significant adverse reactions
in the
subject.
[0008] Provided herein are methods of treating and/or preventing COVID-19
in a
subject by administering the nucleic acid vaccines described herein in an
effective
amount to produce an immune response.
[0009] Provided herein are pharmaceutical compositions and formulations of
the
nucleic acid vaccines for the treatment and prevention of COVID-19. The
nucleic acid
- 2 -
Date Recue/Date Received 2021-04-23
vaccines described herein may be formulated in one or more lipid
nanoparticles. As a
non-limiting example, the nucleic acid vaccines are formulated in a single
lipid
nanoparticle. As a non-limiting example, the nucleic acid vaccines are
formulated in a
more than one lipid nanoparticle. As a non-limiting example, the nucleic acid
vaccines
comprise polynucleotides encoding a single structural protein and the nucleic
acid
vaccines are formulated in a single lipid nanoparticle or formulated in more
than on lipid
nanoparticle. As a non-limiting example, the nucleic acid vaccines comprise
polynucleotides encoding more than one structural protein and the nucleic acid
vaccines
are formulated in a single lipid nanoparticle or formulated in more than on
lipid
nanoparticle. As a non-limiting example, the pharmaceutical compositions may
produce a
dose-responsive immune response upon administration to a subject. The dose-
responsive
immune response may comprise induction of one or more of SARS-CoV-2 spike
protein
specific IgG, IgGl, IgG2a, IgG2b, IgM and IgA antibodies in the subject. As a
further
example, the dose-responsive immune response may comprise induction of one or
more
of IL-2+ T cells, IL-4+ T cells, and IFN-gamma+ T cells in the subject. In
some
embodiments, the pharmaceutical composition does not induce significant
adverse
reactions upon administration to a subject.
[0010] The details of various embodiments are set forth in the description
below.
Other features, objects and advantages will be apparent from the description,
and from
the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows results from a SARS-CoV-2 neutralization assay using
virus
isolated from a patient in Ontario. Groups 1 ¨ 5 correlate to the vaccine
formulation
administered (see Table 6).
[0012] FIG. 2 shows results from a neutralization assay using a SARS-CoV-2
pseudotyped lentivirus that encodes a luciferase gene and can infect HEK293T
cells.
Groups 1 ¨ 5 correlate to the vaccine formulation administered (see Table 6).
[0013] FIG. 3 shows the ID50 (dilution at which 50% inhibition of
infectivity is seen)
for both the SARS-CoV-2 clinical isolate and pseudovirus neutralization
assays.
- 3 -
Date Recue/Date Received 2021-04-23
[0014] FIG. 4 shows IFN-y analysis by ELISpot to determine the T cell
response to
immunization with PTX-B.
[0015] FIG. 5 shows cytokine profiling by Luminex in mice vaccinated with a
prime
and boost of PTX-B at Days 1 and 22.
[0016] FIG. 6A ¨ FIG. 6B show cytokine profiling by flow cytometry in mice
vaccinated with a prime and boost of PTX-B at Days 1 and 22.
[0017] FIG. 7 shows change in body weight in mice challenged with SARS-CoV-2.
[0018] FIG. 8 shows protective efficacy in AAV6-hACE2 transduction mouse
model.
[0019] FIG. 9 shows lung histopathology scores in a AAV6-hACE2 transduction
mouse model.
[0020] FIG. 10 shows IFN-y and IL-4 ELISpot analysis of splenocytes from PTX-B
immunized mice.
[0021] FIG. 11 shows protection from infection with a SARS-CoV-2 clinical
isolate in
a SARS-CoV-2 neutralization assay.
[0022] FIG. 12 shows protection from infection in a pseudovirus
neutralization assay.
[0023] FIG. 13A ¨ FIG. 13C show anti-SARS-CoV-2 anti-Spike protein antibody
profiles.
[0024] FIG. 14 shows levels of infectious virus was significantly lower in
vaccinated
animals in a SARS-CoV-2 challenge study in hamsters.
DETAILED DESCRIPTION
I. INTRODUCTION
[0025] The following description sets forth exemplary methods, parameters
and the
like. It should be recognized, however, that such description is not intended
as a
limitation on the scope of the present disclosure but is instead provided as a
description
of exemplary embodiments.
[0026] Described herein are compositions, methods, formulations, and/or use
of
nucleic acid vaccines, specifically nucleic acid vaccines encoding one or more
proteins,
peptides, fragments or variants thereof of SARS-CoV-2 for the treatment and/or
prevention of COVID-19. In some embodiments, at least one component of the
nucleic
- 4 -
Date Recue/Date Received 2021-04-23
acid vaccine is a polynucleotide encoding at least one of the structural
proteins or the
fragments or variants of the structural proteins of SARS-CoV-2. The
polynucleotide may
be a RNA polynucleotide such as an mRNA polynucleotide. In some embodiments,
the
nucleic acid vaccine includes at least one mRNA polynucleotide encoding at
least one of
the structural proteins or the fragments or variants of the structural
proteins of SARS-
CoV-2.
[0027] In some embodiments, the polynucleotide may be designed to encode
one or
more polypeptides of interest from SARS-CoV-2 or fragments thereof. Such
polypeptide
of interest of SARS-CoV-2 may include, but is not limited to, whole
polypeptides, a
plurality of polypeptides or fragments of polypeptides, which independently
may be
encoded by one or more regions or parts or the whole of a polynucleotide from
SARS-
CoV-2. As used herein, the term "polypeptides of interest" refer to any
polypeptide
which is selected to be encoded within, or whose function is affected by, the
polynucleotides described herein. Any of the peptides or polypeptides
described herein
may be antigenic.
[0028] 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. In some
embodiments, the polypeptides of interest are antigens encoded by the
polynucleotides as
described herein.
[0029] In some embodiments, the polypeptide encoded is smaller than about
50 amino
acids and the polypeptide is then termed a peptide. If the polypeptide is a
peptide, it will
be at least about 2, 3, 4, or at least 5 amino acid residues long. Thus,
polypeptides include
gene products, naturally occurring polypeptides, synthetic polypeptides,
homologs,
orthologs, paralogs, fragments and other equivalents, variants, and analogs of
the
foregoing. A polypeptide may be a single molecule or may be a multi-molecular
complex
such as a dimer, trimer or tetramer. They may also comprise single chain or
multichain
polypeptides such as antibodies or insulin and may be associated or linked.
Most
commonly disulfide linkages are found in multichain polypeptides. The term
polypeptide
- 5 -
Date Recue/Date Received 2021-04-23
may 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.
[0030] The term "polypeptide variant" refers to molecules which differ in
their amino
acid sequence from a native or reference sequence. The amino acid sequence
variants
may possess substitutions, deletions, and/or insertions at certain positions
within the
amino acid sequence, as compared to a native or reference sequence.
Ordinarily, variants
will possess at least about 50% identity (homology) to a native or reference
sequence,
and preferably, they will be at least about 80%, more preferably at least
about 90%
identical (homologous) to a native or reference sequence.
[0031] In some embodiments "variant mimics" are provided. As used herein,
the term
"variant mimic" is one which contains one or more amino acids which would
mimic an
activated sequence. For example, glutamate may serve as a mimic for phosphoro-
threonine and/or phosphoro-serine. Alternatively, variant mimics may result in
deactivation or in an inactivated product containing the mimic, e.g.,
phenylalanine may
act as an inactivating substitution for tyrosine; or alanine may act as an
inactivating
substitution for serine.
[0032] "Homology" as it applies to amino acid sequences is defined as the
percentage
of residues in the candidate amino acid sequence that are identical with the
residues in the
amino acid sequence of a second sequence after aligning the sequences and
introducing
gaps, if necessary, to achieve the maximum percent homology. Methods and
computer
programs for the alignment are well known in the art. It is understood that
homology
depends on a calculation of percent identity but may differ in value due to
gap and
penalties introduced in the calculation.
[0033] By "homologs" as it applies to polypeptide sequences means the
corresponding
sequence of other species having substantial identity to a second sequence of
a second
species.
[0034] "Analogs" is meant to include polypeptide variants which differ by
one or
more amino acid alterations, e.g., substitutions, additions or deletions of
amino acid
- 6 -
Date Recue/Date Received 2021-04-23
residues that still maintain one or more of the properties of the parent or
starting
polypeptide.
[0035] In some embodiments, the present disclosure contemplates several
types of
compositions which are polypeptide based including variants and derivatives.
These
include substitutional, insertional, deletion and covalent variants and
derivatives. The
term "derivative" is used synonymously with the term "variant" but generally
refers to a
molecule that has been modified and/or changed in any way relative to a
reference
molecule or starting molecule.
[0036] As such, polynucleotides encoding peptides or polypeptides
containing
substitutions, insertions and/or additions, deletions and covalent
modifications with
respect to reference sequences, in particular the polypeptide sequences
disclosed herein.
For example, sequence tags or amino acids, such as one or more lysines, can be
added to
the peptide sequences described herein (e.g., at the N-terminal or C-terminal
ends).
Sequence tags can be used for peptide purification or localization. Lysines
can be used to
increase peptide solubility or to allow for biotinylation. Alternatively,
amino acid
residues located at the carboxy and amino terminal regions of the amino acid
sequence of
a peptide or protein may optionally be deleted providing for truncated
sequences. Certain
amino acids (e.g., C-terminal or N-terminal residues) may alternatively be
deleted
depending on the use of the sequence, as for example, expression of the
sequence as part
of a larger sequence which is soluble, or linked to a solid support.
[0037] "Substitutional variants" when referring to polypeptides are those
that have at
least one amino acid residue in a native or starting sequence removed and a
different
amino acid inserted in its place at the same position. The substitutions may
be single,
where only one amino acid in the molecule has been substituted, or they may be
multiple,
where two or more amino acids have been substituted in the same molecule.
[0038] As used herein the term "conservative amino acid substitution"
refers to the
substitution of an amino acid that is normally present in the sequence with a
different
amino acid of similar size, charge, or polarity. Examples of conservative
substitutions
include the substitution of a non-polar (hydrophobic) residue such as
isoleucine, valine
- 7 -
Date Recue/Date Received 2021-04-23
and leucine for another non-polar residue. Likewise, examples of conservative
substitutions include the substitution of one polar (hydrophilic) residue for
another such
as between arginine and lysine, between glutamine and asparagine, and between
glycine
and serine. Additionally, the substitution of a basic residue such as lysine,
arginine or
histidine for another, or the substitution of one acidic residue such as
aspartic acid or
glutamic acid for another acidic residue are additional examples of
conservative
substitutions. Examples of nonconservative substitutions include the
substitution of a
nonpolar (hydrophobic) amino acid residue such as isoleucine, valine, leucine,
alanine,
methionine for a polar (hydrophilic) residue such as cysteine, glutamine,
glutamic acid or
lysine and/or a polar residue for a non-polar residue.
[0039] "Insertional variants" when referring to polypeptides are those with
one or
more amino acids inserted immediately adjacent to an amino acid at a
particular position
in a native or starting sequence. "Immediately adjacent" to an amino acid
means
connected to either the alpha-carboxy or alpha-amino functional group of the
amino acid.
[0040] "Deletional variants" when referring to polypeptides are those with
one or
more amino acids in the native or starting amino acid sequence removed.
Ordinarily,
deletional variants will have one or more amino acids deleted in a particular
region of the
molecule.
[0041] "Covalent derivatives" when referring to polypeptides include
modifications of
a native or starting protein with an organic proteinaceous or non-
proteinaceous
derivatizing agent, and/or post-translational modifications. Covalent
modifications are
traditionally introduced by reacting targeted amino acid residues of the
protein with an
organic derivatizing agent that is capable of reacting with selected side-
chains or terminal
residues, or by harnessing mechanisms of post-translational modifications that
function in
selected recombinant host cells. The resultant covalent derivatives are useful
in programs
directed at identifying residues important for biological activity, for
immunoassays, or for
the preparation of anti-protein antibodies for immunoaffinity purification of
the
recombinant glycoprotein. Such modifications are within the ordinary skill in
the art and
are performed without undue experimentation.
- 8 -
Date Recue/Date Received 2021-04-23
[0042] "Features" when referring to polypeptides are defined as distinct
amino acid
sequence-based components of a molecule. Features of the polypeptides encoded
by the
polynucleotides described herein include surface manifestations, local
conformational
shape, folds, loops, half-loops, domains, half-domains, sites, termini or any
combination
thereof.
[0043] As used herein when referring to polypeptides the term "surface
manifestation"
refers to a polypeptide based component of a protein appearing on an outermost
surface.
[0044] As used herein when referring to polypeptides the term "local
conformational
shape" means a polypeptide based structural manifestation of a protein which
is located
within a definable space of the protein.
[0045] As used herein when referring to polypeptides the term "fold" refers
to the
resultant conformation of an amino acid sequence upon energy minimization. A
fold may
occur at the secondary or tertiary level of the folding process. Examples of
secondary
level folds include beta sheets and alpha helices. Examples of tertiary folds
include
domains and regions formed due to aggregation or separation of energetic
forces.
Regions formed in this way include hydrophobic and hydrophilic pockets, and
the like.
[0046] As used herein the term "turn" as it relates to protein conformation
means a
bend which alters the direction of the backbone of a peptide or polypeptide
and may
involve one, two, three or more amino acid residues.
[0047] As used herein when referring to polypeptides the term "loop" refers
to a
structural feature of a polypeptide which may serve to reverse the direction
of the
backbone of a peptide or polypeptide. Where the loop is found in a polypeptide
and only
alters the direction of the backbone, it may comprise four or more amino acid
residues.
Oliva et al. have identified at least 5 classes of protein loops (J. Mol Biol
266 (4): 814-
830; 1997). Loops may be open or closed. Closed loops or "cyclic" loops may
comprise
2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids between the bridging moieties.
Such bridging
moieties may comprise a cysteine-cysteine bridge (Cys-Cys) typical in
polypeptides
having disulfide bridges or alternatively bridging moieties may be non-protein
based such
as the dibromozylyl agents used herein.
- 9 -
Date Recue/Date Received 2021-04-23
[0048] As used herein when referring to polypeptides the term "half-loop"
refers to a
portion of an identified loop having at least half the number of amino acid
resides as the
loop from which it is derived. It is understood that loops may not always
contain an even
number of amino acid residues. Therefore, in those cases where a loop contains
or is
identified to comprise an odd number of amino acids, a half-loop of the odd-
numbered
loop will comprise the whole number portion or next whole number portion of
the loop
(number of amino acids of the loop/2+/-0.5 amino acids).
[0049] As used herein when referring to polypeptides the term "domain"
refers to a
motif of a polypeptide having one or more identifiable structural or
functional
characteristics or properties (e.g., binding capacity, serving as a site for
protein-protein
interactions).
[0050] As used herein when referring to polypeptides the term "half-domain"
means a
portion of an identified domain having at least half the number of amino acid
resides as
the domain from which it is derived. It is understood that domains may not
always
contain an even number of amino acid residues. Therefore, in those cases where
a domain
contains or is identified to comprise an odd number of amino acids, a half-
domain of the
odd-numbered domain will comprise the whole number portion or next whole
number
portion of the domain (number of amino acids of the domain/2+/-0.5 amino
acids). For
example, a domain identified as a 7 amino acid domain could produce half-
domains of 3
amino acids or 4 amino acids (7/2=3.5+1-0.5 being 3 or 4). It is also
understood that sub-
domains may be identified within domains or half-domains, these subdomains
possessing
less than all of the structural or functional properties identified in the
domains or half
domains from which they were derived. It is also understood that the amino
acids that
comprise any of the domain types herein need not be contiguous along the
backbone of
the polypeptide (i.e., nonadjacent amino acids may fold structurally to
produce a domain,
half-domain or subdomain).
[0051] As used herein when referring to polypeptides the terms "site" as it
pertains to
amino acid based embodiments is used synonymously with "amino acid residue"
and
"amino acid side chain." A site represents a position within a peptide or
polypeptide that
- 10 -
Date Recue/Date Received 2021-04-23
may be modified, manipulated, altered, derivatized or varied within the
polypeptide based
molecules described herein.
[0052] As used herein the terms "termini" or "terminus" when referring to
polypeptides refers to an extremity of a peptide or polypeptide. Such
extremity is not
limited only to the first or final site of the peptide or polypeptide but may
include
additional amino acids in the terminal regions. The poly peptide based
molecules
described herein may be characterized as having both an N-terminus (terminated
by an
amino acid with a free amino group (NH2)) and a C-terminus (terminated by an
amino
acid with a free carboxyl group (COOH)). Proteins described herein are in some
cases
made up of multiple polypeptide chains brought together by disulfide bonds or
by non-
covalent forces (multimers, oligomers). These sorts of proteins will have
multiple N- and
C-termini. Alternatively, the termini of the polypeptides may be modified such
that they
begin or end, as the case may be, with a non-polypeptide based moiety such as
an organic
conjugate.
[0053] Once any of the features have been identified or defined as a
desired
component of a polypeptide to be encoded by a polynucleotide described herein,
any of
several manipulations and/or modifications of these features may be performed
by
moving, swapping, inverting, deleting, randomizing or duplicating.
Furthermore, it is
understood that manipulation of features may result in the same outcome as a
modification to the molecules described herein. For example, a manipulation
which
involved deleting a domain would result in the alteration of the length of a
molecule just
as modification of a nucleic acid to encode less than a full length molecule
would.
[0054] In a polypeptide, the term "modification" refers to a modification
as compared
to the canonical set of 20 amino acids. The modifications may be various
distinct
modifications. In some embodiments, the regions may contain one, two, or more
(optionally different) modifications.
[0055] Modifications and manipulations can be accomplished by methods known in
the art such as, but not limited to, site directed mutagenesis or a priori
incorporation
during chemical synthesis. The resulting modified molecules may then be tested
for
- 1 1 -
Date Recue/Date Received 2021-04-23
activity using in vitro or in vivo assays such as those described herein or
any other
suitable screening assay known in the art.
[0056] In some embodiments, the polypeptides may comprise a consensus
sequence
which is discovered through rounds of experimentation. As used herein a
"consensus"
sequence is a single sequence which represents a collective population of
sequences
allowing for variability at one or more sites.
[0057] As recognized by those skilled in the art, protein fragments,
functional protein
domains, and homologous proteins are also considered to be within the scope of
polypeptides of interest. For example, provided herein is any protein fragment
(meaning a
polypeptide sequence at least one amino acid residue shorter than a reference
polypeptide
sequence but otherwise identical) of a reference protein 10, 20, 30, 40, 50,
60, 70, 80, 90,
100 or greater than 100 amino acids in length. In another example, any protein
that
includes a stretch of about 20, about 30, about 40, about 50, or about 100
amino acids
which are about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,
about
95%, or about 100% identical to any of the sequences described herein can be
utilized in
accordance with the nucleic acid vaccines described herein. In certain
embodiments, a
polypeptide to be utilized in accordance with the nucleic acid vaccines
described herein
includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the
sequences
provided or referenced herein.
[0058] Reference molecules (polypeptides or polynucleotides) may share a
certain
identity with the designed molecules (polypeptides or polynucleotides). The
term
"identity" as known in the art, refers to a relationship between the sequences
of two or
more peptides, polypeptides or polynucleotides, as determined by comparing the
sequences. In the art, identity also means the degree of sequence relatedness
between
them as determined by the number of matches between strings of two or more
amino acid
residues or nucleosides. Identity measures the percent of identical matches
between the
smaller of two or more sequences with gap alignments (if any) addressed by a
particular
mathematical model or computer program (i.e., "algorithms"). Identity of
related peptides
can be readily calculated by known methods. Such methods include, but are not
limited
- 12 -
Date Recue/Date Received 2021-04-23
to, those described in Computational Molecular Biology, Lesk, A. M., ed.,
Oxford
University Press, N.Y., 1988; Biocomputing: Informatics and Genome Projects,
Smith,
D. W., ed., Academic Press, N.Y., 1993; Computer Analysis of Sequence Data,
Part 1,
Griffin, A. M., and Griffin, H. G., eds., Humana Press, N.J., 1994; Sequence
Analysis in
Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis
Primer,
Gribskov, M. and Devereux, J., eds., M. Stockton Press, N.Y, 1991; and Carillo
et al.,
SIAM J. Applied Math. 48, 1073 (1988).
[0059] In some embodiments, the encoded polypeptide variant may have the
same or a
similar activity as the reference polypeptide. Alternatively, the variant may
have an
altered activity (e.g., increased or decreased) relative to a reference
polypeptide.
Generally, variants of a particular polynucleotide or polypeptide described
herein will
have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to
that
particular reference polynucleotide or polypeptide as determined by sequence
alignment
programs and parameters described herein and known to those skilled in the
art. Such
tools for alignment include those of the BLAST suite (Stephen F. Altschul,
Thomas L.
Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and
David J.
Lipman (1997), "Gapped BLAST and PSLBLAST: a new generation of protein
database
search programs", Nucleic Acids Res. 25:3389-3402.) Other tools are described
herein,
specifically in the definition of "Identity."
II. COMPOSITIONS OF THE PRESENT DISCLSOURE
SARS-CoV-2
[0060] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a
new
strain of coronavirus which causes coronavirus disease 2019 termed "COVID-19."
COVID-19 affects mammals in different ways including individuals who are
asymptomatic to individuals who have a wide span of symptoms that range from
mild
symptoms to severe illness or death. To date, about 80% of COVID-19 patients
have mild
to moderate symptoms whereas about 20% may develop complications such as sever
pneumonia, acute respiratory distress syndrome, sepsis and even death. The
list of
- 13 -
Date Recue/Date Received 2021-04-23
symptoms associated with COVID-19 is constantly changing as doctors and
scientists
learn more about COVID-19 and how it affects the body, but some of the
symptoms
recognized to date include fever or chills, cough, shortness of breath or
difficulty
breathing, fatigue, body aches, muscle aches, headaches, sore throat,
congestion or runny
nose, nausea and/or vomiting, diarrhea, and a new loss of taste or smell.
[0061] The genome of SARS-CoV-2 encodes four structural proteins: spike
(S),
envelope (E), membrane (M), and nucleocapsid (N). The spike protein is
generally the
leading mediator for viral entry and is a protective antigen that elicits
neutralizing
antibodies, nonstructural proteins (named nspl to nsp16) and accessory
proteins. Another
feature of the spike protein of SARS-CoV-2 also has a functional furin
cleavage site at
the S1-S2 boundary (Si is the receptor binding unit and S2 is the membrane
fusion unit).
The membrane protein and the envelope protein are for viral assembly. The
nucleocapsid
protein packages the viral genome into a helical ribonucleocapsid (RNP) and
has a role in
viral self-assembly (Chang et al.; The SARS coronavirus nucleocapsid protein ¨
Forms
and functions; Antiviral Res. 2014; the contents of which are herein
incorporated by
reference in their entirety). Additionally, the nucleocapsid protein in SARS-
CoV can
modulate the host cell machinery and may be included in regulatory roles in
the viral life
cycle.
[0062] While not wishing to be bound by theory, it appears that SARS-CoV-2
binds to
the human receptor ACE2. The receptor-binding domain (RBD) in the spike
protein
appears to be the most variable part of the genome for coronavirus genome.
There are six
RBD amino acids have been shown to be critical for binding to ACE2 receptors
and the
SARS-CoV-2 genome appears to have a RBD that has a high affinity binding to
ACE2
for humans, ferrets, cats and other species with high receptor homology
(Anderson et. al.;
The Proximal Origin of SARS-CoV-2; Nature Medicine 2020; the contents of which
are
herein incorporated by reference in their entirety).
[0063] In some embodiments, the polynucleotides of the nucleic acid vaccine
described herein encode a fragment or variant of a structural protein of SARS-
CoV-2,
such as the spike protein, the nucleocapsid protein or the membrane protein.
- 14 -
Date Recue/Date Received 2021-04-23
[0064] In some embodiments, the polynucleotides of the nucleic acid vaccine
described herein encode more than one fragment or variant of a structural
protein of
SARS-CoV-2, such as the spike protein, the nucleocapsid protein and/or the
membrane
protein.
[0065] In some embodiments, the polynucleotides of the nucleic acid vaccine
described herein encode a mutated variant of one of the structural proteins or
a fragment
of the structural proteins of SARS-CoV-2. As a non-limiting example, the
variant may be
a single amino acid change from Aspartic Acid to Glycine in one of the
structural
proteins of SARS-CoV-2. As a non-limiting example, the variant may be a single
amino
acid change from Aspartic Acid to Glycine in the spike protein of SARS-CoV-2.
As a
non-limiting example, the variant may be a single amino acid change from
Aspartic Acid
to Glycine at position 614 (D614G) in the spike protein of SARS-CoV-2 (Korber
et al.;
Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases
Infectivity of
the COVID-19 Virus; Cell; 2020; the contents of which is herein incorporated
by
reference in its entirety).
[0066] In some embodiments, the nucleic acid vaccine described herein may
encode
one or more proteins, peptides, fragments or variants thereof of the
structural proteins of
SARS-CoV-2. Non-limiting examples of proteins, peptides, fragments or variants
of the
structural proteins of SARS-CoV-2 are provided in Table 1. In the table, the
NCBI
reference number is also provided if known.
Table 1. Structural Protein Sequences of SARS-CoV-2
Sequence Description Sequence
Identifier Type
(SEQ ID
NO.)
1 Spike protein (NCBI Ref.: YP 009724390.1) ("S Protein
protein")
2 Spike protein with D614G mutation Protein
3 Envelope protein (NCBI Ref.: YP 009724392.1) Protein
4 Membrane protein (NCBI Ref.: YP 009724393.1) Protein
Nucleocapsid phosphoprotein (NCBI Ref.: Protein
YP 009724397.2)
6 B.1.351 (South African) Variant Spike protein Protein
- 15 -
Date Recue/Date Received 2021-04-23
[0067] In some embodiments, the nucleic acid vaccine may encode at least
one
structural protein with at least 75%, 80%, 85%, 90%, 95%, 99% or 100% of any
of the
sequences in Table 1 or fragments of the sequences in Table 1.
[0068] In some embodiments, the nucleic acid vaccine may be an mRNA vaccine
that,
when translated, produces one or more proteins, peptides, fragments or
variants thereof of
the structural proteins of SARS-CoV-2.
[0069] The coding sequences of mRNA vaccine constructs described herein may be
based on the S protein from the SARS-CoV-2 Wuhan-Hu-1 isolate (GenBank:
MN908947.3). In some embodiments, a change of D614 to G614 is introduced to
match
the amino acid of the current dominant circulating strains.
[0070] Non-limiting examples of a RNA sequence encoding proteins, peptides,
fragments or variants of the structural proteins of SARS-CoV-2 are provided in
Table 2.
Table 2. mRNA Sequence of Structural Proteins of SARS-CoV-2
Sequence Description Sequence
Identifier Type
(SEQ ID
NO.)
7 mRNA encoding Spike protein with D614G mutation RNA
[0071] In some embodiments, the nucleic acid vaccines may comprise a region
encoding any of the sequences listed in Table 1 or a fragment or variant
thereof. The
nucleic acid vaccines may comprise hybrid or chimeric regions, or mimics or
variants.
Table 3. Exemplary Sequences to be used in the Nucleic Acid Vaccines for
treating
or preventing COVID-19
Sequence Description Sequence
Identifier Type
(SEQ ID
NO.)
8 Sequence encoding M protein DNA
9 Sequence encoding N and M protein DNA
Sequence encoding N protein DNA
11 Sequence with a signal peptide and encoding N and DNA
M protein
- 16 -
Date Recue/Date Received 2021-04-23
12 Sequence encoding SARS-CoV-2 variant B.1.351 DNA
(South African variant) Spike protein
[0072] Any of the sequences referred to in Tables 1-3 may also be used in a
memory
booster vaccine described herein.
[0073] In some embodiments, the nucleic acid vaccine described herein
encodes a
protein or fragment thereof that is at least 90%, at least 91%, at least 92%,
at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99%
identical to a protein provided by an amino acid sequence in Table 1. The
terms
"identical" or percent "identity" in the context of two or more polypeptide
sequences
refer to two or more sequences that are the same. The percent identity between
polypeptide sequences may be performed using algorithms known in the art, such
as
BLAST and CLUSTAL.
[0074] The sequence of the SARS-CoV-2 protein or fragment thereof may be
obtained
from any source. In some embodiments, the sequence of the SARS-CoV-2 protein
or
fragment thereof is from a strain that is capable of or at risk of infecting
human subjects.
[0075] In some embodiments, the sequence of the SARS-CoV-2 protein or fragment
thereof may be modified or optimized (such as codon optimized) for expression
in a
particular cell or host organism.
[0076] In some embodiments, the nucleic acid vaccine described herein may
comprise
a multivalent vaccine. The multivalent vaccine may include polynucleotides
that encodes
at least two different one or more proteins, peptides, fragments or variants
thereof of
SARS-CoV-2. As a non-limiting example, the polynucleotides may encode the same
or a
different structural protein. As a non-limiting example, the polynucleotides
may encode
the same structural protein but different variants of the structural protein.
[0077] In some embodiments, the nucleic acid vaccine encodes the full
length S
protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a
fragment of the S protein of SARS-CoV-2. In some embodiments, the nucleic acid
vaccine encodes the receptor binding domain (RBD) fragment of the S protein of
SARS-
CoV-2. In some embodiments, the nucleic acid vaccine encodes a S protein
sequence of
- 17 -
Date Recue/Date Received 2021-04-23
SARS-CoV-2 (e.g., full length, fragment or variant) where the S protein has a
mutated
furin cleavage site. The S protein furin cleavage site mutant construct was
made to
remove or disable the furin cleavage site(s) in S protein (e.g., between the
Si and S2
boundary). In some viral envelope proteins, disruption of a furin cleavage
site was found
to enhance expression and stability. In some embodiments, the nucleic acid
vaccine
encodes a S protein sequence of SARS-CoV-2 (e.g., full length, fragment or
variant)
where the S protein includes the D614G mutation. The nucleic acid vaccine
encoding the
S protein of SARS-CoV-2, a fragment or variant thereof may also include a
signal
peptide and/or at least one linker (e.g., GSG linker) sequence and one or more
sequences
in the nucleic acid vaccine may be codon optimized.
[0078] In some embodiments, the nucleic acid vaccine encodes the full
length M
protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a
fragment of the M protein of SARS-CoV-2. In some embodiments, the nucleic acid
vaccine encodes the topological domain (e.g., virion surface or intravirion
region) of the
M protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes
the
transmembrane domain of the M protein of SARS-CoV-2. The nucleic acid vaccine
encoding the M protein of SARS-CoV-2, a fragment or variant thereof may also
include a
signal peptide and/or at least one linker (e.g., GSG linker) sequence and one
or more
sequences in the nucleic acid vaccine may be codon optimized.
[0079] In some embodiments, the nucleic acid vaccine encodes the full
length N
protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a
fragment of the N protein of SARS-CoV-2. In some embodiments, the nucleic acid
vaccine encodes the RNA binding domain of the N protein of SARS-CoV-2. In some
embodiments, the nucleic acid vaccine encodes the dimerization domain of the N
protein
of SARS-CoV-2. The nucleic acid vaccine encoding the N protein of SARS-CoV-2,
a
fragment or variant thereof may also include a signal peptide and/or at least
one linker
(e.g., GSG linker) sequence and one or more sequences in the nucleic acid
vaccine may
be codon optimized.
- 18 -
Date Recue/Date Received 2021-04-23
[0080] In some embodiments, the nucleic acid vaccine encodes the full
length E
protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes a
fragment of the E protein of SARS-CoV-2. In some embodiments, the nucleic acid
vaccine encodes the topological domain (e.g., virion surface or intravirion
region) of the
E protein of SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes
the
transmembrane domain of the E protein of SARS-CoV-2. The nucleic acid vaccine
encoding the E protein of SARS-CoV-2, a fragment or variant thereof may also
include a
signal peptide and/or at least one linker (e.g., GSG linker) sequence and one
or more
sequences in the nucleic acid vaccine may be codon optimized.
[0081] In some embodiments, the nucleic acid vaccine encodes two different
structural proteins of SARS-CoV-2. In some embodiments, the nucleic acid
vaccine
encodes a S protein, fragment or variant thereof of SARS-CoV-2 and a M
protein,
fragment or variant thereof of SARS-CoV-2. In some embodiments, the nucleic
acid
vaccine encodes a S protein, fragment or variant thereof of SARS-CoV-2 and a N
protein,
fragment or variant thereof of SARS-CoV-2. In some embodiments, the nucleic
acid
vaccine encodes a S protein, fragment or variant thereof of SARS-CoV-2 and an
E
protein, fragment or variant thereof of SARS-CoV-2. In some embodiments, the
nucleic
acid vaccine encodes a M protein, fragment or variant thereof of SARS-CoV-2
and a N
protein, fragment or variant thereof of SARS-CoV-2. In some embodiments, the
nucleic
acid vaccine encodes a M protein, fragment or variant thereof of SARS-CoV-2
and an E
protein, fragment or variant thereof of SARS-CoV-2. In some embodiments, the
nucleic
acid vaccine encodes a N protein, fragment or variant thereof of SARS-CoV-2
and an E
protein, fragment or variant thereof of SARS-CoV-2. The nucleic acid vaccine
encoding
two different structural proteins, fragment or variant thereof of SARS-CoV-2,
may also
include a signal peptide and/or at least one linker (e.g., GSG linker)
sequence and one or
more sequences in the nucleic acid vaccine may be codon optimized.
[0082] In some embodiments, the nucleic acid vaccine encodes at least three
different
sequences of the structural proteins fragment or variant thereof for SARS-CoV-
2. In
some embodiments, the nucleic acid vaccine encodes two different S proteins,
fragments
- 19 -
Date Recue/Date Received 2021-04-23
or variants sequences for SARS-CoV-2 and a M protein, fragment or variant
sequence for
SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two
different S
proteins, fragments or variants sequences for SARS-CoV-2 and a N protein,
fragment or
variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine
encodes two different S proteins, fragments or variants sequences for SARS-CoV-
2 and
an E protein, fragment or variant sequence for SARS-CoV-2. In some
embodiments, the
nucleic acid vaccine encodes two different M proteins, fragments or variants
sequences
for SARS-CoV-2 and a S protein, fragment or variant sequence for SARS-CoV-2.
In
some embodiments, the nucleic acid vaccine encodes two different N proteins,
fragments
or variants sequences for SARS-CoV-2 and a S protein, fragment or variant
sequence for
SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two
different E
proteins, fragments or variants sequences for SARS-CoV-2 and a S protein,
fragment or
variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine
encodes two different M proteins, fragments or variants sequences for SARS-CoV-
2 and
a N protein, fragment or variant sequence for SARS-CoV-2. In some embodiments,
the
nucleic acid vaccine encodes two different M proteins, fragments or variants
sequences
for SARS-CoV-2 and an E protein, fragment or variant sequence for SARS-CoV-2.
In
some embodiments, the nucleic acid vaccine encodes two different N proteins,
fragments
or variants sequences for SARS-CoV-2 and a M protein, fragment or variant
sequence for
SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes two
different N
proteins, fragments or variants sequences for SARS-CoV-2 and an E protein,
fragment or
variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine
encodes two different E proteins, fragments or variants sequences for SARS-CoV-
2 and a
N protein, fragment or variant sequence for SARS-CoV-2. In some embodiments,
the
nucleic acid vaccine encodes a S protein, fragment or variant sequence for
SARS-CoV-2,
a M protein, fragment or variant sequence for SARS-CoV-2, and a N protein,
fragment or
variant sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine
encodes a S protein, fragment or variant sequence for SARS-CoV-2, a M protein,
fragment or variant sequence for SARS-CoV-2, and an E protein, fragment or
variant
- 20 -
Date Recue/Date Received 2021-04-23
sequence for SARS-CoV-2. In some embodiments, the nucleic acid vaccine encodes
a S
protein, fragment or variant sequence for SARS-CoV-2, a N protein, fragment or
variant
sequence for SARS-CoV-2, and an E protein, fragment or variant sequence for
SARS-
CoV-2. In some embodiments, the nucleic acid vaccine encodes a M protein,
fragment or
variant sequence for SARS-CoV-2, a N protein, fragment or variant sequence for
SARS-
CoV-2, and an E protein, fragment or variant sequence for SARS-CoV-2. The
nucleic
acid vaccine encoding at least three different sequences of the structural
proteins
fragment or variant thereof for SARS-CoV-2, may also include a signal peptide
and/or at
least one linker (e.g., GSG linker) sequence and one or more sequences in the
nucleic
acid vaccine may be codon optimized.
SARS-CoV-2 Variants
[0083] SARS-
CoV-2 is a member of the large coronavirus family of viruses. Multiple
variants (sometimes referred to as "strains" or "lineages") of SARS-CoV-2 have
been
identified globally. The nomenclature for SARS-CoV-2 variants used in this
description
is consistent with the PANGO nomenclature for new virus lineages (Rambaut,
Andrew,
et al. "A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist
genomic
epidemiology." Nature microbiology 5.11 (2020): 1403-1407, the contents of
which are
incorporated herein by reference in their entirety). Near real-time data
relating to
PANGO SARS-CoV-2 lineages or variants can be accessed online using
Nextstrain's
SARs-CoV-2 analysis user interface (nextstrain.org/ncov/global).
[0084] As of this filing, numerous PANGO lineage variants of SARS-CoV-2 have
been identified, including the following (number in parentheses represents
number of
cases per each submitted PANGO lineage: A (37); A.1 (8); A.11 (2); A.12 (1);
A.19 (5);
A.2 (6); A.2.2 (9); A.2.4 (5); A.2.5 (12); A.21 (8); A.22 (1); A.23 (2);
A.23.1 (40); A.24
(2); A.25 (1); A.28 (4); A.3 (3); A.5 (5); A.6 (1); AD.2 (1); AE.1 (1); AE.2
(2); AE.4 (1);
AE.5 (1); AE.7 (1); AE.8 (1); AG.1 (1); B (47); B.1 (374); B.1.1 (237);
B.1.1.1 (40);
B.1.1.10 (2); B.1.1.111 (2); B.1.1.121 (1); B.1.1.133 (2); B.1.1.141 (5);
B.1.1.142 (6);
B.1.1.153 (6); B.1.1.157 (1); B.1.1.159 (3); B.1.1.160 (1); B.1.1.161 (1);
B.1.1.163 (8);
B.1.1.170 (1); B.1.1.174 (1); B.1.1.176 (2); B.1.1.180 (1); B.1.1.186 (2);
B.1.1.189 (4);
- 21 -
Date Recue/Date Received 2021-04-23
B.1.1.198 (2); B.1.1.200 (1); B.1.1.204 (2); B.1.1.207 (6); B.1.1.214 (22);
B.1.1.216 (9);
B.1.1.219 (1); B.1.1.222 (32); B.1.1.226 (1); B.1.1.230 (1); B.1.1.231 (4);
B.1.1.232 (1);
B.1.1.241 (1); B.1.1.243 (1); B.1.1.25 (26); B.1.1.263 (2); B.1.1.265 (1);
B.1.1.27 (6);
B.1.1.273 (1); B.1.1.274 (7); B.1.1.28 (34); B.1.1.280 (3); B.1.1.284 (5);
B.1.1.294 (7);
B.1.1.297 (1); B.1.1.300 (1); B.1.1.301 (1); B.1.1.304 (1); B.1.1.306 (5);
B.1.1.312 (3);
B.1.1.315 (2); B.1.1.316 (4); B.1.1.317 (8); B.1.1.318 (1); B.1.1.326 (1);
B.1.1.328 (3);
B.1.1.33 (14); B.1.1.330 (6); B.1.1.331 (1); B.1.1.333 (4); B.1.1.337 (2);
B.1.1.344 (2);
B.1.1.345 (1); B.1.1.348 (29); B.1.1.350 (1); B.1.1.351 (2); B.1.1.354 (7);
B.1.1.355 (2);
B.1.1.359 (2); B.1.1.365 (1); B.1.1.366 (1); B.1.1.368 (1); B.1.1.37 (1);
B.1.1.372 (2);
B.1.1.374 (5); B.1.1.375 (9); B.1.1.381 (1); B.1.1.383 (1); B.1.1.388 (1);
B.1.1.389 (17);
B.1.1.39 (3); B.1.1.394 (3); B.1.1.397 (4); B.1.1.398 (2); B.1.1.40 (2);
B.1.1.404 (1);
B.1.1.410 (3); B.1.1.411 (3); B.1.1.413 (3); B.1.1.416 (6); B.1.1.419 (2);
B.1.1.420 (4);
B.1.1.428 (2); B.1.1.429 (2); B.1.1.430 (1); B.1.1.432 (8); B.1.1.434 (1);
B.1.1.447 (1);
B.1.1.448 (2); B.1.1.451 (1); B.1.1.464 (2); B.1.1.485 (1); B.1.1.487 (5);
B.1.1.50 (16);
B.1.1.514 (2); B.1.1.516 (2); B.1.1.517 (1); B.1.1.519 (106); B.1.1.521 (1);
B.1.1.54 (2);
B.1.1.56 (1); B.1.1.57 (1); B.1.1.63 (7); B.1.1.7 (534); B.1.1.70 (10);
B.1.1.71 (1);
B.1.1.99 (1); B.1.108 (1); B.1.110.3 (1); B.1.111 (29); B.1.116 (1); B.1.126
(2); B.1.128
(3); B.1.13 (1); B.1.139 (2); B.1.146 (1); B.1.149 (1); B.1.153 (2); B.1.160
(65);
B.1.160.14 (1); B.1.160.15 (1); B.1.160.25 (1); B.1.160.28 (1); B.1.160.8 (1);
B.1.160.9
(1); B.1.164 (2); B.1.170 (2); B.1.177 (71); B.1.177.11 (1); B.1.177.12 (1);
B.1.177.15
(1); B.1.177.18 (1); B.1.177.21 (7); B.1.177.32 (4); B.1.177.35 (1); B.1.177.4
(1);
B.1.177.40 (2); B.1.177.42 (1); B.1.177.43 (1); B.1.177.44 (2); B.1.177.46
(3);
B.1.177.49 (1); B.1.177.51 (1); B.1.177.52 (3); B.1.177.53 (1); B.1.177.54
(2);
B.1.177.59 (1); B.1.177.6 (1); B.1.177.60 (23); B.1.177.68 (1); B.1.177.73
(6);
B.1.177.76 (1); B.1.177.77 (1); B.1.177.78 (1); B.1.177.79 (1); B.1.177.81
(5);
B.1.177.82 (1); B.1.177.83 (1); B.1.177.86 (3); B.1.189 (2); B.1.192 (7);
B.1.195 (4);
B.1.2 (222); B.1.210 (2); B.1.214 (6); B.1.214.2 (1); B.1.219 (5); B.1.22 (3);
B.1.22.1
(16); B.1.220 (1); B.1.221 (27); B.1.221.1 (1); B.1.223 (1); B.1.229 (1);
B.1.23 (2);
B.1.232 (1); B.1.234 (20); B.1.236 (3); B.1.237 (3); B.1.240 (7); B.1.240.1
(14); B.1.241
- 22 -
Date Recue/Date Received 2021-04-23
(1); B.1.243 (34); B.1.256 (1); B.1.258 (61); B.1.258.11 (1); B.1.258.17 (17);
B.1.258.2
(1); B.1.258.22 (1); B.1.258.23 (1); B.1.260 (2); B.1.273 (1); B.1.277 (1);
B.1.279 (1);
B.1.281 (4); B.1.289 (1); B.1.291 (2); B.1.3 (1); B.1.306 (1); B.1.308 (1);
B.1.311 (5);
B.1.324 (1); B.1.329 (1); B.1.334 (1); B.1.338 (1); B.1.346 (1); B.1.349 (2);
B.1.351
(199); B.1.356 (3); B.1.357 (1); B.1.36 (56); B.1.36.1 (5); B.1.36.10 (2);
B.1.36.16 (33);
B.1.36.17 (1); B.1.36.18 (12); B.1.36.19 (1); B.1.36.21 (1); B.1.36.22 (6);
B.1.36.29 (6);
B.1.36.31 (5); B.1.36.34 (3); B.1.36.38 (1); B.1.36.8 (3); B.1.360 (1);
B.1.361 (3);
B.1.362 (8); B.1.367 (3); B.1.369 (12); B.1.369.1 (1); B.1.370 (1); B.1.371
(1); B.1.375
(1); B.1.379 (1); B.1.380 (9); B.1.393 (1); B.1.396 (2); B.1.398 (11); B.1.399
(1);
B.1.400 (4); B.1.404 (2); B.1.409 (5); B.1.411 (19); B.1.416 (16); B.1.420
(6); B.1.426
(1); B.1.427 (25); B.1.428 (4); B.1.429 (58); B.1.438 (4); B.1.441 (4);
B.1.451 (1);
B.1.456 (4); B.1.459 (15); B.1.462 (1); B.1.465 (1); B.1.466 (4); B.1.466.1
(1); B.1.466.2
(34); B.1.468 (7); B.1.469 (1); B.1.470 (11); B.1.471 (5); B.1.476 (1);
B.1.478 (1);
B.1.479 (1); B.1.480 (2); B.1.492 (1); B.1.497 (28); B.1.499 (24); B.1.504
(1); B.1.505
(1); B.1.509 (3); B.1.517 (6); B.1.517.1 (16); B.1.523 (3); B.1.524 (21);
B.1.525 (16);
B.1.526 (8); B.1.526.1 (6); B.1.526.2 (2); B.1.527 (2); B.1.530 (9); B.1.535
(2); B.1.540
(1); B.1.541 (1); B.1.544 (8); B.1.547 (1); B.1.551 (1); B.1.558 (3); B.1.560
(1); B.1.561
(6); B.1.564 (2); B.1.565 (6); B.1.568 (3); B.1.575 (4); B.1.576 (1); B.1.577
(5); B.1.581
(1); B.1.582 (7); B.1.587 (1); B.1.588 (4); B.1.595.4 (1); B.1.596 (17);
B.1.596.1 (1);
B.1.600 (8); B.1.603 (2); B.1.605 (1); B.1.609 (6); B.1.67 (1); B.1.84 (1);
B.1.91 (4);
B.1.94 (1); B.12 (1); B.27 (3); B.28 (1); B.3 (6); B.31 (2); B.35 (4); B.4
(13); B.4.1 (1);
B.4.2 (1); B.4.6 (2); B.4.7 (2); B.40 (3); B.42 (2); B.43 (1); B.45 (1); B.53
(2); B.55 (2);
B.56 (1); B.6 (23); B.6.3 (1); B.6.6 (5); B.6.7 (1); B.6.8 (29); C.1 (2);
C.1.1 (1); C.11 (5);
C.12 (3); C.13 (1); C.14 (4); C.16 (18); C.17 (2); C.18 (1); C.2 (5); C.2.1
(11); C.23 (2);
C.26 (5); C.29 (1); C.30 (1); C.32 (1); C.35 (10); C.36 (14); C.36.1 (1); C.4
(3); C.8 (2);
D.2 (33); L.3 (8); N.2 (1); N.3 (4); N.4 (14); N.5 (8); N.6 (3); N.7 (4); N.9
(4); P.1 (57);
P.2 (47); R.1 (9); S.1 (1); U.2 (1); U.3 (1); Y.1 (2); and Z.1 (1).
100851 From an epidemiological perspective, variants are typically
categorized as
Variants of Interest (VOIs), Variants of Concern (VOCs), and Variants of High
- 23 -
Date Recue/Date Received 2021-04-23
Consequence (VOHCs). For information relevant to categorizing specific
variants as
VOIs, VOCs, or VOHCs see, for example, cdc.gov/coronavirus/2019-ncov/cases-
updates/variant-surveillance/variant-info.html.
[0086] VOIs may have certain genetic markers associated with changes to
receptor
binding, reduced neutralization by antibodies generated against previous
infection or
vaccination, reduced efficacy of treatments, potential diagnostic impact, or
predicted
increase in transmissibility or disease severity. In some instances, VOIs have
specific
genetic markers that are predicted to affect transmission, diagnostics,
therapeutics, or
immune escape, or cause an increased proportion of cases or unique outbreak
clusters.
SARS-CoV-2 VOIs include, for example, PANGO lineage B.1.526, B.1.525, and P.2.
[0087] VOCs may include variants for which there is evidence of an increase
in
transmissibility, more severe disease (increased hospitalizations or deaths),
significant
reduction in neutralization by antibodies generated during previous infection
or
vaccination, reduced effectiveness of treatments or vaccines, or diagnostic
detection
failures. In some instances, VOCs have evidence of impact on diagnostics,
treatments,
and vaccines, widespread interference with diagnostic test targets, evidence
of
substantially increased resistance to one or more class of therapies, evidence
of
significant decreased neutralization by antibodies generated during previous
infection or
vaccination, evidence of reduced vaccine-induced protection from severe
disease,
evidence of increased transmissibility, or evidence of increased disease
severity. SARS-
CoV-2 VOCs may include, for example, PANGO lineage B.1.17, P.1, B.1.351,
B.1.427,
and B.1.429.
[0088] VOHCs may have clear evidence that prevention measures or medical
countermeasures (MCMs) have significantly reduced effectiveness relative to
previously
circulating variants. In some instances, VOHCs have impact on Medical
Countermeasures (MCM), demonstrated failure of diagnostics, evidence to
suggest a
significant reduction in vaccine effectiveness, a disproportionately high
number of
vaccine breakthrough cases, very low vaccine-induced protection against severe
disease,
- 24 -
Date Recue/Date Received 2021-04-23
significantly reduced susceptibility to multiple Emergency Use Authorization
(EUA) or
approved therapeutics, more severe clinical disease and increased
hospitalizations.
[0089] The nucleic acid vaccines disclosed herein may encode one or more
polypeptides, e.g., one or more proteins, peptides, fragments or variants
thereof, of the
SARS-CoV-2 variants described herein. In some embodiments, the nucleic acid
vaccines
disclosed herein may encode one or more polypeptides, e.g., one or more
proteins,
peptides, fragments or variants thereof, of a SARS-CoV-2 VOI, VOC, and/or
VOHC. In
some embodiments, the nucleic acid vaccines encode a polypeptide comprising
the
specific mutation called D614G.
[0090] In some embodiments, the nucleic acid vaccines encode one or more
polypeptide comprising one or more mutations or substitutions present in the
B.1.526
SARS-CoV-2 variant, such as one or more of: Spike protein substitutions L5F,
T95I,
D253G, 5477N, E484K, D614G, and/or A701V; ORFla substitutions L3201P, T265I,
and/or A3675/3677; ORF lb substitutions P314L and/or Q1011H; ORF3a
substitutions
P42L, Q57H; ORF8 substitution Till; and/or 5'UTR substitution R81C.
[0091] In some embodiments, the nucleic acid vaccines encode one or more
polypeptide comprising one or more mutations or substitutions present in the
B.1.525
SARS-CoV-2 variant, such as one or more of: Spike protein substitutions A67V,
A69/70,
A144, E484K, D614G, Q677H and/or F888L; ORF lb substitution P314F; ORFla
substitution T20071; M protein substitution I82T; N protein substitutions Al2G
and/or
T2051; and/or 5'UTR substitution R81C.
[0092] In some embodiments, the nucleic acid vaccines encode one or more
polypeptide comprising one or more mutations or substitutions present in the
P.2 SARS-
CoV-2 variant, such as one or more of: Spike protein substitutions E484K,
D614G,
and/or V1176F; ORFla substitutions L3468V and/or L3930F; ORFlb substitution
P314L; N protein substitutions Al 19S, R203K, G204R, and/or M234I; 5'UTR
substitution R81C.
[0093] In some embodiments, the nucleic acid vaccines encode one or more
polypeptide comprising one or more mutations or substitutions present in the
B.1.1.7
- 25 -
Date Recue/Date Received 2021-04-23
SARS-CoV-2 variant, such as one or more of: Spike protein substitutions
A69/70,
A144Y, E484K, S494P, N501Y, A570D, D614G, and/or P681H.
[0094] In some embodiments, the nucleic acid vaccines encode one or more
polypeptide comprising one or more mutations or substitutions present in the
P.1 SARS-
CoV-2 variant, such as one or more of: Spike protein substitutions K417N/T,
E484K,
N501Y, and/or D614G.
[0095] In some embodiments, the nucleic acid vaccines encode one or more
polypeptide comprising one or more mutations or substitutions present in the
B.1.351
SARS-CoV-2 variant, such as one or more of: Spike protein substitutions K417N,
E484K, N501Y, and/or D614G. The B.1.351 variant is also referred to as the
South
African variant, as it first originated in South Africa.
[0096] In some embodiments, the nucleic acid vaccines encode one or more
polypeptide comprising one or more mutations or substitutions present in the
B.1.427
SARS-CoV-2 variant, such as one or more of: Spike protein substitutions L452R
and/or
D614G.
[0097] In some embodiments, the nucleic acid vaccines encode one or more
polypeptide comprising one or more mutations or substitutions present in the
B.1.429
SARS-CoV-2 variant, such as one or more of: Spike protein substitutions S13I,
W152C,
L452R, and/or D614G.
[0098] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2
Spike
protein, e.g., protein, peptide, fragment, or variant, comprising one or more
substitutions
selected from: A570D, A67V, A701V, D253G, D614G, E484K, F888L, K417N/T,
L452R, L5F, N501Y, P681H, Q677H, S13I, 5477N, 5494P, T951, V1176F, W152C,
A144, A144Y, and A69/70.
[0099] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2
ORFla comprising one or more substitutions selected from: L3201P, T2651,
T20071,
L3468V, A3675/3677, and L3930F.
[0100] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2
ORFlb comprising one or more substitutions selected from: P314F, P314L, and
Q1011H.
- 26 -
Date Recue/Date Received 2021-04-23
[0101] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2
ORF3a comprising one or more substitutions selected from: P42L and Q57H.
[0102] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2 ORF8
comprising a T11I substitution.
[0103] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2
5'UTR comprising a R81C substitution.
[0104] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2 M
protein, e.g., protein, peptide, fragment, or variant, comprising I82T
substitution.
[0105] In some embodiments, the nucleic acid vaccines encode a SARS-CoV-2 N
protein, e.g., protein, peptide, fragment, or variant, comprising one or more
substitutions
selected from: Al2G, Al 19S, R203K, G204R, T2051, and M234I.
Components of Nucleic Acid Vaccines
[0106] In some embodiments, the polynucleotides described herein encode at
least one
polypeptide of interest, e.g., one or more proteins, peptides, fragments or
variants thereof
of SARS-CoV-2. The proteins, peptides, fragments or variants thereof of SARS-
CoV-2
of the present disclosure may be wild type where they are derived from the
infectious
agent, or modified (e.g., the structural proteins or fragments and variants
thereof are
engineered, designed or artificial). They may have any combination of the
features
described herein.
[0107] In some embodiments, provided herein are nucleic acid vaccines which
include
polynucleotides which, in some embodiments, encode one or more peptides or
polypeptides of interest. Such peptides or polypeptides are fragments or
variants thereof
of SARS-CoV-2 for the treatment and/or prevention of COVID-19. As a non-
limiting
example, these peptides or polypeptides may serve as an antigen or antigenic
molecule.
The term "nucleic acid," in its broadest sense, includes any compound and/or
substance
that comprise a polymer of nucleotides. These polymers are often referred to
as
polynucleotides.
[0108] Exemplary nucleic acids or polynucleotides include, but are not
limited to,
ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids
(TNAs),
- 27 -
Date Recue/Date Received 2021-04-23
glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic
acids (LNAs,
including LNA having a I3-D-ribo configuration, a-LNA having an a-L-ribo
configuration
(a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and
2'-
amino-a-LNA having a 2'-amino functionalization), ethylene nucleic acids
(ENA),
cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof.
[0109] In some embodiments, in vitro transcription (PIT) enzymatic
synthesis
methods may be used to make linear polynucleotides (referred to as "IVT
polynucleotides") encoding one or more proteins, peptides, fragments or
variants thereof
of SARS-CoV-2 of the present disclosure.
101101 In some embodiment, the nucleic acid vaccines may include "chimeric
polynucleotides" which have portions or regions which differ in size and/or
encoded
protein (e.g., structural protein of SARS-CoV-2). A "chimera" is an entity
having two or
more incongruous or heterogeneous parts or regions. As used herein a "part" or
"region"
of a polynucleotide is defined as any portion of the polynucleotide which is
less than the
entire length of the polynucleotide.
101111 In some embodiments, the nucleic acid vaccine includes from about 30
to
about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to
250, from 30
to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to
5,000, from 30
to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to
70,000,
from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from
100 to
3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to
25,000,
from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to
1,000, from
500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from
500 to
7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to
70,000,
from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to
3,000,
from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to
25,000,
from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500
to 3,000,
from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to
25,000,
from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000
to 3,000,
- 28 -
Date Recue/Date Received 2021-04-23
from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to
25,000,
from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000).
[0112] In some embodiments, the nucleic acid vaccine includes at least one
polynucleotide encoding at least one peptide or polypeptide of interest. In
another
embodiment, the polynucleotides may be non-coding.
[0113] In some embodiments, the length of a region encoding at least one
peptide
polypeptide of interest of the polynucleotides of the nucleic acid vaccine is
greater than
about 30 nucleotides in length (e.g., at least or greater than about 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, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000,
40,000,
50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000
nucleotides). As
used herein, such a region may be referred to as a "coding region" or "region
encoding."
[0114] In some embodiments, the polynucleotides of the nucleic acid vaccine
is or
functions as a messenger RNA (mRNA). As used herein, the term "messenger RNA"
(mRNA) refers to any polynucleotide which encodes at least one peptide or
polypeptide
of interest and which is capable of being translated to produce the encoded
peptide
polypeptide of interest in vitro, in vivo, in situ or ex vivo.
[0115] The shortest length of a region of the polynucleotide of the nucleic
acid
vaccine can be the length of a nucleic acid sequence that is sufficient to
encode for a
dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a
heptapeptide, an
octapeptide, a nonapeptide, or a decapeptide. In another embodiment, the
length may be
sufficient to encode a peptide of 2-30 amino acids, e.g. 5-30, 10-30, 2-25, 5-
25, 10-25, or
10-20 amino acids. The length may be sufficient to encode for a peptide of at
least 11, 12,
13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer than
40 amino
acids, e.g. no 1ongerthan35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino
acids.
Examples of dipeptides that the polynucleotide sequences can encode or
include, but are
not limited to, carnosine and anserine.
- 29 -
Date Recue/Date Received 2021-04-23
[0116] The region of the polynucleotide of the nucleic acid vaccine
encoding one or
more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for the
treatment
and/or prevention of COVID-19 may be greater than about 30 nucleotides in
length. The
length may be, but is not limited to, at least or greater than about 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, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000,
40,000,
50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000
nucleotides. In
some embodiments, the region includes from about 30 to about 100,000
nucleotides (e.g.,
from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to
1,000, from 30
to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to
10,000, from
30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100
to 500,
from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000,
from 100 to
7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to
70,000,
from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000,
from 500
to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500
to 25,000,
from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to
1,500, from
1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000,
from
1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to
70,000, from
1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to
7,000, from
1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to
70,000, from
1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to
7,000, from
2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to
70,000, and
from 2,000 to 100,000).
mRNA Components
[0117] The nucleic acid vaccines described herein may be an mRNA. In
general, an
mRNA molecule generally includes at least a coding region, a 5' untranslated
region
(UTR), a 3' UTR, a 5' cap and a poly-A tail.
- 30 -
Date Recue/Date Received 2021-04-23
mRNA Components: Start Codon and Stop Codon
[0118] In some embodiments, the mRNA includes a region to initiate
translation. This
region may include any translation initiation sequence or signal including a
Start codon.
As a non-limiting example, the region includes a Start codon. In some
embodiments, the
Start codon may be "ATG," "ACG," "AGG," "ATA," "ATT," "CTG," "GTG," "TTG,"
"AUG," "AUA," "AUU," "CUG," "GUG," or
[0119] In some embodiments, the mRNA includes a region to stop translation.
This
region may include any translation termination sequence or signal including a
Stop
codon. As a non-limiting example, the region includes a Stop codon. In some
embodiments, the Stop codon may be "TGA," "TAA," "TGA," "TAG," "UGA," "UAA,"
"UGA" or "UAG."
[0120] In some embodiments, the regions to initiate or terminate
translation may
independently range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30
or fewer
nucleotides in length. Additionally, these regions may comprise, in addition
to a Start
and/or Stop codon, one or more signal and/or restriction sequences.
[0121] In some embodiments, a masking agent may be used to mask a first
start codon
or alternative start codon in order to increase the chance that translation
will initiate on a
start codon or alternative start codon downstream to the masked start codon or
alternative
start codon.
[0122] In some embodiments, the start codon may be removed from the
polynucleotide sequence in order to have the translation of the polynucleotide
begin on a
codon which is not the start codon. Translation of the polynucleotide may
begin on the
codon following the removed start codon or on a downstream start codon or an
alternative start codon. The polynucleotide sequence where the start codon was
removed
may further comprise at least one masking agent for the downstream start codon
and/or
alternative start codons in order to control or attempt to control the
initiation of
translation, the length of the polynucleotide and/or the structure of the
polynucleotide.
-31 -
Date Recue/Date Received 2021-04-23
mRNA Components: Coding Region
[0123] In some embodiments, the coding region of the nucleic acid vaccine
may
encode at least one peptide or polypeptide of interest. Non-limiting examples
of peptides
or polypeptides of interest include one or more proteins, peptides, fragments
or variants
thereof of SARS-CoV-2 for the treatment and/or prevention of COVID-19.
mRNA Components: Untranslated Region
[0124] The polynucleotides of the nucleic acid vaccines described herein
may
comprise one or more regions or parts which act or function as an untranslated
region
(UTR). Wild type UTRs of a gene are transcribed but not translated. In mRNA,
the 5
'UTR starts at the transcription start site and continues to the start codon
but does not
include the start codon; whereas, the 3' UTR starts immediately following the
stop codon
and continues until the transcriptional termination signal. While not wishing
to be bound
by theory, UTRs may have a role in terms of stability and translation of the
nucleic acid
molecule and translation. Variants of UTRs may be utilized wherein one or more
nucleotides are added or removed to the termini, including A, T, C or G.
[0125] In some embodiments, the UTRs of the nucleic acid vaccine may range
independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40,
45, 50, 55,
60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500,
600, 700, 800,
and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120,
140, 160, 180,
200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).
[0126] Wild type 5' UTRs include features which play roles in translation
initiation as
these 5' UTRs include sequences such as Kozak sequences which are known to be
involved in how the ribosome initiates translation of many genes. 5' UTRs also
have been
known to form secondary structures which are involved in elongation factor
binding.
Other non-UTR sequences (e.g., introns or portions of intron sequences) may
also be
used as regions or subregions which may increase protein production as well as
polynucleotide levels.
[0127] Natural or wild type 3' UTRs are known to have stretches of Adenosines
and
Uridines embedded in them. These AU rich signatures are particularly prevalent
in genes
- 32 -
Date Recue/Date Received 2021-04-23
with high rates of turnover. Introduction, removal or modification of 3' UTR
AU rich
elements (AREs) can be used to modulate the stability of polynucleotides of
the nucleic
acid vaccines.
[0128] The UTR from any gene may be incorporated into the regions of the
polynucleotides of the nucleic acid vaccines. Alternatively, artificial UTRs,
which are not
variants of wild type regions, may also be used in the polynucleotides of the
nucleic acid
vaccines. These UTRs or portions thereof may be placed in the same orientation
as in the
transcript from which they were selected or may be altered in orientation or
location. As
used herein, the term "altered" as it relates to a UTR sequence, means that
the UTR has
been changed in some way in relation to a reference sequence. As a non-
limiting
example, a 5' or 3' UTR may be inverted, shortened, lengthened, made with one
or more
other 5' UTRs or 3' UTRs from a different parental sequence.
[0129] In some embodiments, flanking regions are selected from a family of
transcripts whose proteins share a common function, structure, feature of
property. For
example, polypeptides of interest may belong to a family of proteins which are
expressed
in a particular cell, tissue or at some time during development. The UTRs from
any of
these genes may be swapped for any other UTR of the same or different family
of
proteins to create a new polynucleotide. As used herein, a "family of
proteins" is used in
the broadest sense to refer to a group of two or more polypeptides of interest
which share
at least one function, structure, feature, localization, origin, or expression
pattern.
[0130] The polynucleotides of the nucleic acid vaccines disclosed herein
may
comprise a 5'UTR having a sequence of SEQ ID NO: 13. In some embodiments, the
5'
UTR of the polynucleotides of the nucleic acid vaccines disclosed herein
consist of the
nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the 5'UTR is
directly 5'
of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the
nucleic
acid vaccine. In some embodiments, the 5'UTR is 1, 2, 3, 4, 5, 6 or more
nucleotides 5'
of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the
nucleic
acid vaccine; i.e., a spacer sequence of 1, 2, 3, 4, 5, 6 or more nucleotides
separates the
5'UTR from the start codon of the sequence encoding the SARs-CoV-2 polypeptide
of
- 33 -
Date Recue/Date Received 2021-04-23
the nucleic acid vaccine. The polynucleotides of the nucleic acid vaccines
disclosed
herein may comprise a 5'UTR having a sequence with at least 80% sequence
identity to
the nucleic acid sequence of SEQ ID NO: 13. The polynucleotides of the nucleic
acid
vaccines disclosed herein may comprise a 5'UTR having a sequence with at least
85%
sequence identity to the nucleic acid sequence of SEQ ID NO: 13. The
polynucleotides of
the nucleic acid vaccines disclosed herein may comprise a 5'UTR having a
sequence with
at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 13.
The
polynucleotides of the nucleic acid vaccines disclosed herein may comprise a
5'UTR
having a sequence with at least 91% sequence identity to the nucleic acid
sequence of
SEQ ID NO: 13. The polynucleotides of the nucleic acid vaccines disclosed
herein may
comprise a 5'UTR having a sequence with at least 92% sequence identity to the
nucleic
acid sequence of SEQ ID NO: 13. The polynucleotides of the nucleic acid
vaccines
disclosed herein may comprise a 5'UTR having a sequence with at least 93%
sequence
identity to the nucleic acid sequence of SEQ ID NO: 13. The polynucleotides of
the
nucleic acid vaccines disclosed herein may comprise a 5'UTR having a sequence
with at
least 94% sequence identity to the nucleic acid sequence of SEQ ID NO: 13. The
polynucleotides of the nucleic acid vaccines disclosed herein may comprise a
5'UTR
having a sequence with at least 95% sequence identity to the nucleic acid
sequence of
SEQ ID NO: 13. The polynucleotides of the nucleic acid vaccines disclosed
herein may
comprise a 5'UTR having a sequence with at least 96% sequence identity to the
nucleic
acid sequence of SEQ ID NO: 13. The polynucleotides of the nucleic acid
vaccines
disclosed herein may comprise a 5'UTR having a sequence with at least 97%
sequence
identity to the nucleic acid sequence of SEQ ID NO: 13. The polynucleotides of
the
nucleic acid vaccines disclosed herein may comprise a 5'UTR having a sequence
with at
least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 13. The
polynucleotides of the nucleic acid vaccines disclosed herein may comprise a
5'UTR
having a sequence with at least 99% sequence identity to the nucleic acid
sequence of
SEQ ID NO: 13. The polynucleotides of the nucleic acid vaccines disclosed
herein may
- 34 -
Date Recue/Date Received 2021-04-23
comprise a 5'UTR having a sequence with at least 100% sequence identity to the
nucleic
acid sequence of SEQ ID NO: 13.
[0131] The polynucleotides of the nucleic acid vaccines disclosed herein
may
comprise a 3'UTR having a sequence of SEQ ID NO: 14. In some embodiments, the
3'
UTR of the polynucleotides of the nucleic acid vaccines disclosed herein
consist of the
nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the 3'UTR is
directly 3'
of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the
nucleic
acid vaccine. In some embodiments, the 3'UTR is 1, 2, 3, 4, 5, 6 or more
nucleotides 3'
of the start codon of the sequence encoding the SARs-CoV-2 polypeptide of the
nucleic
acid vaccine; i.e., a spacer sequence of 1, 2, 3, 4, 5, 6 or more nucleotides
separates the
3'UTR from the start codon of the sequence encoding the SARs-CoV-2 polypeptide
of
the nucleic acid vaccine. The polynucleotides of the nucleic acid vaccines
disclosed
herein may comprise a 3'UTR having a sequence with at least 80% sequence
identity to
the nucleic acid sequence of SEQ ID NO: 14. The polynucleotides of the nucleic
acid
vaccines disclosed herein may comprise a 3'UTR having a sequence with at least
85%
sequence identity to the nucleic acid sequence of SEQ ID NO: 14. The
polynucleotides of
the nucleic acid vaccines disclosed herein may comprise a 3'UTR having a
sequence with
at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.
The
polynucleotides of the nucleic acid vaccines disclosed herein may comprise a
3'UTR
having a sequence with at least 91% sequence identity to the nucleic acid
sequence of
SEQ ID NO: 14. The polynucleotides of the nucleic acid vaccines disclosed
herein may
comprise a 3'UTR having a sequence with at least 92% sequence identity to the
nucleic
acid sequence of SEQ ID NO: 14. The polynucleotides of the nucleic acid
vaccines
disclosed herein may comprise a 3'UTR having a sequence with at least 93%
sequence
identity to the nucleic acid sequence of SEQ ID NO: 14. The polynucleotides of
the
nucleic acid vaccines disclosed herein may comprise a 3'UTR having a sequence
with at
least 94% sequence identity to the nucleic acid sequence of SEQ ID NO: 14. The
polynucleotides of the nucleic acid vaccines disclosed herein may comprise a
3'UTR
having a sequence with at least 95% sequence identity to the nucleic acid
sequence of
- 35 -
Date Recue/Date Received 2021-04-23
SEQ ID NO: 14. The polynucleotides of the nucleic acid vaccines disclosed
herein may
comprise a 3'UTR having a sequence with at least 96% sequence identity to the
nucleic
acid sequence of SEQ ID NO: 14. The polynucleotides of the nucleic acid
vaccines
disclosed herein may comprise a 3'UTR having a sequence with at least 97%
sequence
identity to the nucleic acid sequence of SEQ ID NO: 14. The polynucleotides of
the
nucleic acid vaccines disclosed herein may comprise a 3'UTR having a sequence
with at
least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 14. The
polynucleotides of the nucleic acid vaccines disclosed herein may comprise a
3'UTR
having a sequence with at least 99% sequence identity to the nucleic acid
sequence of
SEQ ID NO: 14. The polynucleotides of the nucleic acid vaccines disclosed
herein may
comprise a 3'UTR having a sequence with at least 100% sequence identity to the
nucleic
acid sequence of SEQ ID NO: 14.
mRNA Components: Cap and IRES Sequences
[0132] The 5' cap structure of a natural mRNA is involved in nuclear
export,
increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which
is
responsible for mRNA stability in the cell and translation competency through
the
association of CBP with poly(A) binding protein to form the mature cyclic mRNA
species. The cap further assists the removal of 5' proximal introns removal
during mRNA
splicing.
[0133] In some embodiments, the capping region of the nucleic acid vaccine
may
comprise a single cap or a series of nucleotides forming the cap. The capping
region may
be from 1 to 10, e.g. 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer
nucleotides in
length. In some embodiments, the cap is absent.
[0134] In some embodiments, cap analogs, which herein are also referred to
as
synthetic cap analogs, chemical caps, chemical cap analogs, or structural or
functional
cap analogs may be used in the nucleic acid vaccines. Cap analogs, which may
be
chemically (e.g., non-enzymatically) or enzymatically synthesized, differ from
natural
(e.g., endogenous, wild-type or physiological) 5'-caps in their chemical
structure, but they
retain cap function.
- 36 -
Date Recue/Date Received 2021-04-23
[0135] In some embodiments, the 5' terminal caps of the polynucleotides of
the
nucleic acid vaccines may include endogenous caps or cap analogs. As a non-
limiting
example, 5' terminal caps may comprise a guanine analog. Useful guanine
analogs
include, but are not limited to, inosine, Ni-methyl-guanosine (m1G), 2'fluoro-
guanosine,
7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-
azido-
guanosine.
[0136] The 5' terminal capping region may comprise a single cap or a series
of
nucleotides forming the cap. In this embodiment the capping region may be from
1 to 10
nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. The 5' cap
structure of an
mRNA is involved in increasing mRNA stability and binding the mRNA Cap Binding
Protein (CBP), which is responsible for mRNA stability in the cell and
translation
competency through the association of CBP with poly(A) binding protein to form
the
mature cyclic mRNA species.
[0137] The skilled artisan will appreciate that 5' capping can be generated
via
enzymatic or other synthetic processes. Endogenous mRNA molecules are 5'-end
capped
generating a 5'-ppp-5'-triphosphate linkage between a terminal guanosine cap
residue and
the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-
guanylate
cap can then be methylated to generate an N7-methyl-guanylate residue. The
ribose
sugars of the terminal and/or ante-terminal transcribed nucleotides of the 5'
end of the
mRNA can optionally also be 2'-0-methylated. 5'-decapping through hydrolysis
and
cleavage of the guanylate cap structure can target a nucleic acid molecule,
such as an
mRNA molecule, for degradation.
[0138] Polynucleotides, e.g., mRNAs, of the present invention may be
modified to
include a non-hydrolyzable cap structure preventing decapping and thus
increasing
mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp-
5'
phosphorodiester linkages, modified nucleotides may be used during the capping
reaction. For example, a vaccinia virus capping enzyme available from, e.g.,
New
England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides
according to the manufacturer's instructions to create a phosphorothioate
linkage in the
- 37 -
Date Recue/Date Received 2021-04-23
5'-ppp-5' cap. Additional modified guanosine nucleotides may be used such as a-
methyl-
phosphonate and seleno-phosphate nucleotides.
[0139] Additional modifications include, but are not limited to, 2'-0-
methylation of
the ribose sugars of 5'-terminal and/or 51-ante-terminal nucleotides of the
mRNA (as
mentioned above) on the 2'-hydroxyl group of the sugar ring. Multiple distinct
5'-cap
structures can be used to generate the 5'-cap of a nucleic acid molecule, such
as an
mRNA molecule.
[0140] Cap analogs, which herein are also referred to as synthetic cap
analogs,
chemical caps, chemical cap analogs, or structural or functional cap analogs,
differ from
natural (i.e., endogenous, wild-type or physiological) 5'-caps in their
chemical structure,
while retaining cap function. Cap analogs may be chemically (i.e., non-
enzymatically) or
enzymatically synthesized and linked to a nucleic acid molecule.
[0141] For example, the Anti-Reverse Cap Analog (ARCA) cap contains two
guanines linked by a 5'-5'-triphosphate group, wherein one guanine contains an
N7
methyl group as well as a 3'-0-methyl group (i.e., N7,3'-0-dimethyl-guanosine-
5'-
triphosphate-5'-guanosine (m7G-3'mppp-G; which may equivalently be designated
3' 0-
Me-m7G(51)ppp(5')G). The 3'-0 atom of the other, unmodified, guanine becomes
linked
to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., an
mRNA). The
N7- and 31-0-methlyated guanine provide the terminal moiety of the capped
nucleic acid
molecule (e.g., mRNA).
[0142] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-0-
methyl group on guanosine (i.e., N7,21-0-dimethyl-guanosine-51-triphosphate-51-
guanosine, m7Gm-ppp-G).
[0143] While cap analogs allow for the concomitant capping of a nucleic
acid
molecule in an in vitro transcription reaction, up to 20% of transcripts can
remain
uncapped. This, as well as the structural differences of cap analogs from
endogenous 5'-
cap structures may lead to reduced translational competency and reduced
cellular
stability.
- 38 -
Date Recue/Date Received 2021-04-23
[0144] In exemplary aspects of the invention, polynucleotides, e.g., mRNAs,
can be
capped post-transcriptionally, using enzymes. For example, recombinant
Vaccinia Virus
Capping Enzyme and recombinant T-0-methyltransferase enzyme can create a
canonical
51-5'-triphosphate linkage between the 51-terminal nucleotide of an mRNA and a
guanine
cap nucleotide wherein the cap guanine contains an N7 methylation and the 5'-
terminal
nucleotide of the mRNA contains a T-0-methyl. Such a structure is termed the
Cap 1
structure. In some embodiments, the Cap 1 structure provides a higher
translational-
competency and cellular stability and a reduced activation of cellular pro-
inflammatory
cytokines, as compared, e.g., to other 5'cap analog structures known in the
art. Cap
structures include 7mG(5')ppp(5')N,pN2p (Cap 0), 7mG(51)ppp(5')NlmpNp (Cap 1),
and
7mG(5')-ppp(5')N1mpN2mp (Cap 2).
[0145] Because the polynucleotides, e.g., mRNA, may be capped post-
transcriptionally, and because this process is more efficient, up to 100% of
the
polynucleotides, e.g., mRNA, may be capped. This is in contrast to ¨80% when a
cap
analog is linked to an mRNA in the course of an in vitro transcription
reaction.
[0146] According to the present invention, 5' terminal caps may include
endogenous
caps or cap analogs. According to the present invention, a 5' terminal cap may
comprise a
guanine analog. Useful guanine analogs include inosine, Ni-methyl-guanosine,
2'fluoro-
guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-
guanosine,
and 2-azido-guanosine.
[0147] In some embodiments, the polynucleotides of the nucleic acid
vaccines may
contain an internal ribosome entry site (IRES) sequence. While not wishing to
be bound
by theory, IRES plays an important role in initiating protein synthesis in
absence of the 5'
cap structure. An IRES may act as the sole ribosome binding site, or may serve
as one of
multiple ribosome binding sites of an mRNA.
mRNA Components: Tailing Region
[0148] In some embodiments, the mRNA includes a tailing region. Non-liming
examples of a tailing region include a poly-A sequence, a poly-C sequence,
and/or a
polyA-G quartet.
- 39 -
Date Recue/Date Received 2021-04-23
[0149] In some embodiments the mRNA includes a chain terminating
nucleoside.
Non-limiting examples of chain terminating nucleosides include 2'-0 methyl, F
and
locked nucleic acids (LNA).
[0150] In some embodiments, the sequence of the tailing region of the
nucleic acid
vaccine may range from absent to 500 nucleotides in length (e.g., at least 60,
70, 80, 90,
120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). If the
tailing region
is a polyA tail, the length may be described in units of or as a function of
polyA Binding
Protein binding.
[0151] In some embodiments, polyA tails may also be added after the
construct is
exported from the nucleus.
[0152] In some embodiments, a long chain of adenine nucleotides (poly-A
tail) may
be added to a polynucleotide such as an mRNA molecule during RNA processing in
order to increase stability. Immediately after transcription, the 3' end of
the transcript may
be cleaved to free a 3' hydroxyl. Then poly-A polymerase adds a chain of
adenine
nucleotides to the RNA. The process, called polyadenylation, adds a poly-A
tail that can
be between, for example, approximately 80 to approximately 250 residues long,
including
approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,
210, 220,
230, 240 or 250 residues long.
[0153] In some embodiments, the length of a poly-A tail, when present, is
greater than
30 nucleotides in length (e.g., at least or greater than about 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 polynucleotide or region thereof
includes
from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100,
from 30 to
250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from
30 to
2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from
50 to 750,
from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from
50 to 3,000,
from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from
100 to
2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to
1,000, from
-40 -
Date Recue/Date Received 2021-04-23
500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from
1,000 to
1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from
1,500 to
2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from
2,000 to
2,500, and from 2,500 to 3,000).
[0154] In some embodiments, the poly-A tail is designed relative to the
length of the
overall polynucleotide or the length of a particular region of the
polynucleotide. This
design may be based on the length of a coding region, the length of a
particular feature or
region or based on the length of the ultimate product expressed from the
polynucleotides.
[0155] In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70,
80, 90, or
100% greater in length than the polynucleotide or feature thereof. The poly-A
tail may
also be designed as a fraction of the polynucleotides to which it belongs. In
this context,
the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the
total length of
the construct, a construct region or the total length of the construct minus
the poly-A tail.
Further, engineered binding sites and conjugation of polynucleotides for Poly-
A binding
protein may enhance expression.
Signal Sequences
[0156] In some embodiments, the polynucleotides of the nucleic acid
vaccines may
also encode additional features which may facilitate the trafficking of the
polypeptides to
therapeutically relevant sites. One such feature which aids in protein
trafficking is the
signal sequence. As used herein, a "signal sequence" or "signal peptide" is a
polynucleotide or polypeptide, respectively, which is from about 9 to 200
nucleotides (3-
60 amino acids) in length which is incorporated at the 5' (or N-termi- nus) of
the coding
region or polypeptide encoded, respectively. Addition of these sequences
result in
trafficking of the encoded polypeptide to the endoplasmic reticulum through
one or more
secretory pathways. Some signal peptides are cleaved from the protein by
signal
peptidase after the proteins are transported.
Codon Optimization
[0157] The polynucleotides of the nucleic acid vaccines, their regions or
parts or
subregions may be codon optimized. Codon optimization methods are known in the
art
- 41 -
Date Recue/Date Received 2021-04-23
and may be useful in efforts to achieve one or more of several goals. These
goals include,
but are not limited to, match codon frequencies in target and host organisms
to ensure
proper folding, alter GC content to increase mRNA stability or reduce
secondary
structures, minimize tandem repeat codons or base runs that may impair gene
construction or expression, customize transcriptional and translational
control regions,
insert or remove protein trafficking sequences, remove/add post translation
modification
sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle
protein
domains, insert or delete restriction sites, modify ribosome binding sites and
mRNA
degradation sites, to adjust translational rates to allow the various domains
of the protein
to fold properly, or to reduce or eliminate problem secondary structures
within the
polynucleotide. Codon optimization tools, algorithms and services are known in
the art,
non-limiting examples include, but are not limited to, services from GeneArt
(Life
Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods. In some
embodiments, the ORF sequence is optimized using optimization algorithms.
Codon
options for each amino acid are given in Table 4.
Table 4. Codon Options
Single Letter Amino Acid Name Codon Options
Nomenclature
A Alanine GCT, GCC, GCA, GCG
C Cysteine TGT, TGC
D Aspartic acid GAT, GAC
E Glutamic acid GAA, GAG
F Phenylalanine TTT, TTC
G Glycine GGT, GGC, GGA, GGG
H Histidine CAT, CAC
I Isoleucine ATT, ATC, ATA
K Lysine AAA, AAG
L Leucine CTT, CTC, CTA, CTG, TTA, TTG
M Methionine ATG
N Asparagine AAT, AAC
P Proline CCT, CCC, CCA, CCG
Q Glutamine CAA, CAG
R Arginine CGT, CGC, CGA, CGG, AGA, AGG
s Serine TCT, TCC, TCA, TCG, AGT, AGC
-42 -
Date Recue/Date Received 2021-04-23
UGA in mRNA in presence of
Sec Selenocysteine Selenocysteine insertion element
(SECTS)
Stop Stop codons TAA, TAG, TGA
T Threonine ACT, ACC, AC A, ACG
V Valine GTT, GTC, GTA, GTG
w Tryptophan TGG
7 Tyrosine TAT, TAC
[0158] In some embodiments, the nucleic acid vaccine is vectorized after
codon
optimization. Non-limiting examples of vectors include, but are not limited
to, plasmids,
viruses, cosmids, and artificial chromosomes.
Modifications
[0159] Nucleic acid vaccines of the present disclosure, including mRNA
vaccines,
may include one or more modifications. The terms "modification" or, as
appropriate,
"modified" refer to modification with respect to A, G, U or C ribonucleotides.
Generally,
herein, these terms are not intended to refer to the ribonucleotide
modifications in
naturally occurring 5'-terminal mRNA cap moieties. In a polypeptide, the term
"modification" refers to a modification as compared to the canonical set of 20
amino
acids.
[0160] As described herein "nucleoside" is defined as a compound containing
a sugar
molecule (e.g., a pentose or ribose) or a derivative thereof in combination
with an organic
base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to
herein as
"nucleobase"). As described herein, "nucleotide" is defined as a nucleoside
including a
phosphate group or other backbone linkage (internucleoside linkage).
[0161] The modifications may be various distinct modifications. In some
embodiments, the coding region(s), the untranslated region(s), the flanking
region(s),
and/or the terminal or tailing regions may contain one, two, or more
(optionally different)
nucleoside or nucleotide modifications. In some embodiments, nucleic acid
vaccines of
the present disclosure comprise one or more modifications which render the
nucleic acid
molecules, when introduced to a cell, more resistant to degradation in the
cell and/or
more stable in the cell as compared to unmodified polynucleotides.
-43 -
Date Recue/Date Received 2021-04-23
[0162] The polynucleotides can include any useful modification, such as to
the sugar,
the nucleobase, or the internucleoside linkage (e.g., to a linking phosphate /
to a
phosphodiester linkage / to the phosphodiester backbone). One or more atoms of
a
pyrimidine nucleobase may be replaced or substituted, for example, with
optionally
substituted amino, optionally substituted thiol, optionally substituted alkyl
(e.g., methyl
or ethyl), optionally substituted or halo (e.g., chloro or fluoro) atoms or
groups. In certain
embodiments, modifications (e.g., one or more modifications) are present in
each of the
sugar and the internucleoside linkage. Modifications according to the present
disclosure
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.
[0163] Modifications according to the present disclosure 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. In some embodiments, the modifications include 2'-0-
Methyl-modified or 2'-0-Methoxyethyl-modified nucleotides (2'-0Me and 2'-MOE
modifications, respectively).
[0164] In some embodiments, the polynucleotides of the present disclosure
may
comprise at least one modification described herein.
[0165] The polynucleotides of the present disclosure can include a
combination of
modifications to the sugar, the nucleobase, and/or the internucleoside
linkage.
[0166] Modifications of polynucleotides (e.g., RNA polynucleotides, such as
mRNA
polynucleotides) that are useful in the vaccines of the present disclosure
include, but are
not limited to, any modifications as described in PCT Publication
W02017070626, the
contents of which are incorporated herein by reference in their entirety,
including, for
example, modification or deletion of nucleotides (or codons) encoding one or
more N-
linked glycosylation site in a translated polypeptide. Modifications that are
useful in the
vaccines of the present disclosure may also comprise any modifications as
described in
-44 -
Date Recue/Date Received 2021-04-23
PCT Publication W02018200892, the contents of which are incorporated herein by
reference in their entirety. The vaccines of the present disclosure may
further comprise
features or modifications as described in PCT Publication W02020255063, PCT
Publication W02020182869, United States Publication 20200254086, United States
Publication 20200206362, United States Publication 20180311336, United States
Publication 20180303929, PCT Publication W02016011222, PCT Publication
W02016011226, PCT Publication W02016005004, PCT Publication W02016000792,
PCT Publication W02015176737, PCT Publication W02015085318, PCT Publication
W02015048744, and PCT Publication W02015034925, the contents of each of which
are incorporated herein by reference in their entireties.
[0167] For example, the polynucleotides, including mRNA vaccines described
herein,
can include modifications as follows. The internucleoside linkages of the
polynucleotides
may be partially or fully modified. The polynucleotides may comprise
modifications to
one or more nucleobases. The polynucleotides may comprise 5-methylcytosines in
place
of all cytosine nucleobases/cytidine nucleotides. Further the polynucleotides
may have
one or more modifications to one or more of the sugar subunits of a
nucleoside. The
sugar modification can be one or more locked nucleic acids (LNAs) or 2'-0-
Methoxyethyl-modified ("2'-MOE") modifications. The oligonucleotides can be
designed with a patterned array of sugar, nucleobase or linkage modifications.
In some
embodiments, the polynucleotides can comprise modifications to maximize
stability. In
some embodiments, the polynucleotides can be fully 2'-M0E-sugar modified.
Modified Nucleobases
[0168] The modified nucleosides and nucleotides can include a modified
nucleobase.
Examples of nucleobases found in RNA include, but are not limited to, adenine,
guanine,
cytosine, and uracil. Examples of nucleobases found in DNA include, but are
not limited
to, adenine, guanine, cytosine, and thymine.
[0169] In some embodiments, the modified nucleobase is a modified uracil.
Exemplary nucleobases and nucleosides having a modified uracil include
pseudouridine
(w), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-
uridine, 2-
-45 -
Date Recue/Date Received 2021-04-23
thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-
pseudouridine, 5-
hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-
uridine or 5-
bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-
oxyacetic
acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-
uridine
(cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U),
5-
carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-
uridine (mcm5U), 5-methoxycarbonylmethy1-2-thio-uridine (mcm5s2U), 5-
aminomethy1-
2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-
methylaminomethy1-
2-thio-uridine (mnm5s2U), 5-methylaminomethy1-2-seleno-uridine (mnm5se2U), 5-
carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U),
5-
carboxymethylaminomethy1-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-
propynyl-
pseudouridine, 5-taurinomethyl-uridine ('rm5U), 1-taurinomethyl-pseudouridine,
5-
taurinomethy1-2-thio-uridine(rm5s2U), 1-taurinomethy1-4-thio-pseudouridine, 5-
methyl-
uridine (m5U, i.e., having the nucleobase deoxythymine), 1-methylpseudouridine
(m1w),
5-methyl-2-thio-uridine (m 5 S2 t1), 1 -methy1-4-thio-pseudouridine (m' s4), 4-
thio-1-
methyl-pseudouridine, 3-methyl-pseudouridine (m3w), 2-thio-1-methyl-
pseudouridine, 1-
methyl-l-deaza-pseudouridine, 2-thio-1-methy1-1-deaza-pseudouridine,
dihydrouridine
(D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D),
2-thio-
dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-
thio-
uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-
pseudouridine (also known as 1-methylpseudouridine (m1w)), 3-(3-amino-3-
carboxypropyl)uridine (acp3U), 1-methy1-3-(3-amino-3-
carboxypropyl)pseudouridine
(acp3 w), 5-(isopentenylaminomethyl)uridine (inm5U), 5-
(isopentenylaminomethyl)-2-
thio-uridine (inm5s2U), a-thio-uridine, 21-0-methyl-uridine (Urn), 5,2'-0-
dimethyl-
uridine (m5Um), 21-0-methyl-pseudouri dine (m), 2-thio-21-0-methyl-uridine
(s2Um), 5-
methoxycarbonylmethy1-21-0-methyl-uridine (mcm5Um), 5-carbamoylmethy1-21-0-
methyl-uridine (ncm5Um), 5-carboxymethylaminomethy1-21-0-methyl-uridine
(cmnm5Um), 3,21-0-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-0-
methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-
F-uridine,
-46 -
Date Recue/Date Received 2021-04-23
2'-011-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-
propenylamino)uridine.
[0170] In some embodiments, the modified nucleobase is a modified cytosine.
Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-
cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-
acetyl-cytidine
(ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine
(m5C), 5-
halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-
methyl-
pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-
cytidine (s2C), 2-
thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-
pseudoisocytidine, 4-
thi o- 1 -methyl- 1 -deaza-ps eudoisocytidine, 1 -methyl- 1 -deaza-ps eudoi
socytidine,
zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-
thio-
zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-
pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), a-
thio-
cytidine, 21-0-methyl-cytidine (Cm), 5,2'-0-dimethyl-cytidine (m5Cm), N4-
acety1-21-0-
methyl-cytidine (ac4Cm), N4,2'-0-dimethyl-cytidine (m4Cm), 5-formy1-21-0-
methyl-
cytidine (f5Cm), N4,N4,21-0-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-
ara-
cytidine, 2'-F-cytidine, and 2'-011-ara-cytidine.
[0171] In some embodiments, the modified nucleobase is a modified adenine.
Exemplary nucleobases and nucleosides having a modified adenine include 2-
amino-
purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-
purine), 6-
halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-
adenosine, 7-
deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-
amino-
purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-
adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-
methylthio-
N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-
isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-
methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-
glycinylcarbamoyl-
adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-
on6t6A),
threonylcarbamoyl-adenosine 2-methylthio-N6-threonylcarbamoyl-adenosine
-47 -
Date Recue/Date Received 2021-04-23
(ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-
adenosine
(hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-
acetyl-
adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-
thio-
adenosine, 21-0-methyl-adenosine (Am), N6,21-0-dimethyl-adenosine (m6Am),
N6,N6,21-0-trimethyl-adenosine (m62Am), 1,21-0-dimethyl-adenosine (m 'Am), 2'-
0-
ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-
adenosine, 8-
azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-011-ara-adenosine, and
N6-(19-
amino-pentaoxanonadecy1)-adenosine.
[0172] In some embodiments, the modified nucleobase is a modified guanine.
Exemplary nucleobases and nucleosides having a modified guanine include
inosine (I), 1-
methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine
(imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW),
hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-
guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ),
mannosyl-
queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethy1-7-deaza-
guanosine (preQi), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-
guanosine, 6-thio-
7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-
thio-
7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine
(ml G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-
dimethyl-
guanosine (m2'7G), N2, N2,7-dimethyl-guanosine (m2,2,7u)z, \ ,
8-oxo-guanosine, 7-methyl-
8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-
dimethy1-6-thio-guanosine, a-thio-guanosine, 21-0-methyl-guanosine (Gm), N2-
methy1-
21-0-methyl-guanosine (m2Gm), N2,N2-dimethy1-21-0-methyl-guanosine (m22Gm), 1-
methy1-21-0-methyl-guanosine (m 'Gm), N2,7-dimethy1-21-0-methyl-guanosine
(m2'7Gm), 2'-0-methyl-inosine (Im), 1,21-0-dimethyl-inosine (mlIm), and 2'-0-
ribosylguanosine (phosphate) (Gr(p)).
[0173] The nucleobase of the nucleotide can be independently selected from
a purine,
a pyrimidine, a purine or pyrimidine analog. For example, the nucleobase can
each be
independently selected from adenine, cytosine, guanine, uracil, or
hypoxanthine. In
-48 -
Date Recue/Date Received 2021-04-23
another embodiment, the nucleobase can also include, for example, naturally-
occurring
and synthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines, 5-
methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-
aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-
propyl and
other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine
and 2-
thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and
thymine, 5-uracil
(pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-
thioalkyl, 8-
hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-
bromo, 5-
trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine
and 7-
methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-
deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-
d]pyrimidine, imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines, imidazo[4,5-
d]pyrazines,
thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine, pyridazine; and
1,3,5 triazine.
[0174]
Different sugar modifications, nucleotide modifications, and/or
internucleoside
linkages (e.g., backbone structures) may be introduced at various positions in
a
polynucleotide described herein. 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 a
polynucleotide such that the function of the polynucleotide is not
substantially decreased.
The polynucleotides of the present disclosure may contain 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, T/U or C) or any
intervening
percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to
60%,
from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to
20%,
from 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%
-49 -
Date Recue/Date Received 2021-04-23
to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to
100%).
[0175] In some embodiments, polynucleotides of the present disclosure may
be
modified to be a circular nucleic acid. The termini of the polynucleotides of
the present
disclosure may be linked by chemical reagents or enzymes, producing circular
polynucleotides that have no free ends. Circular polynucleotides are expected
to be more
stable than linear counterparts and to be resistant to digestion with
exonucleases. Circular
polynucleotides may further comprise other structural and/or chemical
modifications with
respect to A, G, T/U or C ribonucleotides/deoxyribonucleotides.
[0176] In some embodiments, the polynucleotides are at least 50% modified,
e.g., at
least 50% of the nucleotides are modified. In some embodiments, the
polynucleotides are
at least 75% modified, e.g., at least 75% of the nucleotides are modified. It
is to be
understood that since a nucleotide (sugar, base and phosphate moiety, e.g.,
linkage) may
each be modified, any modification to any portion of a nucleotide, or
nucleoside, will
constitute a modification.
[0177] In some embodiments, the polynucleotides are at least 10% modified
in only
one component of the nucleotide, with such component being the nucleobase,
sugar, or
linkage between nucleosides. For example, modifications may be made to at
least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the nucleobases, sugars, or
linkages of a polynucleotide described herein.
Sugar Modifications
[0178] The modified nucleosides and nucleotides which may be incorporated
into
polynucleotides (e.g., RNA or mRNA, as described herein), can be modified on
the sugar
of the ribonucleic acid. For example, the 2' hydroxyl group (OH) can be
modified or
replaced with a number of different substituents. Exemplary substitutions at
the 2'-
position include, but are not limited to, H, halo, optionally substituted C1-6
alkyl;
optionally substituted C1-6 alkoxy; optionally substituted C6-10 aryloxy;
optionally
substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy;
optionally
substituted C6-10 aryloxy; optionally substituted C6-10 aryl-C1-6 alkoxy,
optionally
- 50 -
Date Recue/Date Received 2021-04-23
substituted C1-12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any
described
herein); a polyethyleneglycol (PEG), -0(CH2CH20)nal2CH2OR, where R is H or
optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0
to 4, from 0 to
8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1
to 16, from 1
to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20,
from 4 to 8, from
4 to 10, from 4 to 16, and from 4 to 20); "locked" nucleic acids (LNA) in
which the 2'-
hydroxyl is connected by a C1-6 alkylene or C1-6 heteroalkylene bridge to the
4'-carbon
of the same ribose sugar, where exemplary bridges include methylene,
propylene, ether,
or amino bridges; aminoalkyl; aminoalkoxy; amino; and amino acid.
[0179] In some embodiments, a polynucleotide, such as a mRNA, disclosed
herein
comprises at least one sugar modification. Generally, RNA includes the sugar
group
ribose, which is a 5-membered ring having an oxygen. Exemplary, non-limiting
modified
nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or
alkylene,
such as methylene or ethylene); addition of a double bond (e.g., to replace
ribose with
cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-
membered
ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6-
or 7-
membered ring having an additional carbon or heteroatom, such as for
anhydrohexitol,
altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a
phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and "unlocked"
forms,
such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is
replaced by
glycol units attached to phosphodiester bonds), threose nucleic acid (TNA,
where ribose
is replace with a-L-threofuranosyl-(3'¨>2)) , and peptide nucleic acid (PNA,
where 2-
amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone).
The sugar
group can also contain one or more carbons that possess the opposite
stereochemical
configuration than that of the corresponding carbon in ribose. Thus,
polynucleotide
molecules as described herein, including mRNAs, can include nucleotides
containing,
e.g., arabinose, as the sugar.
[0180] Nonlimiting examples of the sugar modification may include the
modifications
provided in Table 5. The polynucleotides of the present disclosure can have
one or more
-51 -
Date Recue/Date Received 2021-04-23
nucleotides carrying a modification as provided in Table 5. In some
embodiments, each
of the nucleotides of a polynucleotide described herein carries any one of the
modifications as provided in Table 5, or none of the modifications as provided
in Table 5.
Table 5. Nucleotide Sugar Modifications
Nucleotide Structure Depiction
DNA
*
Pas*
0,..._
0
2'-0-Methyl (2'-0Me)
1117
..'/.....,10,....._. 1 -ae0:1 i- !
I'l 11
? OCI-13
2'F-RNA
IL.
Be se
--.
2 rr
t
2'F-ANA
1).
rA le
= 0 - r
ICcirinertivi
I
- 52 -
Date Recue/Date Received 2021-04-23
4'S-RNA
1)3'1/2 ,s;11150
0 OH
UNA
11/21311c101.3.
0 OH
i
LNA
111:kirisf:0,.,1001Base
6-----6
i
4'S-FANA
,1/2,,, Bine
lti.;s31
6.
- 53 -
Date Recue/Date Received 2021-04-23
2'-0-Methoxyethyl (2'-M0E)
Nictilitio?
0 0
1 'hl
õ. la
I
2'-0-Ally1 .
'
1131ciase
i
2'-0-Ethylamine
1/41) 0 .nto:iam lc
0 0.
1
NH2
2'-0-Cyanoethyl ,
Si
'1
04)
- 54 -
Date Recue/Date Received 2021-04-23
2'-0-Acetalester ti
0 l 0
i f'al
Ft
4'-C-aminomethy1-2'-0-methyl -.,... _
RNA -0 easo
0 OCIS
,
2'-azido
0.
0 No,
1
Methylene-cLNA
1 Bes
''..." .......,.?.._ L44
-- - ..
.,
N-Me0-amino BNA
'Is
0 ' N
4r 0 C his
- 55 -
Date Recue/Date Received 2021-04-23
N-Me-aminooxy BNA
140
,..Ø_.....F3 IrisT
.0 H
2',4'-BNANc[NMe] L,
,......-.00-.,6 12111/4õkt
MC
MI
.õe....:ieyoliase
11410
dr:
4.
ONA
IteN,(70.),carige
'
tc-DNA 111/2
0 H
Ja So
Z
$11
- 56 -
Date Recue/Date Received 2021-04-23
CeNA
Bass
Ct
ANA
4rsuir¨CD¨NBase
C(
HNA
0-whBase
14?
[0181]
[0182] In some embodiments, at least one of the 2' positions of the sugar
(OH in RNA
or H in DNA) of a nucleotide of the polynucleotides is substituted with -0Me,
referred to
as 2'-0Me. In some embodiments, at least one of the 2' positions of the sugar
(OH in
RNA or H in DNA) of a nucleotide of the polynucleotides is substituted with -
F, referred
to as 2'-F.
Internucleoside Linkages
[0183] The polynucleotides of the present disclosure can include any
modification to
the intemucleoside linkage (e.g., to a linking phosphate / to a phosphodiester
linkage / to
the phosphodiester backbone). 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 intemucleoside
linkage as
- 57 -
Date Recue/Date Received 2021-04-23
described herein. Examples of modified phosphate groups include, but are not
limited to,
phosphorothioate, methylphosphonates 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
methylene-phosphonates).
[0184] 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
polynucleotide
molecules are expected to also reduce the innate immune response through
weaker
binding/activation of cellular innate immune molecules.
[0185] In specific embodiments, a modified nucleoside includes an alpha-
thio-
nucleoside (e.g., 5'-041-thiophosphate)-adenosine, 5'-041-thiophosphate)-
cytidine (a-
thio-cytidine), 5'-041-thiophosphate)-guanosine, 5'-041-thiophosphate)-
uridine, or 5'-
041-thiophosphate)-pseudouridine).
[0186] In some embodiments, the polynucleotides comprise at least one
phosphorothioate linkage or methylphosphonate linkage between nucleotides.
[0187] In some embodiments, the polynucleotides comprise at least one 5'-
(E)-
vinylphosphonate (5'-E-VP), a phosphate mimic, as a modification.
Valency
[0188] Nucleic acid vaccines of the present disclosure may vary in their
valency.
"Valency" refers to the number of antigenic components in the nucleic acid
vaccine or
the polynucleotide of the nucleic acid vaccines. The antigenic components of
the nucleic
acid vaccine may be on the same polynucleotide or they may be on different
polynucleotides. In some embodiments, the nucleic acid vaccine may be
monovalent. In
some embodiments, the nucleic acid vaccine may be divalent. In some
embodiments, the
- 58 -
Date Recue/Date Received 2021-04-23
nucleic acid vaccine may be trivalent. In some embodiments, the nucleic acid
vaccine
may be multivalent which may comprise 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25 or more than 25 antigens or antigenic
moieties such as,
but not limited to, antigenic peptides. As a non-limiting example, antigenic
peptides may
be one or more fragments or variants of the structural proteins of SARS-CoV-2.
Synthesis
Enzymatic Methods
In Vitro Transcription-Enzymatic Synthesis
[0189] cDNA encoding the polynucleotides described herein may be
transcribed using
an in vitro transcription (IVT) system. The system typically comprises a
transcription
buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase.
The NTPs
may be manufactured in house, may be selected from a supplier, or may be
synthesized
as described herein. The NTPs may be selected from, but are not limited to,
those
described herein including natural and unnatural (modified) NTPs. The
polymerase may
be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase
and
mutant polymerases.
[0190] Any number of RNA polymerases or variants may be used in the synthesis
of
the polynucleotides of the nucleic acid vaccine described herein. RNA
polymerases may
be modified by inserting or deleting amino acids of the RNA polymerase
sequence.
[0191] Polynucleotide or nucleic acid synthesis reactions may be carried
out by
enzymatic methods utilizing polymerases. Polymerases catalyze the creation of
phosphodiester bonds between nucleotides in a polynucleotide or nucleic acid
chain.
Currently known DNA polymerases can be divided into different families based
on
amino acid sequence comparison and crystal structure analysis. DNA polymerase
I (pol I)
or A polymerase family, including the Klenow fragments of E. Coli, Bacillus
DNA
polymerase I, Thermus aquaticus (Taq) DNA polymerases, and the T7 RNA and DNA
polymerases, is among the best studied of these families. Another large family
is DNA
polymerase a (pol a) or B polymerase family, including all eukaryotic
replicating DNA
polymerases and polymerases from phages T4 and RB69. Although they employ
similar
- 59 -
Date Recue/Date Received 2021-04-23
catalytic mechanism, these families of polymerases differ in substrate
specificity,
substrate analog-incorporating efficiency, degree and rate for primer
extension, mode of
DNA synthesis, exonuclease activity, and sensitivity against inhibitors.
Solid-Phase Chemical Synthesis
[0192] In some embodiments, polynucleotides of the nucleic acid vaccines
described
herein may be manufactured in whole or in part using solid phase techniques.
Solid-phase
chemical synthesis of polynucleotides or nucleic acids is an automated method
wherein
molecules are immobilized on a solid support and synthesized step by step in a
reactant
solution. Impurities and excess reagents are washed away and no purification
is required
after each step. The automation of the process is amenable on a computer-
controlled
solid-phase synthesizer. Solid-phase synthesis allows rapid production of
polynucleotides
or nucleic acids in a relatively large scale that leads to the commercial
availability of
some polynucleotides or nucleic acids.
[0193] In some embodiments, automated solid-phase synthesis is used where
the chain
is synthesized in 3' to 5' direction. The hydroxyl group in the 3' end of a
nucleoside is
tethered to a solid support via a chemically cleavable or light-cleavable
linker. Activated
nucleoside monomers, such as 2'-deoxynucleosides (dA, dC, dG and dT),
ribonucleosides
(A, C, G, and U), or chemically modified nucleosides, are added to the support-
bound
nucleoside sequentially. At the end of the synthesis, a cleaving agent such as
ammonia or
ammonium hydroxide is added to remove all the protecting groups and release
the
polynucleotide chains from the solid support. Light may also be applied to
cleave the
polynucleotide chain. The product can then be further purified with high
pressure liquid
chromatography (HPLC) or electrophoresis.
Liquid Phase Chemical Synthesis
[0194] The synthesis of polynucleotides of the nucleic acid vaccines
described herein
by the sequential addition of monomer building blocks may be carried out in a
liquid
phase. A covalent bond is formed between the monomers or between a terminal
functional group of the growing chain and an incoming monomer. Functional
groups not
involved in the reaction must be temporarily protected. After the addition of
each
- 60 -
Date Recue/Date Received 2021-04-23
monomer building block, the reaction mixture has to be purified before adding
the next
monomer building block. The functional group at one terminal of the chain has
to be
deprotected to be able to react with the next monomer building blocks. A
liquid phase
synthesis is labor- and time-consuming and cannot not be automated. Despite
the
limitations, liquid phase synthesis is still useful in preparing short
polynucleotides in a
large scale. Because the system is homogenous, it does not require a large
excess of
reagents and is cost- effective in this respect.
Quantification and Purification
[0195] In some embodiments, the polynucleotides of the nucleic acid
vaccines
described herein may be quantified in exosomes or when derived from one or
more
bodily fluid. As used herein "bodily fluids" include peripheral blood, serum,
plasma,
ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow,
synovial fluid,
aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage
fluid,
semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal
matter, hair,
tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph,
chyme, chyle, bile,
interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal
secretion, stool
water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary
aspirates,
blastocyl cavity fluid, and umbilical cord blood. Alter natively, exosomes may
be
retrieved from an organ selected from the group consisting of lung, heart,
pancreas,
stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast,
prostate, brain,
esophagus, liver, and placenta.
[0196] In the exosome quantification method, a sample of not more than 2 mL
is
obtained from the subject and the exosomes isolated by size exclusion
chromatography,
density gradient centrifugation, differential centrifugation, nanomembrane
ultrafiltration,
immunosorbent capture, affinity purification, microfluidic separation, or
combinations
thereof. In the analysis, the level or concentration of a polynucleotide may
be an
expression level, presence, absence, truncation or alteration of the
administered construct.
It is advantageous to correlate the level with one or more clinical phenotypes
or with an
assay for a human disease biomarker. The assay may be performed using
construct
- 61 -
Date Recue/Date Received 2021-04-23
specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry,
electrophoresis, mass spectrometry, or combinations thereof while the exosomes
may be
isolated using immunohistochemical methods such as enzyme linked immunosorbent
assay (ELISA) methods. Exosomes may also be isolated by size exclusion
chromatography, density gradient centrifugation, differential centrifugation,
nanomembrane ultrafiltration, immunosorbent capture, affinity purification,
microfluidic
separation, or combinations thereof.
[0197] These methods afford the investigator the ability to monitor, in
real time, the
level of polynucleotides remaining or delivered. This is possible because the
polynucleotides described herein differ from the endogenous forms due to the
structural
modifications.
[0198] In some embodiments, the polynucleotide may be quantified using
methods
such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis). Anon-
limiting
example of a UV/Vis spectrometer is a NANODROPO spectrometer (ThermoFisher,
Waltham, Mass.). The quantified polynucleotide may be analyzed in order to
determine if
the polynucleotide may be of proper size, check that no degradation of the
polynucleotide
has occurred. Degradation of the polynucleotide may be checked by methods such
as, but
not limited to, agarose gel electrophoresis, HPLC based purification methods
such as, but
not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse
phase
HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC- HPLC), liquid
chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and
capillary
gel electrophoresis (CGE).
[0199] Purification of the polynucleotides of the nucleic acid vaccines
described
herein may include, but is not limited to, polynucleotide clean-up, quality
assurance and
quality control. Clean-up may be performed by methods known in the arts such
as, but
not limited to, AGEN- COURT beads (Beckman Coulter Genomics, Danvers, Mass.),
poly-T beads, LNATM oligo-T capture probes (EX- IQONO Inc, Vedbaek, Denmark)
or
HPLC based purification methods such as, but not limited to, strong anion
exchange
HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic
- 62 -
Date Recue/Date Received 2021-04-23
interaction HPLC (HIC-HPLC). The term "purified" when used in relation to a
polynucleotide such as a "purified polynucleotide" refers to one that is
separated from at
least one contaminant. As used herein, a "contaminant" is any substance which
makes
another unfit, impure or inferior. Thus, a purified polynucleotide (e.g., DNA
and RNA) is
present in a form or setting different from that in which it is found in
nature, or a form or
setting different from that which existed prior to subjecting it to a
treatment or
purification method.
[0200] A quality assurance and/or quality control check may be conducted
using
methods such as, but not limited to, gel electrophoresis, UV absorbance, or
analytical
HPLC.
III. PHARMACEUTICAL COMPOSITIONS AND DELIVERY
[0201] The nucleic acid vaccines described herein may be used as
therapeutic or
prophylactic agents. In some embodiments, the present disclosure provides
pharmaceutical compositions comprising at least one pharmaceutically
acceptable carrier
and a nucleic acid vaccine, i.e., a nucleic acid vaccine for COVID-19.
[0202] Provided herein are nucleic acid vaccines and pharmaceutical
composition
thereof which may be used in combination with one or more pharmaceutically
acceptable
excipients. Pharmaceutical compositions may optionally comprise one or more
additional
active substances, e.g. therapeutically and/or prophylactically active
substances.
Pharmaceutical compositions of the nucleic acid vaccines described herein may
be sterile
and/or pyrogen-free.
[0203] In some embodiments, compositions are administered to humans, human
patients or subjects. For the purposes of the present disclosure, the phrase
"active
ingredient" generally refers to the nucleic acid vaccines or the
polynucleotides contained
therein, e.g., polynucleotides encoding one or more proteins, peptides,
fragments or
variants thereof of SARS-CoV-2 for the treatment and/or prevention of COVID-
19, to be
delivered as described herein.
[0204] Although the descriptions of pharmaceutical compositions provided
herein are
principally directed to pharmaceutical compositions which are suitable for
administration
- 63 -
Date Recue/Date Received 2021-04-23
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 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. 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
[0205] Pharmaceutical formulations may additionally comprise a
pharmaceutically
acceptable excipient, which, as used herein, includes, but is not limited to,
any and all
solvents, dispersion media, diluents, or other liquid vehicles, dispersion or
suspension
aids, surface active agents, isotonic agents, thickening or emulsifying
agents,
preservatives, and the like, as suited to the particular dosage form desired.
Various
excipients for formulating pharmaceutical compositions and techniques for
preparing the
composition are known in the art (see Remington: The Science and Practice of
Pharmacy,
20 Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD,
2006;
incorporated herein by reference in its entirety). The use of a conventional
excipient
medium may be contemplated within the scope of the present disclosure, except
insofar
as any conventional excipient medium may be incompatible with a substance or
its
derivatives, such as by producing any undesirable biological effect or
otherwise
interacting in a deleterious manner with any other component(s) of the
pharmaceutical
composition.
[0206] In some embodiments, compositions are administered to humans, human
patients or subjects. For the purposes of the present disclosure, the phrase
"active
- 64 -
Date Recue/Date Received 2021-04-23
ingredient" generally refers to a nucleic acid vaccine composition to be
delivered as
described herein.
[0207] 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 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. 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.
[0208] 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
into a desired
single- or multi-dose unit.
[0209] A pharmaceutical composition in accordance with the disclosure may
be
prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a
plurality of
single unit doses. As used herein, a "unit dose" is discrete amount of the
pharmaceutical
composition comprising a predetermined amount of the active ingredient. The
amount of
the active ingredient is generally equal to the dosage of the active
ingredient which would
be administered to a subject and/or a convenient fraction of such a dosage
such as, for
example, one-half or one-third of such a dosage.
- 65 -
Date Recue/Date Received 2021-04-23
[0210] Relative amounts of the active ingredient, the pharmaceutically
acceptable
excipient, and/or any additional ingredients in a pharmaceutical composition
in
accordance with the disclosure will vary, depending upon the identity, size,
and/or
condition of the subject treated and further depending upon the route by which
the
composition is to be administered. By way of example, the composition may
comprise
between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%,
at
least 80% (w/w) active ingredient.
[0211] In some embodiments, the formulations described herein may contain
at least
one nucleic acid vaccine composition, e.g., nucleic acid vaccine for COVID-19.
As a
non-limiting example, the formulations may contain 1, 2, 3, 4 or 5 nucleic
acid vaccine
compositions with different sequences. In some embodiments, the formulation
contains at
least three nucleic acid vaccine compositions with different sequences. In
some
embodiments, the formulation contains at least five nucleic acid vaccine
compositions
with different sequences.
[0212] The nucleic acid vaccine compositions of the present disclosure can
be
formulated using one or more excipients to: (1) increase stability; (2)
increase cell
transfection; (3) permit the sustained or delayed release (e.g., from a depot
formulation of
the nucleic acid vaccine composition); (4) alter the biodistribution (e.g.,
target the nucleic
acid vaccine composition to specific tissues or cell types); (5) increase the
translation of
encoded protein in vivo; and/or (6) alter the release profile of encoded
protein in vivo.
[0213] In addition to traditional excipients such as any and all solvents,
dispersion
media, diluents, or other liquid vehicles, dispersion or suspension aids,
surface active
agents, isotonic agents, thickening or emulsifying agents, preservatives,
excipients of the
present disclosure can include, without limitation, lipidoids, liposomes,
lipid
nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides,
proteins, cells
transfected with nucleic acid vaccine compositions (e.g., for transplantation
into a
subject), hyaluronidase, nanoparticle mimics and combinations thereof.
Accordingly, the
formulations of the disclosure can include one or more excipients, each in an
amount that
together increases the stability of the nucleic acid vaccine compositions
and/or increases
- 66 -
Date Recue/Date Received 2021-04-23
cell transfection by the nucleic acid vaccine compositions. Further, the
nucleic acid
vaccine compositions of the present disclosure may be formulated using self-
assembled
nucleic acid nanoparticles. Pharmaceutically acceptable carriers, excipients,
and delivery
agents for nucleic acids that may be used in the formulation with the nucleic
acid vaccine
compositions of the present disclosure are disclosed in International
Publication WO
2013/090648, the contents of which are incorporated herein by reference in
their entirety.
Lipidoids
[0214] The synthesis of lipidoids has been extensively described and
formulations
containing these compounds are particularly suited for delivery of
oligonucleotides or
nucleic acids (see Mahon et al., Bioconjug Chem. 2010 21:1448-1454; Schroeder
et al., J
Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love
et al.,
Proc Natl Acad Sci U S A. 2010 107:1864-1869; Siegwart et al., Proc Natl Acad
Sci U S
A. 2011108:12996-3001; all of which are incorporated herein in their
entireties).
[0215] While these lipidoids have been used to effectively deliver double-
stranded
small interfering RNA molecules in rodents and non-human primates (see Akinc
et al.,
Nat Biotechnol. 2008 26:561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci U
S A.
2008 105:11915-11920; Akinc et al., Mol Ther. 2009 17:872-879; Love et al.,
Proc Natl
Acad Sci USA. 2010 107:1864-1869; Leuschner et al., Nat Biotechnol. 2011
29:1005-
1010; all of which is incorporated herein in their entirety), the present
disclosure
contemplates their formulation and use in delivering at least one
pharmaceutically
acceptable carrier, including nucleic acid vaccines. Complexes, micelles,
liposomes or
particles can be prepared containing these lipidoids and therefore, can result
in an
effective delivery of the nucleic acid vaccine compositions following the
injection of a
lipidoid formulation via localized and/or systemic routes of administration.
Lipidoid
complexes of nucleic acid vaccine compositions can be administered by various
means
including, but not limited to, intravenous (IV), intramuscular (IM),
subcutaneous (SC),
intraparenchymal (IPa), intrathecal (IT), or intracerebroventricular (ICV)
administration.
[0216] In vivo delivery of nucleic acids may be affected by many
parameters,
including, but not limited to, the formulation composition, nature of particle
PEGylation,
- 67 -
Date Recue/Date Received 2021-04-23
degree of loading, oligonucleotide to lipid ratio, and biophysical parameters
such as, but
not limited to, particle size (Akinc et al., Mol Ther. 2009 17:872-879; the
contents of
which are herein incorporated by reference in its entirety). As an example,
small changes
in the anchor chain length of poly(ethylene glycol) (PEG) lipids may result in
significant
effects on in vivo efficacy. Formulations with the different lipidoids,
including, but not
limited to penta[3-(1-laurylaminopropiony1)]-triethylenetetramine
hydrochloride (TETA¨
SLAP; aka 98N12-5, see Murugaiah etal., Analytical Biochemistry, 401:61
(2010); the
contents of which are herein incorporated by reference in its entirety), C12-
200
(including derivatives and variants), and MD1, can be tested for in vivo
activity.
[0217] The lipidoid referred to herein as "98N12-5" is disclosed by Akinc
et al., Mol
Ther. 2009 17:872-879 and the contents of which is incorporated by reference
in its
entirety.
[0218] The lipidoid referred to herein as "C12-200" is disclosed by Love et
al., Proc
Nat! Acad Sci U S A. 2010 107:1864-1869 and Liu and Huang, Molecular Therapy.
2010
669-670; the contents of both of which are herein incorporated by reference in
their
entirety. The lipidoid formulations can include particles comprising either 3
or 4 or more
components in addition to the nucleic acid vaccine compositions. As an
example,
formulations with certain lipidoids, include, but are not limited to, 98N12-5
and may
contain 42% lipidoid, 48% cholesterol and 10% PEG (C14 alkyl chain length). As
another example, formulations with certain lipidoids, include, but are not
limited to, C12-
200 and may contain 50% lipidoid, 10% disteroylphosphatidyl choline, 38.5%
cholesterol, and 1.5% PEG-DMG.
[0219] In some embodiments, nucleic acid vaccine compositions formulated
with a
lipidoid for systemic intravenous administration. For example, a final
optimized
intravenous formulation using nucleic acid vaccine compositions and comprising
a lipid
molar composition of 42% 98N12-5, 48% cholesterol, and 10% PEG-lipid with a
final
weight ratio of about 7.5 to 1 total lipid to nucleic acid vaccine
compositions and a C14
alkyl chain length on the PEG lipid, with a mean particle size of roughly 50-
60 nm, can
result in the distribution of the formulation to be greater than 90% to the
liver. (see,
- 68 -
Date Recue/Date Received 2021-04-23
Akinc etal., Mol Ther. 2009 17:872-879; the contents of which are herein
incorporated
by reference in its entirety). In another example, an intravenous formulation
using a C12-
200 (see published international application W02010129709, the contents of
which is
herein incorporated by reference in their entirety) lipidoid may have a molar
ratio of
50/10/38.5/1.5 of C12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG,
with a
weight ratio of 7 to 1 total lipid to nucleic acid and a mean particle size of
80 nm may be
effective to deliver nucleic acid vaccine compositions (see, Love et al., Proc
Nat! Acad
Sci U S A. 2010 107:1864-1869, the contents of which are herein incorporated
by
reference in its entirety).
[0220] In some embodiments, an MD1 lipidoid-containing formulation may be
used
to effectively deliver nucleic acid vaccine compositions to hepatocytes in
vivo. The
characteristics of optimized lipidoid formulations for intramuscular or
subcutaneous
routes may vary significantly depending on the target cell type and the
ability of
formulations to diffuse through the extracellular matrix into the blood
stream. While a
particle size of less than 150 nm may be desired for effective hepatocyte
delivery due to
the size of the endothelial fenestrae (see, Akinc et al., Mol Ther. 2009
17:872-879, the
contents of which are herein incorporated by reference in its entirety), use
of a lipidoid-
formulated nucleic acid vaccine compositions to deliver the formulation to
other cells
types including, but not limited to, endothelial cells, myeloid cells, and
muscle cells may
not be similarly size-limited.
[0221] Use of lipidoid formulations to deliver siRNA in vivo to other non-
hepatocyte
cells such as myeloid cells and endothelium has been reported (see Akinc et
al., Nat
Biotechnol. 2008 26:561-569; Leuschner etal., Nat Biotechnol. 2011 29:1005-
1010; Cho
etal. Adv. Funct. Mater. 2009 19:3112-3118; 8th International Judah Folkman
Conference, Cambridge, MA October 8-9, 2010; the contents of each of which is
herein
incorporated by reference in its entirety). Effective delivery to myeloid
cells, such as
monocytes, lipidoid formulations may have a similar component molar ratio.
Different
ratios of lipidoids and other components including, but not limited to,
disteroylphosphatidyl choline, cholesterol and PEG-DMG, may be used to
optimize the
- 69 -
Date Recue/Date Received 2021-04-23
formulation of nucleic acid vaccine compositions for delivery to different
cell types
including, but not limited to, hepatocytes, myeloid cells, muscle cells, etc.
For example,
the component molar ratio may include, but is not limited to, 50% C12-200, 10%
disteroylphosphatidyl choline, 38.5% cholesterol, and %1.5 PEG-DMG (see
Leuschner et
al., Nat Biotechnol 2011 29:1005-1010; the contents of which are herein
incorporated by
reference in its entirety). The use of lipidoid formulations for the localized
delivery of
nucleic acids to cells via either subcutaneous or intramuscular delivery, may
not require
all of the formulation components desired for systemic delivery, and as such
may
comprise only the lipidoid and nucleic acid vaccine compositions.
Liposomes, Lzpoplexes, and Lipid Nanoparticles
[0222] The nucleic acid vaccine compositions of the disclosure can be
formulated
using one or more liposomes, lipoplexes, or lipid nanoparticles. In some
embodiments,
pharmaceutical compositions of nucleic acid vaccine compositions include
liposomes.
Liposomes are artificially-prepared vesicles which may primarily be composed
of a lipid
bilayer and may be used as a delivery vehicle for the administration of
nutrients and
pharmaceutical formulations. Liposomes can be of different sizes such as, but
not limited
to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in
diameter and
may contain a series of concentric bilayers separated by narrow aqueous compai
intents, a
small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter,
and a
large unilamellar vesicle (LUV) which may be between 50 and 500 nm in
diameter.
Liposome design may include, but is not limited to, opsonins or ligands in
order to
improve the attachment of liposomes to unhealthy tissue or to activate events
such as, but
not limited to, endocytosis. Liposomes may contain a low or a high pH in order
to
improve the delivery of the pharmaceutical formulations.
[0223] The formation of liposomes may depend on the physicochemical
characteristics such as, but not limited to, the pharmaceutical formulation
entrapped and
the liposomal ingredients, the nature of the medium in which the lipid
vesicles are
dispersed, the effective concentration of the entrapped substance and its
potential
toxicity, any additional processes involved during the application and/or
delivery of the
- 70 -
Date Recue/Date Received 2021-04-23
vesicles, the optimization size, polydispersity and the shelf-life of the
vesicles for the
intended application, and the batch-to-batch reproducibility and possibility
of large-scale
production of safe and efficient liposomal products.
[0224] In some embodiments, pharmaceutical compositions described herein
may
include, without limitation, liposomes such as those formed from 1,2-
dioleyloxy-/V,N-
dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech
(Bothell, WA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-
dilinoley1-
4-(2-dimethylaminoethy1)41,3]-dioxolane (DLin-KC2-DMA), and MC3
(US20100324120; the contents of which are herein incorporated by reference in
its
entirety) and liposomes which may deliver small molecule drugs such as, but
not limited
to, DOXILO from Janssen Biotech, Inc. (Horsham, PA).
[0225] In some embodiments, pharmaceutical compositions described herein
may
include, without limitation, liposomes such as those formed from the synthesis
of
stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid
particle (SNALP)
that have been previously described and shown to be suitable for
oligonucleotide delivery
in vitro and in vivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang
et al. Gene
Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey
et al.,
Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441:111-114;
Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010
28:172-
176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene
Ther. 2008
19:125-132; the contents of each of which are incorporated herein in their
entireties). The
original manufacture method by Wheeler et al. was a detergent dialysis method,
which
was later improved by Jeffs et al. and is referred to as the spontaneous
vesicle formation
method. The liposome formulations may be composed of 3 to 4 lipid components
in
addition to the nucleic acid vaccine compositions. As a non-limiting example,
a liposome
can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl
choline
(DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-/V,N-dimethylaminopropane
(DODMA), as described by Jeffs et al. In another example, certain liposome
formulations
may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA,
and
- 71 -
Date Recue/Date Received 2021-04-23
30% cationic lipid, where the cationic lipid can be 1,2-distearloxy-/V,N-
dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-
dimethylaminopropane (DLenDMA), as described by Heyes et al. In another
example,
the nucleic acid-lipid particle may comprise a cationic lipid comprising from
about 50
mol % to about 85 mol % of the total lipid present in the particle; a non-
cationic lipid
comprising from about 13 mol % to about 49.5 mol % of the total lipid present
in the
particle; and a conjugated lipid that inhibits aggregation of particles
comprising from
about 0.5 mol % to about 2 mol % of the total lipid present in the particle as
described in
W02009127060 to Maclachlan et al, the contents of which are incorporated
herein by
reference in their entirety. In another example, the nucleic acid-lipid
particle may be any
nucleic acid-lipid particle disclosed in US2006008910 to Maclachlan et al.,
the contents
of which are incorporated herein by reference in their entirety. As a non-
limiting
example, the nucleic acid-lipid particle may comprise a cationic lipid of
Formula I, a non-
cationic lipid, and a conjugated lipid that inhibits aggregation of particles.
[0226] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated in a lipid vesicle which may have crosslinks
between
functionalized lipid bilayers.
[0227] In some embodiments, the liposome may contain a sugar-modified lipid
disclosed in US5595756 to Bally et al., the contents of which are incorporated
herein by
reference in their entirety. The lipid may be a ganglioside and cerebroside in
an amount
of about 10 mol percent.
[0228] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated in a liposome comprising a cationic lipid. The
liposome
may have a molar ratio of nitrogen atoms in the cationic lipid to the
phosphates in the
nucleic acid vaccine compositions (N:P ratio) of between 1:1 and 20:1 as
described in
International Publication No. W02013006825, the contents of which are herein
incorporated by reference in its entirety. In some embodiments, the liposome
may have a
N:P ratio of greater than 20:1 or less than 1:1.
- 72 -
Date Recue/Date Received 2021-04-23
[0229] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated in a lipid-polycation complex. The formation of
the lipid-
polycation complex may be accomplished by methods known in the art and/or as
described in U.S. Pub. No. 20120178702, the contents of which are herein
incorporated
by reference in its entirety. As a non-limiting example, the polycation may
include a
cationic peptide or a polypeptide such as, but not limited to, polylysine,
polyornithine
and/or polyarginine and the cationic peptides described in International Pub.
No.
W02012013326; herein incorporated by reference in its entirety. In some
embodiments,
the nucleic acid vaccine compositions may be formulated in a lipid-polycation
complex
which may further include a neutral lipid such as, but not limited to,
cholesterol or
dioleoyl phosphatidylethanolamine (DOPE).
[0230] The liposome formulation may be influenced by, but not limited to,
the
selection of the cationic lipid component, the degree of cationic lipid
saturation, the
nature of the PEGylation, ratio of all components and biophysical parameters
such as
size. In one example by Semple et al. (Semple et al. Nature Biotech. 2010
28:172-176;
the contents of which are herein incorporated by reference in its entirety),
the liposome
formulation was composed of 57.1 % cationic lipid, 7.1%
dipalmitoylphosphatidylcholine, 34.3 % cholesterol, and 1.4% PEG-c-DMA.
[0231] In some embodiments, the ratio of PEG in the lipid nanoparticle
(LNP)
formulations may be increased or decreased and/or the carbon chain length of
the PEG
lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or
biodistribution of the LNP formulations. As a non-limiting example, LNP
formulations
may contain 1-5% of the lipid molar ratio of PEG-c-DOMG as compared to the
cationic
lipid, DSPC and cholesterol. In some embodiments, the PEG-c-DOMG may be
replaced
with a PEG lipid such as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-
glycerol,
methoxypolyethylene glycol) or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol,
methoxypolyethylene glycol). The cationic lipid may be selected from any lipid
known in
the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-
KC2-DMA.
- 73 -
Date Recue/Date Received 2021-04-23
[0232] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated in a lipid nanoparticle such as the lipid
nanoparticles
described in International Publication No. W02012170930, the contents of which
are
herein incorporated by reference in its entirety.
[0233] In some embodiments, the cationic lipid which may be used in
formulations of
the present disclosure may be selected from, but not limited to, a cationic
lipid described
in International Publication Nos. W02012040184, W02011153120, W02011149733,
W02011090965, W02011043913, W02011022460, W02012061259, W02012054365,
W02012044638, W02010080724, W0201021865 and W02008103276, US Patent Nos.
7,893,302, 7,404,969 and 8,283,333 and US Patent Publication No. US20100036115
and
US20120202871; the contents of each of which is herein incorporated by
reference in
their entirety. The cationic lipid may be selected from, but not limited to,
formula A
described in International Publication Nos. W02012040184, W02011153120,
W02011149733, W02011090965, W02011043913, W02011022460, W02012061259,
W02012054365 and W02012044638; the contents of each of which is herein
incorporated by reference in their entirety. Alternatively, the cationic lipid
may be
selected from, but not limited to, formula CLI-CLXXIX of International
Publication No.
W02008103276, formula CLI-CLXXIX of US Patent No. 7,893,302, formula CLI-
CLXXXXII of US Patent No. 7,404,969 and formula I-VI of US Patent Publication
No.
U520100036115; the contents of each of which are herein incorporated by
reference in
their entirety. The cationic lipid may be a multivalent cationic lipid such as
the cationic
lipid disclosed in US Patent No. 7223887 to Gaucheron et al., the contents of
which are
incorporated herein by reference in their entirety. The cationic lipid may
have a
positively-charged head group including two quaternary amine groups and a
hydrophobic
portion including four hydrocarbon chains as described in US Patent No.
7223887 to
Gaucheron et al., the contents of which are incorporated herein by reference
in their
entirety. The cationic lipid may be biodegradable as the biodegradable lipids
disclosed in
U520130195920 to Maier et al., the contents of which are incorporated herein
by
reference in their entirety. The cationic lipid may have one or more
biodegradable groups
- 74 -
Date Recue/Date Received 2021-04-23
located in a lipidic moiety of the cationic lipid as described in formula I-TV
in US
20130195920 to Maier et al., the contents of which are incorporated herein by
reference
in their entirety.
[0234] As a
non-limiting example, the cationic lipid may be selected from (20Z,23Z)-
N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimemylhexacosa-17,20-
dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-1 6, 19-dien-8-amine, (13Z,16Z)-
N,N-
dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylhenicosa-12,15-dien-4-
amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N-
dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-
dien-
10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-
dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)-N,N-dimeihyloctacosa-19,22-dien-
9-
amine, (18Z,21 Z)-N,N-dimethylheptacosa- 18 ,21 -dien-8 ¨amine, (17Z,20Z)-N,N-
dimethylhexacosa- 17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-
dien-
6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21 Z ,24Z)-
N,N-
dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimetylheptacos-18-en-10-
amine,
(17Z)-N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-
dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-
methylnonacosa-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicosa-11,14-dien-l-yl]
pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-1 0-amine, (15Z)-N,N-dimethyl
eptacos-
15-en-1 0-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)-N,N-
dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine,
(20Z)-
N,N-dimethylnonacos-20-en-1 0-amine, (22Z)-N,N-dimethylhentriacont-22-en-10-
amine,
(16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethy1-2-
nonylhenicosa-
12,15-dien-1¨amine, (13Z,16Z)-N,N-dimethy1-3-nonyldocosa-13,16-dien-l¨amine,
N,N-
dimethy1-1-[(1S,2R)-2-octylcyclopropyl] eptadecan-8-amine, 1-[(1S,2R)-2-
hexylcyclopropy1]-N,N-dimethylnonadecan-10-amine, N,N-dimethy1-1-[(1S ,2R)-2-
octylcyclopropyl]nonadecan-10-amine, N,N-dimethy1-21-[(1S,2R)-2-
octylcyclopropyl]henicosan-10-amine,N,N-dimethy1-1-[(1S,25)-2- {[(1R,2R)-2-
pentylcycIopropyl]methylIcyclopropyl]nonadecan-10-amine,N,N-dimethy1-1-
[(1S,2R)-
- 75 -
Date Recue/Date Received 2021-04-23
2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(1R,2S)-2-
undecyIcyclopropyl]tetradecan-5-amine, N,N-dimethy1-3- {7- [(1S,2R)-2-
octylcyclopropyl]heptyll dodecan- 1¨amine, 1-[(1R,2S)-2-hepty lcyclopropy1]-
N,N-
dimethyloctadecan-9¨amine, 1-[(1S,2R)-2-decylcyclopropy1]-N,N-dimethylpentadec
an-
6-amine, N,N-dimethy1-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, R-N,N-
dimethy1-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, S-
N,N-
dimethy1-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-
{2-
[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyll pyrrolidine,
(2S)-N,N-
dimethyl-1- [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3- [(5Z)-oct-5-en-1-
yloxy]propan-2-
amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyll
azetidine,
(2S)-1-(hexyloxy)-N,N-dimethy1-3- [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-
2-
amine, (2S)-1-(heptyloxy)-N,N-dimethy1-3-[(9Z,12Z)-octadeca-9,12-dien-1-
yloxy]propan-2-amine, N,N-dim ethy1-1 -(nonyl oxy)-3 - [(9Z,12Z)-octadec a-
9,12-di en-1 -
yl oxy]propan-2-ami ne, N,N-dimethy1-1-[(9Z)-octadec-9-en-1-yloxy]-3-
(octyloxy)propan-2-amine; (2S)-N,N-dimethy1-1-[(6Z,9Z,12Z)-octadeca-6,9,12-
trien-1-
yloxy]-3-(octyloxy)propan-2-amine, (2S)-1 -[(11Z,14Z)-icosa-11,14-di en-1 -
yloxy]-N,N-
dimethy1-3 -(penty1oxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-
11,14-
dien-l-yloxy]-N,N-dimethylpropan-2-amine, 1 -[(11Z,14Z)-icosa-11,14-dien- 1 -
yloxy]-
N,N-dimethy1-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-
yloxy]-
N,N-dimethy1-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-
yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docos-13-en-l-
yloxy]-
3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docos-13-en-l-yloxy]-N,N-
dimethy1-3-(octyloxy)propan-2-amine, 1- [(9Z)-hexadec-9-en-1-yloxy]-N,N-
dimethy1-3-
(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-metoylo ctyl)oxy]-3-[(9Z,12Z)-
octadeca-9,12-dien-l-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-
dimethy1-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethy1-1-
(octyloxy)-3-({8-[(1S,2S)-2- { [(1R,2R)-2-
pentylcyclopropyl]methyl 1 cyclopropyl]octyll oxy)propan-2-amine, N,N-dimethy1-
1- {[8-
(2-oc1y1cyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11E,20Z,23Z)-
N,N-
- 76 -
Date Recue/Date Received 2021-04-23
dimethylnonacosa-11,20,2-trien-10-amine or a pharmaceutically acceptable salt
or
stereoisomer thereof.
[0235] In some embodiments, the lipid may be a cleavable lipid such as
those
described in International Publication No. W02012170889, the contents of which
are
herein incorporated by reference in their entirety.
[0236] In some embodiments, the nanoparticles described herein may comprise
at
least one cationic polymer described herein and/or known in the art.
[0237] In some embodiments, the cationic lipid may be synthesized by
methods
known in the art and/or as described in International Publication Nos.
W02012040184,
W02011153120, W02011149733, W02011090965, W02011043913, W02011022460,
W02012061259, W02012054365, W02012044638, W02010080724 and
W0201021865; the contents of each of which is herein incorporated by reference
in their
entirety.
[0238] In some embodiments, the LNP formulations of the nucleic acid
vaccine
compositions may contain PEG-c-DOMG at 3% lipid molar ratio. In some
embodiments,
the LNP formulations of the nucleic acid vaccine compositions may contain PEG-
c-
DOMG at 1.5% lipid molar ratio.
[0239] In some embodiments, the pharmaceutical compositions of the nucleic
acid
vaccine compositions may include at least one of the PEGylated lipids
described in
International Publication No. 2012099755, the contents of which is herein
incorporated
by reference in its entirety.
[0240] In some embodiments, the LNP formulation may contain PEG-DMG 2000
(1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethylene
glycol)-
2000). In some embodiments, the LNP formulation may contain PEG-DMG 2000, a
cationic lipid known in the art and at least one other component. In some
embodiments,
the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the
art,
DSPC and cholesterol. As a non-limiting example, the LNP formulation may
contain
PEG-DMG 2000, DLin-DMA, DSPC and cholesterol. As another non-limiting example
the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol
- 77 -
Date Recue/Date Received 2021-04-23
in a molar ratio of 2:40:10:48 (see e.g., Geall etal., Non-viral delivery of
self-amplifying
RNA vaccines, PNAS 2012; PMID: 22908294; herein incorporated by reference in
its
entirety). As another non-limiting example, the nucleic acid vaccine
compositions
described herein may be formulated in a nanoparticle to be delivered by a
parenteral route
as described in U.S. Pub. No. 20120207845; the contents of which is herein
incorporated
by reference in its entirety. The cationic lipid may also be the cationic
lipids disclosed in
US20130156845 to Manoharan et al. and US 20130129785 to Manoharan et al., WO
2012047656 to Wasan et al., WO 2010144740 to Chen et al., WO 2013086322 to
Ansell
etal., or WO 2012016184 to Manoharan etal., the contents of each of which are
incorporated herein by reference in their entirety.
[0241] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated with a plurality of cationic lipids, such as a
first and a
second cationic lipid as described in US20130017223 to Hope et al., the
contents of
which are incorporated herein by reference in their entirety. The first
cationic lipid can be
selected on the basis of a first property and the second cationic lipid can be
selected on
the basis of a second property, where the properties may be determined as
outlined in
U520130017223, the contents of which are herein incorporated by reference in
its
entirety. In some embodiments, the first and second properties are
complementary.
[0242] The nucleic acid vaccine compositions described herein may be
formulated
with a lipid particle comprising one or more cationic lipids and one or more
second
lipids, and one or more nucleic acids, wherein the lipid particle comprises a
solid core, as
described in US Patent Publication No. US20120276209 to Cullis et al., the
contents of
which are incorporated herein by reference in their entirety.
[0243] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be complexed with a cationic amphiphile in an oil-in-water
(o/w)
emulsion such as described in EP2298358 to Satishchandran et al., the contents
of which
are incorporated herein by reference in their entirety. The cationic
amphiphile may be a
cationic lipid, modified or unmodified spermine, bupivacaine, or benzalkonium
chloride
and the oil may be a vegetable or an animal oil. As a non-limiting example, at
least 10%
- 78 -
Date Recue/Date Received 2021-04-23
of the nucleic acid-cationic amphiphile complex is in the oil phase of the oil-
in-water
emulsion (see e.g., the complex described in European Publication No.
EP2298358 to
Satishchandran et al., the contents of which are herein incorporated by
reference in its
entirety).
[0244] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated with a composition comprising a mixture of
cationic
compounds and neutral lipids. As a non-limiting example, the cationic
compounds may
be formula (I) disclosed in WO 1999010390 to Anse11 et al., the contents of
which are
described herein by reference in their entirety, and the neutral lipid may be
selected from
the group consisting of diacylphosphatidylcholine,
diacylphosphatidylethanolamine,
ceramide and sphingomyelin. In another non-limiting example, the lipid
formulation may
comprise a cationic lipid of formula A, a neutral lipid, a sterol and a PEG or
PEG-
modified lipid disclosed in US 20120101148 to Akinc et al., the contents of
which are
incorporated herein by reference in their entirety.
[0245] In some embodiments, the LNP formulation may be formulated by the
methods described in International Publication Nos. W02011127255 or
W02008103276,
each of which are herein incorporated by reference in their entirety. As a non-
limiting
example, the nucleic acid vaccine compositions of the present disclosure may
be
encapsulated in any of the lipid nanoparticle (LNP) formulations described in
W02011127255 and/or W02008103276; the contents of each of which are herein
incorporated by reference in their entirety.
[0246] In some embodiments, the LNP formulations described herein may
comprise a
polycationic composition. As a non-limiting example, the polycationic
composition may
be selected from formula 1-60 of US Patent Publication No. U520050222064; the
contents of which is herein incorporated by reference in its entirety. The LNP
formulations comprising a polycationic composition may be used for the
delivery of the
nucleic acid vaccine compositions described herein in vivo and/or in vitro.
[0247] In some embodiments, the LNP formulations described herein may
additionally comprise a permeability enhancer molecule. Non-limiting
permeability
- 79 -
Date Recue/Date Received 2021-04-23
enhancer molecules are described in US Patent Publication No. US20050222064;
the
contents of which is herein incorporated by reference in its entirety.
[0248] In some embodiments, the pharmaceutical compositions may be
formulated in
liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech,
Bothell, WA),
SMARTICLESO/N0V340 (Marina Biotech, Bothell, WA), neutral DOPC (1,2-dioleoyl-
sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian
cancer
(Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); the contents of
which
is herein incorporated by reference in its entirety) and hyaluronan-coated
liposomes
(Quiet Therapeutics, Israel).
[0249] In some embodiments, the pharmaceutical compositions may be
formulated
with any amphoteric liposome disclosed in WO 2008/043575 to Panzner and US
8580297 to Essler et al. (Marina Biotech), the contents of which are
incorporated herein
by reference in their entirety. The amphoteric liposome may comprise a mixture
of lipids
including a cationic amphiphile, an anionic amphiphile and optional one or
more neutral
amphiphiles. The amphoteric liposome may comprise amphoteric compounds based
on
amphiphilic molecules, the head groups of which being substituted with one or
more
amphoteric groups. In some embodiments, the pharmaceutical compositions may be
formulated with an amphoteric lipid comprising one or more amphoteric groups
having
an isoelectric point between 4 and 9, as disclosed in US 20140227345 to Essler
et al.
(Marina Biotech), the contents of which are incorporated herein by reference
in their
entirety.
[0250] In some embodiments, the pharmaceutical composition may be
formulated
with liposomes comprising a sterol derivative as disclosed in US 7312206 to
Panzner et
al. (Novosom), the contents of which are incorporated herein by reference in
their
entirety. In some embodiments, the pharmaceutical composition may be
formulated with
amphoteric liposomes comprising at least one amphipathic cationic lipid, at
least one
amphipathic anionic lipid, and at least one neutral lipid, or liposomes
comprise at least
one amphipathic lipid with both a positive and a negative charge, and at least
one neutral
lipid, wherein the liposomes are stable at pH 4.2 and pH 7.5, as disclosed in
US Pat. No.
- 80 -
Date Recue/Date Received 2021-04-23
7780983 to Panzner et al. (Novosom), the contents of which are incorporated
herein by
reference in their entirety. In some embodiments, the pharmaceutical
composition may be
formulated with liposomes comprising a serum-stable mixture of lipids taught
in US
20110076322 to Panzner et al, the contents of which are incorporated herein by
reference
in their entirety, capable of encapsulating the nucleic acid vaccine
compositions of the
present disclosure. The lipid mixture comprises phosphatidylcholine and
phosphatidylethanolamine in a ratio in the range of about 0.5 to about 8. The
lipid
mixture may also include pH sensitive anionic and cationic amphiphiles, such
that the
mixture is amphoteric, being negatively charged or neutral at pH 7.4 and
positively
charged at pH 4. The drug/lipid ratio may be adjusted to target the liposomes
to particular
organs or other sites in the body. In some embodiments, liposomes loaded with
the
nucleic acid vaccine compositions of the present disclosure as cargo, are
prepared by the
method disclosed in US 20120021042 to Panzner et al., the contents of which
are
incorporated herein by reference in their entirety. The method comprises steps
of
admixing an aqueous solution of a polyanionic active agent and an alcoholic
solution of
one or more amphiphiles and buffering said admixture to an acidic pH, wherein
the one
or more amphiphiles are susceptible of forming amphoteric liposomes at the
acidic pH,
thereby to form amphoteric liposomes in suspension encapsulating the active
agent.
[0251] The nanoparticle formulations may be a carbohydrate nanoparticle
comprising
a carbohydrate carrier and a nucleic acid vaccine composition (e.g., a nucleic
acid
vaccine for COVID-19). As a non-limiting example, the carbohydrate carrier may
include, but is not limited to, an anhydride-modified phytoglycogen or
glycogen-type
material, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin,
anhydride-
modified phytoglycogen beta-dextrin. (See e.g., International Publication No.
W02012109121; the contents of which is herein incorporated by reference in its
entirety).
[0252] Lipid nanoparticle formulations may be improved by replacing the
cationic
lipid with a biodegradable cationic lipid which is known as a rapidly
eliminated lipid
nanoparticle (reLNP). Ionizable cationic lipids, such as, but not limited to,
DLinDMA,
- 81 -
Date Recue/Date Received 2021-04-23
DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and
tissues over time and may be a potential source of toxicity. The rapid
metabolism of the
rapidly eliminated lipids can improve the tolerability and therapeutic index
of the lipid
nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose
in rat.
Inclusion of an enzymatically degraded ester linkage can improve the
degradation and
metabolism profile of the cationic component, while still maintaining the
activity of the
reLNP formulation. The ester linkage can be internally located within the
lipid chain or it
may be terminally located at the terminal end of the lipid chain. The internal
ester linkage
may replace any carbon in the lipid chain.
[0253] In some embodiments, the nucleic acid vaccine compositions may be
formulated as a lipoplex, such as, without limitation, the ATUPLEXTm system,
the
DACC system, the DBTC system and other siRNA-lipoplex technology from Silence
Therapeutics (London, United Kingdom), STEMFECTTm from STEMGENTO
(Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and
non-
targeted delivery of nucleic acids (Aleku et al. Cancer Res. 2008 68:9788-
9798;
Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene
Ther 2006
13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm
Pharmacol. Ther. 2010 23:334-344; Kaufmann et al. Microvasc Res 2010 80:286-
293Weide et al. J Immunother. 2009 32:498-507; Weide et al. J Immunother. 2008
31:180-188; Pascolo Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et
al., 2011 J.
Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et
al., Proc
Natl Acad Sci U S A. 2007 6;104:4095-4100; deFougerolles Hum Gene Ther. 2008
19:125-132; the contents of each of which are incorporated herein by reference
in its
entirety).
[0254] In some embodiments such formulations may also be constructed or
compositions altered such that they passively or actively are directed to
different cell
types in vivo, including but not limited to immune cells, endothelial cells,
antigen
presenting cells, and leukocytes (Akinc et al. Mol Ther. 2010 18:1357-1364;
Song et al.,
Nat Biotechnol. 2005 23:709-717; Judge et al., J Clin Invest. 2009 119:661-
673;
- 82 -
Date Recue/Date Received 2021-04-23
Kaufmann et al., Microvasc Res 2010 80:286-293; Santel et al., Gene Ther 2006
13:1222-1234; Santel etal., Gene Ther 2006 13:1360-1370; Gutbier etal., Pulm
Pharmacol. Ther. 2010 23:334-344; Basha etal., Mol. Ther. 201119:2186-2200;
Fenske
and Cullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer etal., Science. 2008
319:627-
630; Peer and Lieberman, Gene Ther. 201118:1127-1133; the contents of each of
which
are incorporated herein by reference in its entirety). One example of passive
targeting of
formulations to liver cells includes the DLin-DMA, DLin-KC2-DMA and DLin-MC3-
DMA-based lipid nanoparticle formulations which have been shown to bind to
apolipoprotein E and promote binding and uptake of these formulations into
hepatocytes
in vivo (Akinc etal. Mol Ther. 2010 18:1357-1364; the contents of which is
herein
incorporated by reference in its entirety). Formulations can also be
selectively targeted
through expression of different ligands on their surface as exemplified by,
but not limited
by, folate, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeted
approaches (Kolhatkar etal., Curr Drug Discov Technol. 2011 8:197-206;
Musacchio and
Torchilin, Front Biosci. 201116:1388-1412; Yu et al., Mol Membr Biol. 2010
27:286-
298; Patil etal., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit etal.,
Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv.
2008
5:309-319; Akinc etal., Mol Ther. 2010 18:1357-1364; Srinivasan etal., Methods
Mol
Biol. 2012 820:105-116; Ben-Arie etal., Methods Mol Biol. 2012 757:497-507;
Peer
2010 J Control Release. 20:63-68; Peer et al., Proc Nat! Acad Sci U S A. 2007
104:4095-
4100; Kim etal., Methods Mol Biol. 2011 721:339-353; Subramanya etal., Mol
Ther.
2010 18:2028-2037; Song etal., Nat Biotechnol. 2005 23:709-717; Peer etal.,
Science.
2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; the
contents of
each of which are incorporated herein by reference in its entirety).
[0255] In
some embodiments, the nucleic acid vaccine compositions is formulated as
a solid lipid nanoparticle. A solid lipid nanoparticle (SLN) may be spherical
with an
average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix
that can
solubilize lipophilic molecules and may be stabilized with surfactants and/or
emulsifiers.
The lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see
Zhang et
- 83 -
Date Recue/Date Received 2021-04-23
al., ACS Nano, 2008, 2 (8), pp 1696-1702; the contents of which are herein
incorporated
by reference in its entirety).
[0256] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure can be formulated for controlled release and/or targeted delivery.
As used
herein, "controlled release" refers to a pharmaceutical composition or
compound release
profile that conforms to a particular pattern of release to affect a
therapeutic outcome. In
some embodiments, the nucleic acid vaccine compositions may be encapsulated
into a
delivery agent described herein and/or known in the art for controlled release
and/or
targeted delivery. As used herein, the term "encapsulate" means to enclose,
surround or
encase. As it relates to the formulation of the compounds of the disclosure,
encapsulation
may be substantial, complete or partial. The term "substantially encapsulated"
means that
at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9
or greater than
99.999% of the pharmaceutical composition or compound of the disclosure may be
enclosed, surrounded or encased within the delivery agent. "Partially
encapsulated"
means that less than 10, 10, 20, 30, 40 50 or less of the pharmaceutical
composition or
compound of the disclosure may be enclosed, surrounded or encased within the
delivery
agent. Advantageously, encapsulation may be determined by measuring the escape
or the
activity of the pharmaceutical composition or compound of the disclosure using
fluorescence and/or electron micrograph. For example, at least 1, 5, 10, 20,
30, 40, 50,
60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of
the
pharmaceutical composition or compound of the disclosure are encapsulated in
the
delivery agent.
[0257] The nucleic acid vaccine compositions may be encapsulated into a
lipid
nanoparticle or a rapidly eliminated lipid nanoparticle and the lipid
nanoparticles or a
rapidly eliminated lipid nanoparticle may then be encapsulated into a polymer,
hydrogel
and/or surgical sealant described herein and/or known in the art. As a non-
limiting
example, the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl
acetate
(EVAc), poloxamer, GELSITEO (Nanotherapeutics, Inc. Alachua, FL), HYLENEXO
(Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen
polymers
- 84 -
Date Recue/Date Received 2021-04-23
(Ethicon Inc. Cornelia, GA), TISSELLO (Baxter International, Inc., Deerfield,
IL), PEG-
based sealants, and COSEALO (Baxter International, Inc., Deerfield, IL).
[0258] In some embodiments, the lipid nanoparticle may be encapsulated into
any
polymer known in the art which may form a gel when injected into a subject. As
another
non-limiting example, the lipid nanoparticle may be encapsulated into a
polymer matrix
which may be biodegradable.
[0259] In some embodiments, the nucleic acid vaccine compositions
formulation for
controlled release and/or targeted delivery may also include at least one
controlled release
coating. Controlled release coatings include, but are not limited to, OPADRYO,
polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone,
hydroxypropyl
methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT
RLO,
EUDRAGIT RS and cellulose derivatives such as ethylcellulose aqueous
dispersions
(AQUACOATO and SURELEASEO).
[0260] In some embodiments, the controlled release and/or targeted delivery
formulation may comprise at least one degradable polyester which may contain
polycationic side chains. Degradable polyesters include, but are not limited
to,
poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline
ester), and
combinations thereof. In some embodiments, the degradable polyesters may
include a
PEG conjugation to form a PEGylated polymer.
[0261] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated with a targeting lipid with a targeting moiety
such as the
targeting moieties disclosed in US20130202652 to Manoharan et al., the
contents of
which are incorporated herein by reference in their entirety. As a non-
limiting example,
the targeting moiety of formula I of US 20130202652 to Manoharan et al. may
selected in
order to favor the lipid being localized with a desired organ, tissue, cell,
cell type or
subtype, or organelle. Non-limiting targeting moieties that are contemplated
in the
present disclosure include transferrin, anisamide, an RGD peptide, prostate
specific
membrane antigen (PSMA), fucose, an antibody, or an aptamer.
- 85 -
Date Recue/Date Received 2021-04-23
[0262] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be encapsulated in a therapeutic nanoparticle. Therapeutic
nanoparticles
may be formulated by methods described herein and known in the art such as,
but not
limited to, International Pub Nos. W02010005740, W02010030763, W02010005721,
W02010005723, W02012054923, US Pub. Nos. US20110262491, US20100104645,
US20100087337, US20100068285, US20110274759, US20100068286 and
US20120288541 and US Pat No. 8,206,747, 8,293,276, 8,318,208 and 8,318,211;
the
contents of each of which are herein incorporated by reference in their
entirety.
Therapeutic polymer nanoparticles may be identified by the methods described
in US Pub
No. US20120140790, the contents of which are herein incorporated by reference
in its
entirety.
[0263] In some embodiments, the therapeutic nanoparticle may be formulated
for
sustained release. As used herein, "sustained release" refers to a
pharmaceutical
composition or compound that conforms to a release rate over a specific period
of time.
The period of time may include, but is not limited to, hours, days, weeks,
months and
years. As a non-limiting example, the sustained release nanoparticle may
comprise a
polymer and a therapeutic agent such as, but not limited to, the nucleic acid
vaccine
compositions of the present disclosure (see International Pub No. 2010075072
and US
Pub No. U520100216804, U520110217377 and U520120201859, the contents of each
of
which are herein incorporated by reference in their entirety).
[0264] In some embodiments, the therapeutic nanoparticles may be formulated
to be
target specific. As a non-limiting example, the therapeutic nanoparticles may
include a
corticosteroid (see International Pub. No. W02011084518; the contents of which
are
herein incorporated by reference in its entirety). In some embodiments, the
therapeutic
nanoparticles may be formulated to be cancer specific. As a non-limiting
example, the
therapeutic nanoparticles may be formulated in nanoparticles described in
International
Pub No. W02008121949, W02010005726, W02010005725, W02011084521 and US
Pub No. U520100069426, U520120004293 and U520100104655, the contents of each
of
which are herein incorporated by reference in their entirety.
- 86 -
Date Recue/Date Received 2021-04-23
[0265] In some embodiments, the nanoparticles of the present disclosure may
comprise a polymeric matrix. As a non-limiting example, the nanoparticle may
comprise
two or more polymers such as, but not limited to, polyethylenes,
polycarbonates,
polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones,
polyamides,
polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates,
polyvinyl
alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,
polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,
poly(ethylene
imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-
proline ester)
or combinations thereof.
[0266] In some embodiments, the therapeutic nanoparticle comprises a
diblock
copolymer. In some embodiments, the diblock copolymer may include PEG in
combination with a polymer such as, but not limited to, polyethylenes,
polycarbonates,
polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones,
polyamides,
polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates,
polyvinyl
alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,
polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,
poly(ethylene
imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-
proline ester)
or combinations thereof.
[0267] As a non-limiting example, the therapeutic nanoparticle comprises a
PLGA-
PEG block copolymer (see US Pub. No. US20120004293 and US Pat No. 8,236,330,
each of which is herein incorporated by reference in their entirety). In
another non-
limiting example, the therapeutic nanoparticle is a stealth nanoparticle
comprising a
diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No 8,246,968 and
International Publication No. W02012166923, the contents of each of which is
herein
incorporated by reference in its entirety).
[0268] In some embodiments, the therapeutic nanoparticle may comprise a
multiblock
copolymer such as, but not limited to the multiblock copolymers described in
U.S. Pat.
No. 8,263,665 and 8,287,910; the contents of each of which are herein
incorporated by
reference in its entirety.
- 87 -
Date Recue/Date Received 2021-04-23
[0269] In some embodiments, the block copolymers described herein may be
included
in a polyion complex comprising a non-polymeric micelle and the block
copolymer. (See
e.g., U.S. Pub. No. 20120076836; the contents of which are herein incorporated
by
reference in its entirety).
[0270] In some embodiments, the nanoparticles for delivery of the nucleic
acid
vaccines described herein include block co-polymers. Non-limiting examples of
block
co-polymers include those of formula I, formula II, formula III, formula IV,
formula V,
formula VI and formula VII of International PCT Publication No. W02015017519,
the
contents of which are herein incorporated by reference in their entirety.
[0271] In some embodiments, the therapeutic nanoparticle may comprise at
least one
acrylic polymer. Acrylic polymers include but are not limited to, acrylic
acid, methacrylic
acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate
copolymers,
ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate
copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and
combinations thereof.
[0272] In some embodiments, the therapeutic nanoparticles may comprise at
least one
amine-containing polymer such as, but not limited to polylysine, polyethylene
imine,
poly(amidoamine) dendrimers, poly(beta-amino esters) (See e.g., U.S. Pat. No.
8,287,849; the contents of which are herein incorporated by reference in its
entirety) and
combinations thereof.
[0273] In some embodiments, the therapeutic nanoparticles may comprise at
least one
degradable polyester which may contain polycationic side chains. Degradable
polyesters
include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-
lysine), poly(4-
hydroxy-L-proline ester), and combinations thereof. The degradable polyesters
may
include a PEG conjugation to form a PEGylated polymer.
[0274] In some embodiments, the therapeutic nanoparticle may include a
conjugation
of at least one targeting ligand. The targeting ligand may be any ligand known
in the art
such as, but not limited to, a monoclonal antibody. (Kirpotin et al, Cancer
Res. 2006
66:6732-6740; the contents of which are herein incorporated by reference in
its entirety).
- 88 -
Date Recue/Date Received 2021-04-23
[0275] In some embodiments, the therapeutic nanoparticle may be formulated
in an
aqueous solution which may be used to target cancer (see International Pub No.
W02011084513 and US Pub No. US20110294717, the contents of each of which is
herein incorporated by reference in their entirety).
[0276] In some embodiments, the nucleic acid vaccine compositions may be
encapsulated in, linked to and/or associated with synthetic nanocarriers.
Synthetic
nanocarriers include, but are not limited to, those described in International
Pub. Nos.
W02010005740, W02010030763, W0201213501, W02012149252, W02012149255,
W02012149259, W02012149265, W02012149268, W02012149282, W02012149301,
W02012149393, W02012149405, W02012149411, W02012149454 and
W02013019669, and US Pub. Nos. US20110262491, U520100104645, U520100087337
and US20120244222, the contents of each of which are herein incorporated by
reference
in their entirety. The synthetic nanocarriers may be formulated using methods
known in
the art and/or described herein. As a non-limiting example, the synthetic
nanocarriers
may be formulated by the methods described in International Pub Nos.
W02010005740,
W02010030763 and W0201213501and US Pub. Nos. U520110262491,
U520100104645, U520100087337 and U52012024422, the contents of each of which
are
herein incorporated by reference in their entirety. The synthetic nanocarrier
formulations
may be lyophilized by methods described in International Pub. No. W02011072218
and
US Pat No. 8,211,473; the contents of each of which are herein incorporated by
reference
in their entirety.
[0277] In some embodiments, the synthetic nanocarriers may contain reactive
groups
to release the nucleic acid vaccine compositions described herein (see
International Pub.
No. W020120952552 and US Pub No. U520120171229, the contents of each of which
are herein incorporated by reference in their entirety).
[0278] In some embodiments, the synthetic nanocarriers may be formulated
for
targeted release. In some embodiments, the synthetic nanocarrier may be
formulated to
release the nucleic acid vaccine compositions at a specified pH and/or after a
desired time
interval. As a non-limiting example, the synthetic nanoparticle may be
formulated to
- 89 -
Date Recue/Date Received 2021-04-23
release the nucleic acid vaccine compositions after 24 hours and/or at a pH of
4.5 (see
International Pub. Nos. W02010138193 and W02010138194 and US Pub Nos.
US20110020388 and US20110027217, the contents of each of which is herein
incorporated by reference in their entireties).
[0279] In some embodiments, the synthetic nanocarriers may be formulated
for
controlled and/or sustained release of the nucleic acid vaccine compositions
described
herein. As a non-limiting example, the synthetic nanocarriers for sustained
release may
be formulated by methods known in the art, described herein and/or as
described in
International Pub No. W02010138192 and US Pub No. 20100303850, the contents
each
of which is herein incorporated by reference in their entirety.
[0280] In some embodiments, the nanoparticle may be optimized for oral
administration. The nanoparticle may comprise at least one cationic biopolymer
such as,
but not limited to, chitosan or a derivative thereof. As a non-limiting
example, the
nanoparticle may be formulated by the methods described in U.S. Pub. No.
20120282343; the contents of which are herein incorporated by reference in its
entirety.
[0281] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated in a modular composition such as described in US
8575123
to Manoharan et al., the contents of which are herein incorporated by
reference in their
entirety. As a non-limiting example, the modular composition may comprise a
nucleic
acid, e.g., the nucleic acid vaccine compositions of the present disclosure,
at least one
endosomolytic component, and at least one targeting ligand. The modular
composition
may have a formula such as any formula described in US 8575123 to Manoharan et
al.,
the contents of which are herein incorporated by reference in their entirety.
[0282] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be encapsulated in the lipid formulation to form a stable
nucleic acid-lipid
particle (SNALP) such as described in U58546554 to de Fougerolles et al., the
contents
of which are incorporated here by reference in their entirety. The lipid may
be cationic or
non-cationic. In one non-limiting example, the lipid to nucleic acid ratio
(mass/mass
ratio) (e.g., lipid to nucleic acid vaccine compositions ratio) will be in the
range of from
- 90 -
Date Recue/Date Received 2021-04-23
about 1:1 to about 50:1, from about 1:1 to about 25:1, 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, or 5:1, 6:1,
7:1, 8:1, 9:1, 10:1, or 11:1. In another example, the SNALP includes 40% 2,2-
Dilinoley1-
4-dimethylaminoethyl-[1,3]-dioxolane (Lipid A), 10%
dioleoylphosphatidylcholine
(DSPC), 40% cholesterol, 10% polyethylene glycol (PEG)-C-DOMG (mole percent)
with
a particle size of 63.0 20 nm and a 0.027 nucleic acid/lipid ratio. The
nucleic acid
vaccine compositions of the present disclosure may be formulated with a
nucleic acid-
lipid particle comprising an endosomal membrane destabilizer as disclosed in
US
7189705 to Lam et al., the contents of which are incorporated herein by
reference in their
entirety. As a non-limiting example, the endosomal membrane destabilizer may
be a Ca2+
ion.
[0283] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated with formulated lipid particles (FLiPs) disclosed
in US
8148344 to Akinc et al., the contents of which are herein incorporated by
reference in
their entirety. Akinc et al. teach that FLiPs may comprise at least one of a
single or
double-stranded oligonucleotide, where the oligonucleotide has been conjugated
to a
lipophile and at least one of an emulsion or liposome to which the conjugated
oligonucleotide has been aggregated, admixed or associated. These particles
have
surprisingly been shown to effectively deliver oligonucleotides to heart, lung
and muscle
disclosed in US 8148344 to Akinc et al., the contents of which are herein
incorporated by
reference in their entirety.
[0284] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be delivered to a cell using a composition comprising an
expression
vector in a lipid formulation as described in US 6086913 to Tam et al., the
contents of
which are incorporated herein by reference in their entirety. The composition
disclosed
by Tam is serum-stable and comprises an expression vector comprising first and
second
inverted repeated sequences from an adeno associated virus (AAV), a rep gene
from
AAV, and a nucleic acid fragment. The expression vector in Tam is complexed
with
lipids.
- 91 -
Date Recue/Date Received 2021-04-23
[0285] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated with a lipid formulation disclosed in US
20120270921 to
de Fougerolles et al., the contents of which are incorporated herein by
reference in their
entirety. In one non-limiting example, the lipid formulation may include a
cationic lipid
having the formula A described in US 20120270921, the contents of which are
herein
incorporated by reference in its entirety. In another non-limiting example,
the
compositions of exemplary nucleic acid-lipid particles disclosed in Table A of
US20120270921, the contents of which are incorporated herein by reference in
their
entirety, may be used with the nucleic acid vaccine compositions of the
present
disclosure.
[0286] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be fully encapsulated in a lipid particle disclosed in US
20120276207 to
Maurer et al., the contents of which are incorporated herein by reference in
their entirety.
The particles may comprise a lipid composition comprising preformed lipid
vesicles, a
charged therapeutic agent, and a destabilizing agent to form a mixture of
preformed
vesicles and therapeutic agent in a destabilizing solvent, wherein the
destabilizing solvent
is effective to destabilize the membrane of the preformed lipid vesicles
without disrupting
the vesicles.
[0287] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated with a conjugated lipid. In a non-limiting
example, the
conjugated lipid may have a formula such as described in US 20120264810 to Lin
et al.,
the contents of which are incorporated herein by reference in their entirety.
The conjugate
lipid may form a lipid particle which further comprises a cationic lipid, a
neutral lipid,
and a lipid capable of reducing aggregation.
[0288] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated in a neutral liposomal formulation such as
disclosed in US
20120244207 to Fitzgerald et al., the contents of which are incorporated
herein by
reference in their entirety. The phrase "neutral liposomal formulation" refers
to a
liposomal formulation with a near neutral or neutral surface charge at a
physiological pH.
- 92 -
Date Recue/Date Received 2021-04-23
Physiological pH can be, e.g., about 7.0 to about 7.5, or, e.g., about 7.5,
or, e.g., 7.0, 7.1,
7.2, 7.3, 7.4, or 7.5, or, e.g., 7.3, or, e.g., 7.4. An example of a neutral
liposomal
formulation is an ionizable lipid nanoparticle (iLNP). A neutral liposomal
formulation
can include an ionizable cationic lipid, e.g., DLin-KC2-DMA.
[0289] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated with a charged lipid or an amino lipid. As used
herein, the
term "charged lipid" is meant to include those lipids having one or two fatty
acyl or fatty
alkyl chains and a quaternary amino head group. The quaternary amine carries a
permanent positive charge. The head group can optionally include an ionizable
group,
such as a primary, secondary, or tertiary amine that may be protonated at
physiological
pH. The presence of the quaternary amine can alter the pKa of the ionizable
group
relative to the pKa of the group in a structurally similar compound that lacks
the
quaternary amine (e.g., the quaternary amine is replaced by a tertiary amine)
In some
embodiments, a charged lipid is referred to as an "amino lipid." In a non-
limiting
example, the amino lipid may be any amino lipid described in US20110256175 to
Hope
et al., the contents of which are incorporated herein by reference in their
entirety. For
example, the amino lipids may have the structure disclosed in Tables 3-7 of
Hope, such
as structure (II), DLin-K-C2-DMA, DLin-K2-DMA, DLin-K6-DMA, etc. The resulting
pharmaceutical preparations may be lyophilized according to Hope. In another
non-
limiting example, the amino lipids may be any amino lipid described in US
20110117125
to Hope et al., the contents of which are incorporated herein by reference in
their entirety,
such as a lipid of structure (I), DLin-K-DMA, DLin-C-DAP, DLin-DAC, DLin-MA,
DLin-S-DMA, etc. In another non-limiting example, the amino lipid may have the
structure (I), (II), (III), or (IV), or 4-(R)-DLin-K-DMA (VI), 4-(S)-DLin-K-
DMA (V) as
described in W02009132131 to Manoharan et al., the contents of which are
incorporated
herein by reference in their entirety. In another non-limiting example, the
charged lipid
used in any of the formulations described herein may be any charged lipid
described in
EP2509636 to Manoharan et al., the contents of which are incorporated herein
by
reference in their entirety.
- 93 -
Date Recue/Date Received 2021-04-23
[0290] In some embodiments, the nucleic acid vaccine composition s of the
present
disclosure may be formulated with an association complex containing lipids,
liposomes,
or lipoplexes. In a non-limiting example, the association complex comprises
one or more
compounds each having a structure defined by formula (I), a PEG-lipid having a
structure
defined by formula (XV), a steroid and a nucleic acid disclosed in US8034376
to
Manoharan et al., the contents of which are incorporated herein by reference
in their
entirety. The nucleic acid vaccine compositions may be formulated with any
association
complex described in US8034376, the contents of which are herein incorporated
by
reference in its entirety.
[0291] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated with reverse head group lipids. As a non-limiting
example,
the nucleic acid vaccine compositions may be formulated with a zwitterionic
lipid
comprising a headgroup wherein the positive charge is located near the acyl
chain region
and the negative charge is located at the distal end of the head group, such
as a lipid
having structure (A) or structure (I) described in W02011056682 to Leung et
al., the
contents of which are incorporated herein by reference in their entirety.
[0292] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated in a lipid bilayer carrier. As a non-limiting
example, the
nucleic acid vaccine compositions may be combined with a lipid-detergent
mixture
comprising a lipid mixture of an aggregation-preventing agent in an amount of
about 5
mol% to about 20 mol%, a cationic lipid in an amount of about 0.5 mol% to
about 50
mol%, and a fusogenic lipid and a detergent, to provide a nucleic acid-lipid-
detergent
mixture; and then dialyzing the nucleic acid-lipid-detergent mixture against a
buffered
salt solution to remove the detergent and to encapsulate the nucleic acid in a
lipid bilayer
carrier and provide a lipid bilayer-nucleic acid composition, wherein the
buffered salt
solution has an ionic strength sufficient to encapsulate of from about 40 % to
about 80 %
of the nucleic acid, described in W01999018933 to Cullis et al., the contents
of which
are incorporated herein by reference in their entirety.
- 94 -
Date Recue/Date Received 2021-04-23
[0293] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may comprise (a) a nucleic acid; (b) 1.0 mole % to 45 mole % of a
cationic
lipid; (c) 0,0 mole % to 90 mole % of another lipid; (d) 1,0 mole % to 10 mole
% of a
bilayer stabilizing component; (e) 0,0 mole % to 60 mole % cholesterol; and
(f) 0,0 mole
% to 10 mole % of cationic polymer lipid as described in EP1328254 to Cullis
et al., the
contents of which are incorporated herein by reference in their entirety.
[0294] In some embodiments, the nucleic acid vaccine may be delivered using
smaller
LNPs. Such particles may comprise a diameter from below 0.1 um up to 100 nm
such as,
but not limited to, less than 0.1 um, less than 1.0 um, less than Sum, less
than 10 um, less
than 15 um, less than 20 um, less than 25 um, less than 30 um, less than 35
um, less than
40 um, less than 50 um, less than 55 um, less than 60 um, less than 65 um,
less than 70
um, less than 75 um, less than 80 um, less than 85 um, less than 90 um, less
than 95 um,
less than 100 um, less than 125 um, less than 150 um, less than 175 um, less
than 200 um,
less than 225 um, less than 250 um, less than 275 um, less than 300 um, less
than 325 um,
less than 350 um, less than 375 um, less than 400 um, less than 425 um, less
than 450 um,
less than 475 um, less than 500 um, less than 525 um, less than 550 um, less
than 575 um,
less than 600 um, less than 625 um, less than 650 um, less than 675 um, less
than 700 um,
less than 725 um, less than 750 um, less than 775 um, less than 800 um, less
than 825 um,
less than 850 um, less than 875 um, less than 900 um, less than 925 um, less
than 950 um,
less than 975 um,
[0295] In another embodiment, nucleic acid vaccine may be delivered using
smaller
LNPs which may comprise a diameter from about 1 nm to about 100 nm, from about
1
nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm,
from
about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to
about
60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from
about 1
nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to
about 10
nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm
to
about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm,
from
about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to
about
- 95 -
Date Recue/Date Received 2021-04-23
90 nm, about 10 to about 50 nM, from about 20 to about 50 nm, from about 30 to
about
50 nm, from about 40 to about 50 nm, from about 20 to about 60 nm, from about
30 to
about 60 nm, from about 40 to about 60 nm, from about 20 to about 70 nm, from
about
30 to about 70 nm, from about 40 to about 70 nm, from about 50 to about 70 nm,
from
about 60 to about 70 nm, from about 20 to about 80 nm, from about 30 to about
80 nm,
from about 40 to about 80 nm, from about 50 to about 80 nm, from about 60 to
about 80
nm, from about 20 to about 90 nm, from about 30 to about 90 nm, from about 40
to about
90 nm, from about 50 to about 90 nm, from about 60 to about 90 nm and/or from
about
70 to about 90 nm.
[0296] In some embodiments, the nucleic acid vaccine may be formulated in
lipid
nanoparticles having a diameter from about 10 to about 100 nm such as, but not
limited
to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm,
about 10
to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to
about 80
nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm,
about 20
to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to
about 80
nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40
nm, about
30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30
to about
80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50
nm,
about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm,
about 40 to
about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to
about 70
nm about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm,
about
60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60
to about
100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about
100 nm,
about 80 to about 90 nm, about 80 to about 100 nm and/or about 90 to about 100
nm.
[0297] In some embodiments, the lipid nanoparticles may have a diameter
from about
to 500 nm.
[0298] In some embodiments, the lipid nanoparticle may have a diameter
greater than
100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater
than 300
nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater
than 500 nm,
- 96 -
Date Recue/Date Received 2021-04-23
greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than
700 nm,
greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than
900 nm,
greater than 950 nm or greater than 1000 nm.
Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles
[0299] The nucleic acid vaccine compositions of the disclosure can be
formulated
using natural and/or synthetic polymers. Non-limiting examples of polymers
which may
be used for delivery include, but are not limited to, DYNAMIC POLYCONJUGATEO
(Arrowhead Research Corp., Pasadena, CA) formulations from MIRUSO Bio
(Madison,
WI) and Roche Madison (Madison, WI), PHASERXTM polymer formulations such as,
without limitation, SMARTT POLYMER TECHNOLOGYTm (PHASERXO, Seattle,
WA), DMRI/DOPE, poloxamer, VAXFECTINO adjuvant from Vical (San Diego, CA),
chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, CA), dendrimers
and
poly(lactic-co-glycolic acid) (PLGA) polymers. RONDELTM (RNAi/Oligonucleotide
Nanoparticle Delivery) polymers (Arrowhead Research Corporation, Pasadena, CA)
and
pH responsive co-block polymers such as, but not limited to, PHASERXO
(Seattle, WA).
[0300] A non-limiting example of chitosan formulation includes a core of
positively
charged chitosan and an outer portion of negatively charged substrate (U.S.
Pub. No.
20120258176; herein incorporated by reference in its entirety). Chitosan
includes, but is
not limited to N-trimethyl chitosan, mono-N-carboxymethyl chitosan (MCC), N-
palmitoyl chitosan (NPCS), EDTA-chitosan, low molecular weight chitosan,
chitosan
derivatives, or combinations thereof.
[0301] In some embodiments, the polymers used in the present disclosure
have
undergone processing to reduce and/or inhibit the attachment of unwanted
substances
such as, but not limited to, bacteria, to the surface of the polymer. The
polymer may be
processed by methods known and/or described in the art and/or described in
International
Pub. No. W02012150467, herein incorporated by reference in its entirety.
[0302] A non-limiting example of PLGA formulations include, but are not
limited to,
PLGA injectable depots (e.g., ELIGARDO which is formed by dissolving PLGA in
66%
N-methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and
leuprolide.
- 97 -
Date Recue/Date Received 2021-04-23
Once injected, the PLGA and leuprolide peptide precipitates into the
subcutaneous
space).
[0303] Many of these polymer approaches have demonstrated efficacy in
delivering
oligonucleotides in vivo into the cell cytoplasm (reviewed in de Fougerolles
Hum Gene
Ther. 2008 19:125-132; herein incorporated by reference in its entirety). Two
polymer
approaches that have yielded robust in vivo delivery of nucleic acids, i.e.,
in the case of
small interfering RNA (siRNA), are dynamic polyconjugates and cyclodextrin-
based
nanoparticles. The first of these delivery approaches uses dynamic
polyconjugates and
has been shown in vivo in mice to effectively deliver siRNA and silence
endogenous
target mRNA in hepatocytes (Rozema et al., Proc Natl Acad Sci U S A. 2007
104:12982-
12887; herein incorporated by reference in its entirety). This particular
approach is a
multicomponent polymer system whose key features include a membrane-active
polymer
to which nucleic acid, in this case siRNA, is covalently coupled via a
disulfide bond and
where both PEG (for charge masking) and N-acetylgalactosamine (for hepatocyte
targeting) groups are linked via pH-sensitive bonds (Rozema et al., Proc Natl
Acad Sci U
S A. 2007 104:12982-12887; herein incorporated by reference in its entirety).
On binding
to the hepatocyte and entry into the endosome, the polymer complex
disassembles in the
low-pH environment, with the polymer exposing its positive charge, leading to
endosomal escape and cytoplasmic release of the siRNA from the polymer.
Through
replacement of the N-acetylgalactosamine group with a mannose group, it was
shown one
could alter targeting from asialoglycoprotein receptor-expressing hepatocytes
to
sinusoidal endothelium and Kupffer cells. Another polymer approach involves
using
transferrin-targeted cyclodextrin-containing polycation nanoparticles. These
nanoparticles have demonstrated targeted silencing of the EWS-FLI1 gene
product in
transferrin receptor-expressing Ewing's sarcoma tumor cells (Hu-Lieskovan et
al.,
Cancer Res.2005 65: 8984-8982; herein incorporated by reference in its
entirety) and
siRNA formulated in these nanoparticles was well tolerated in non-human
primates
(Heidel et al., Proc Natl Acad Sci USA 2007 104:5715-21; herein incorporated
by
- 98 -
Date Recue/Date Received 2021-04-23
reference in its entirety). Both of these delivery strategies incorporate
rational approaches
using both targeted delivery and endosomal escape mechanisms.
[0304] The polymer formulation can permit the sustained or delayed release
of nucleic
acid vaccine compositions (e.g., following intramuscular, subcutaneous,
intraparenchymal, intrathecal, intracerebroventricular administration). The
altered release
profile for the nucleic acid vaccine compositions can result in, for example,
translation of
an encoded protein over an extended period of time. Biodegradable polymers
have been
previously used to protect nucleic acids from degradation and been shown to
result in
sustained release of payloads in vivo (Rozema et al., Proc Natl Acad Sci U S
A. 2007
104:12982-12887; Sullivan et al., Expert Opin Drug Deliv. 2010 7:1433-1446;
Convertine et al., Biomacromolecules. 2010 Oct 1; Chu et al., Acc Chem Res.
2012 Jan
13; Manganiello et al., Biomaterials. 2012 33:2301-2309; Benoit et al.,
Biomacromolecules. 2011 12:2708-2714; Singha et al., Nucleic Acid Ther. 2011
2:133-
147; de Fougerolles Hum Gene Ther. 2008 19:125-132; Schaffert and Wagner, Gene
Ther. 2008 16:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 2011
8:1455-1468;
Davis, Mol Pharm. 2009 6:659-668; Davis, Nature 2010 464:1067-1070; each of
which is
herein incorporated by reference in its entirety).
[0305] In some embodiments, the pharmaceutical compositions may be
sustained
release formulations. In further embodiments, the sustained release
formulations may be
for subcutaneous delivery. Sustained release formulations may include, but are
not
limited to, PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer,
GELSITEO
(Nanotherapeutics, Inc. Alachua, FL), HYLENEXO (Halozyme Therapeutics, San
Diego
CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia,
GA),
TISSELLO (Baxter International, Inc Deerfield, IL), PEG-based sealants, and
COSEALO (Baxter International, Inc Deerfield, IL).
[0306] As a non-limiting example, nucleic acid vaccine compositions may be
formulated in PLGA microspheres by preparing the PLGA microspheres with
tunable
release rates (e.g., days and weeks) and encapsulating the nucleic acid
vaccine
compositions in the PLGA microspheres while maintaining the integrity of the
nucleic
- 99 -
Date Recue/Date Received 2021-04-23
acid vaccine compositions during the encapsulation process. EVAc are non-
biodegradable, biocompatible polymers which are used extensively in pre-
clinical
sustained release implant applications. Poloxamer F-407 NF is a hydrophilic,
non-ionic
surfactant triblock copolymer of polyoxyethylene-polyoxypropylene-
polyoxyethylene
having a low viscosity at temperatures less than 5 C and forms a solid gel at
temperatures
greater than 15 C. PEG-based surgical sealants comprise two synthetic PEG
components
mixed in a delivery device which can be prepared in one minute, seals in 3
minutes and is
reabsorbed within 30 days. GELSITEO and natural polymers are capable of in-
situ
gelation at the site of administration. They have been shown to interact with
protein and
peptide therapeutic candidates through ionic interaction to provide a
stabilizing effect.
[0307] Polymer formulations can also be selectively targeted through
expression of
different ligands as exemplified by, but not limited by, folate, transferrin,
and N-
acetylgalactosamine (GalNAc) (Benoit et al., Biomacromolecules. 201112:2708-
2714;
Rozema et al., Proc Natl Acad Sci U S A. 2007 104:12982-12887; Davis, Mol
Pharm.
2009 6:659-668; Davis, Nature 2010 464:1067-1070; each of which is herein
incorporated by reference in its entirety).
[0308] The nucleic acid vaccine compositions of the disclosure may be
formulated
with or in a polymeric compound. The polymer may include at least one polymer
such as,
but not limited to, polyethenes, polyethylene glycol (PEG), poly(1-
lysine)(PLL), PEG
grafted to PLL, cationic lipopolymer, biodegradable cationic lipopolymer,
polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), a
polyamine
derivative, a modified poloxamer, a biodegradable polymer, elastic
biodegradable
polymer, biodegradable block copolymer, biodegradable random copolymer,
biodegradable polyester copolymer, biodegradable polyester block copolymer,
biodegradable polyester block random copolymer, multiblock copolymers, linear
biodegradable copolymer, poly[a-(4-aminobuty1)-L-glycolic acid) (PAGA),
biodegradable cross-linked cationic multi-block copolymers, polycarbonates,
polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones,
polyamides,
polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates,
polyvinyl
- 100 -
Date Recue/Date Received 2021-04-23
alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,
polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,
poly(ethylene
imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-
proline ester),
acrylic polymers, amine-containing polymers, dextran polymers, dextran polymer
derivatives or combinations thereof.
[0309] As a non-limiting example, the nucleic acid vaccine compositions of
the
disclosure may be formulated with the polymeric compound of PEG grafted with
PLL as
described in U.S. Pat. No. 6,177,274; herein incorporated by reference in its
entirety. The
formulation may be used for transfecting cells in vitro or for in vivo
delivery of the
nucleic acid vaccine compositions. In another example, the nucleic acid
vaccine
compositions may be suspended in a solution or medium with a cationic polymer,
in a dry
pharmaceutical composition or in a solution that is capable of being dried as
described in
U.S. Pub. Nos. 20090042829 and 20090042825; each of which are herein
incorporated
by reference in their entireties.
[0310] As another non-limiting example, the nucleic acid vaccine
compositions of the
disclosure may be formulated with a PLGA-PEG block copolymer (see US Pub. No.
US20120004293 and US Pat No. 8,236,330, herein incorporated by reference in
their
entireties) or PLGA-PEG-PLGA block copolymers (See U.S. Pat. No. 6,004,573,
herein
incorporated by reference in its entirety). As a non-limiting example, the
nucleic acid
vaccine compositions of the disclosure may be formulated with a diblock
copolymer of
PEG and PLA or PEG and PLGA (see US Pat No 8,246,968, herein incorporated by
reference in its entirety).
[0311] In some embodiments, the nucleic acid vaccines compositions may be
formulated with branched PEG molecules as described in or made by the methods
described in International PCT Publication No. W020180126084, the contents of
which
are herein incorporated by reference in its entirety. As a non-limiting
example, the
branched PEG which may be used in the formulations described herein may have
the
formula I, formula II, formula III, formula IV, formula V, formula VI of PCT
Publication
- 101 -
Date Recue/Date Received 2021-04-23
No. W020180126084, the contents of which are herein incorporated by reference
in its
entirety.
[0312] A polyamine derivative may be used to deliver nucleic acids or to
treat and/or
prevent a disease or to be included in an implantable or injectable device
(U.S. Pub. No.
20100260817 herein incorporated by reference in its entirety). As a non-
limiting
example, a pharmaceutical composition may include the nucleic acid vaccine
compositions and the polyamine derivative described in U.S. Pub. No.
20100260817, the
contents of which are incorporated herein by reference in their entirety. As a
non-limiting
example the nucleic acid vaccine compositions of the present disclosure may be
delivered
using a polyamide polymer such as, but not limited to, a polymer comprising a
1,3-
dipolar addition polymer prepared by combining a carbohydrate diazide monomer
with a
dialkyne unite comprising oligoamines (U.S. Pat. No. 8,236,280; herein
incorporated by
reference in its entirety).
[0313] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be formulated with at least one polymer and/or derivatives
thereof
described in International Publication Nos. W02011115862, W02012082574 and
W02012068187 and U.S. Pub. No. 20120283427, the contents of each of which are
herein incorporated by reference in their entireties. The nucleic acid vaccine
compositions of the present disclosure may be formulated with a polymer of
formula Z as
described in W02011115862, herein incorporated by reference in its entirety.
The
nucleic acid vaccine compositions may be formulated with a polymer of formula
Z, Z' or
Z" as described in International Pub. Nos. W02012082574 or W02012068187 and
U.S.
Pub. No. 2012028342, the contents of each of which are herein incorporated by
reference
in their entireties. The polymers formulated with the nucleic acid vaccine
compositions of
the present disclosure may be synthesized by the methods described in
International Pub.
Nos. W02012082574 or W02012068187, the contents of each of which are herein
incorporated by reference in their entireties.
[0314] The nucleic acid vaccine compositions of the disclosure may be
formulated
with at least one acrylic polymer. Acrylic polymers include but are not
limited to, acrylic
- 102 -
Date Recue/Date Received 2021-04-23
acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl
methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate,
amino
alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),
polycyanoacrylates and combinations thereof.
[0315] Formulations of nucleic acid vaccine compositions of the disclosure
may
include at least one amine-containing polymer such as, but not limited to
polylysine,
polyethylene imine, poly(amidoamine) dendrimers or combinations thereof.
[0316] For example, the nucleic acid vaccine compositions of the disclosure
may be
formulated in a pharmaceutical compound including a poly(alkylene imine), a
biodegradable cationic lipopolymer, a biodegradable block copolymer, a
biodegradable
polymer, or a biodegradable random copolymer, a biodegradable polyester block
copolymer, a biodegradable polyester polymer, a biodegradable polyester random
copolymer, a linear biodegradable copolymer, PAGA, a biodegradable cross-
linked
cationic multi-block copolymer or combinations thereof. The biodegradable
cationic
lipopolymer may be made by methods known in the art and/or described in U.S.
Pat. No.
6,696,038, U.S. App. Nos. 20030073619 and 20040142474 each of which is herein
incorporated by reference in their entireties. The poly(alkylene imine) may be
made using
methods known in the art and/or as described in U.S. Pub. No. 20100004315,
herein
incorporated by reference in its entirety. The biodegradable polymer,
biodegradable block
copolymer, the biodegradable random copolymer, biodegradable polyester block
copolymer, biodegradable polyester polymer, or biodegradable polyester random
copolymer may be made using methods known in the art and/or as described in
U.S. Pat.
Nos. 6,517,869 and 6,267,987, the contents of which are each incorporated
herein by
reference in their entirety. The linear biodegradable copolymer may be made
using
methods known in the art and/or as described in U.S. Pat. No. 6,652,886. The
PAGA
polymer may be made using methods known in the art and/or as described in U.S.
Pat.
No. 6,217,912 herein incorporated by reference in its entirety. The PAGA
polymer may
be copolymerized to form a copolymer or block copolymer with polymers such as
but not
limited to, poly-L-lysine, polyargine, polyornithine, histones, avidin,
protamines,
- 103 -
Date Recue/Date Received 2021-04-23
polylactides and poly(lactide-co-glycolides). The biodegradable cross-linked
cationic
multi-block copolymers may be made my methods known in the art and/or as
described
in U.S. Pat. No. 8,057,821 or U.S. Pub. No. 2012009145 each of which are
herein
incorporated by reference in their entireties. For example, the multi-block
copolymers
may be synthesized using linear polyethyleneimine (LPEI) blocks which have
distinct
patterns as compared to branched polyethyleneimines. Further, the composition
or
pharmaceutical composition may be made by the methods known in the art,
described
herein, or as described in U.S. Pub. No. 20100004315 or U.S. Pat. Nos.
6,267,987 and
6,217,912 each of which are herein incorporated by reference in their
entireties.
[0317] The nucleic acid vaccine compositions of the disclosure may be
formulated
with at least one degradable polyester which may contain polycationic side
chains.
Degradable polyesters include, but are not limited to, poly(serine ester),
poly(L-lactide-
co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In
some
embodiments, the degradable polyesters may include a PEG conjugation to form a
PEGylated polymer.
[0318] The nucleic acid vaccine compositions of the disclosure may be
formulated
with at least one crosslinkable polyester. Crosslinkable polyesters include
those known in
the art and described in US Pub. No. 20120269761, herein incorporated by
reference in
its entirety.
[0319] In some embodiments, the polymers described herein may be conjugated
to a
lipid-terminating PEG. As a non-limiting example, PLGA may be conjugated to a
lipid-
terminating PEG forming PLGA-DSPE-PEG. As another non-limiting example, PEG
conjugates for use with the present disclosure are described in International
Publication
No. W02008103276, herein incorporated by reference in its entirety. The
polymers may
be conjugated using a ligand conjugate such as, but not limited to, the
conjugates
described in U.S. Pat. No. 8,273,363, herein incorporated by reference in its
entirety.
[0320] In some embodiments, the nucleic acid vaccine compositions described
herein
may be conjugated with another compound. Non-limiting examples of conjugates
are
described in US Patent Nos. 7,964,578 and 7,833,992, each of which are herein
- 104 -
Date Recue/Date Received 2021-04-23
incorporated by reference in their entireties. The nucleic acid vaccine
compositions of the
present disclosure may be conjugated with conjugates of formula 1-122 as
described in
US Patent Nos. 7,964,578 and 7,833,992, each of which are herein incorporated
by
reference in their entireties. The nucleic acid vaccine compositions described
herein may
be conjugated with a metal such as, but not limited to, gold. (See e.g.,
Giljohann et al.
Journ. Amer. Chem. Soc. 2009 131(6): 2072-2073; herein incorporated by
reference in its
entirety). In some embodiments, the nucleic acid vaccine compositions
described herein
may be conjugated and/or encapsulated in gold-nanoparticles. (International
Pub. No.
W0201216269 and U.S. Pub. No. 20120302940; each of which is herein
incorporated by
reference in its entirety).
[0321] As described in U.S. Pub. No. 20100004313, herein incorporated by
reference
in its entirety, a gene delivery composition may include a nucleotide sequence
and a
poloxamer. For example, the nucleic acid vaccine compositions of the present
disclosure
may be used in a gene delivery composition with the poloxamer described in
U.S. Pub.
No. 20100004313.
[0322] In some embodiments, the polymer formulation of the present
disclosure may
be stabilized by contacting the polymer formulation, which may include a
cationic
carrier, with a cationic lipopolymer which may be covalently linked to
cholesterol and
polyethylene glycol groups. The polymer formulation may be contacted with a
cationic
lipopolymer using the methods described in U.S. Pub. No. 20090042829 herein
incorporated by reference in its entirety.
[0323] The cationic carrier may include, but is not limited to,
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-
- 105 -
Date Recue/Date Received 2021-04-23
propanaminium trifluoroacetate (DOSPA), 3B-[N¨(N1,N1-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) and combinations thereof.
[0324] The nucleic acid vaccine compositions of the disclosure may be
formulated in
a polyplex of one or more polymers (U.S. Pub. No. 20120237565 and 20120270927;
each of which is herein incorporated by reference in its entirety). In some
embodiments,
the polyplex comprises two or more cationic polymers. The cationic polymer may
comprise a poly(ethylene imine) (PEI) such as linear PEI.
[0325] The nucleic acid vaccine compositions of the disclosure can also be
formulated
as a nanoparticle using a combination of polymers, lipids, and/or other
biodegradable
agents, such as, but not limited to, calcium phosphate. Components may be
combined in a
core-shell, hybrid, and/or layer-by-layer architecture, to allow for fine-
tuning of the
nanoparticle so delivery of the nucleic acid vaccine compositions may be
enhanced
(Wang et al., Nat Mater. 2006 5:791-796; Fuller et al., Biomaterials. 2008
29:1526-1532;
DeKoker et al., Adv Drug Deliv Rev. 2011 63:748-761; Endres et al.,
Biomaterials. 2011
32:7721-7731; Su et al., Mol Pharm. 2011 Jun 6;8(3):774-87; herein
incorporated by
reference in its entirety). As a non-limiting example, the nanoparticle may
comprise a
plurality of polymers such as, but not limited to hydrophilic-hydrophobic
polymers (e.g.,
PEG-PLGA), hydrophobic polymers (e.g., PEG) and/or hydrophilic polymers
(International Pub. No. W020120225129; herein incorporated by reference in its
entirety).
[0326] Biodegradable calcium phosphate nanoparticles in combination with
lipids
and/or polymers may be used to deliver nucleic acid vaccine compositions in
vivo. In
some embodiments, a lipid coated calcium phosphate nanoparticle, which may
also
contain a targeting ligand such as anisamide, may be used to deliver the
nucleic acid
vaccine compositions of the present disclosure. For example, to effectively
deliver
siRNA in a mouse metastatic lung model a lipid coated calcium phosphate
nanoparticle
- 106 -
Date Recue/Date Received 2021-04-23
was used (Li etal., J Contr Rel. 2010 142: 416-421; Li etal., J Contr Rel.
2012 158:108-
114; Yang et al., Mol Ther. 2012 20:609-615; herein incorporated by reference
in its
entirety). This delivery system combines both a targeted nanoparticle and a
component to
enhance the endosomal escape, calcium phosphate, in order to improve delivery
of the
siRNA.
[0327] In some embodiments, calcium phosphate with a PEG-polyanion block
copolymer may be used to delivery nucleic acid vaccine compositions (Kazikawa
et al., J
Contr Rel. 2004 97:345-356; Kazikawa et al., J Contr Rel. 2006 111:368-370;
herein
incorporated by reference in its entirety).
[0328] In some embodiments, a PEG-charge-conversional polymer (Pitella et
al.,
Biomaterials. 2011 32:3106-3114) may be used to form a nanoparticle to deliver
the
nucleic acid vaccine compositions of the present disclosure. The PEG-charge-
conversional polymer may improve upon the PEG-polyanion block copolymers by
being
cleaved into a polycation at acidic pH, thus enhancing endosomal escape.
[0329] The use of core-shell nanoparticles has additionally focused on a
high-
throughput approach to synthesize cationic cross-linked nanogel cores and
various shells
(Siegwart etal., Proc Nat! Acad Sci U S A. 2011108:12996-13001). The
complexation,
delivery, and internalization of the polymeric nanoparticles can be precisely
controlled by
altering the chemical composition in both the core and shell components of the
nanoparticle. For example, the core-shell nanoparticles may efficiently
deliver nucleic
acid vaccine compositions to mouse hepatocytes after they covalently attach
cholesterol
to the nanoparticle.
[0330] In some embodiments, the nanoparticles described herein may be
nanoparticles
which include at least one ligand, and the ligand may be a peptide, an
aptamer, which is a
small molecular weight (8-13 Kda) single-stranded RNA or DNA with low
nanomolar
binding affinities toward their targets, an antibody, a small molecule ligand
such as, but
not limited to, folate, anisamide, and galactose. (Leng et al. Journal of Drug
Delivery.
Vo. 17, Article ID 6971297; the contents of which are herein incorporated by
reference in
their entirety).
- 107 -
Date Recue/Date Received 2021-04-23
[0331] In some embodiments, a hollow lipid core comprising a middle PLGA
layer
and an outer neutral lipid layer containing PEG may be used to delivery of the
nucleic
acid vaccine compositions of the present disclosure. As a non-limiting
example, in mice
bearing a luciferase-expressing tumor, it was determined that the lipid-
polymer-lipid
hybrid nanoparticle significantly suppressed luciferase expression, as
compared to a
conventional lipoplex (Shi et al, Angew Chem Int Ed. 2011 50:7027-7031; herein
incorporated by reference in its entirety).
[0332] In some embodiments, the lipid nanoparticles may comprise a core of
the
nucleic acid vaccine compositions described herein and a polymer shell. The
polymer
shell may be any of the polymers described herein and are known in the art. In
an
additional embodiment, the polymer shell may be used to protect the modified
nucleic
acids in the core.
[0333] Core¨shell nanoparticles for use with the nucleic acid vaccine
compositions of
the present disclosure may be formed by the methods described in U.S. Pat. No.
8,313,777 herein incorporated by reference in its entirety.
[0334] In some embodiments, the core-shell nanoparticles may comprise a
core of the
nucleic acid vaccine compositions described herein and a polymer shell. The
polymer
shell may be any of the polymers described herein and are known in the art. In
an
additional embodiment, the polymer shell may be used to protect the nucleic
acid vaccine
compositions in the core. As a non-limiting example, the core-shell
nanoparticle may be
used to treat an eye disease or disorder (see, e.g., US Publication No.
20120321719,
herein incorporated by reference in its entirety).
[0335] In some embodiments, the polymer used with the formulations
described
herein may be a modified polymer (such as, but not limited to, a modified
polyacetal) as
described in International Publication No. W02011120053, herein incorporated
by
reference in its entirety.
[0336] In some embodiments, the nucleic acid vaccine compositions may be
delivered
to the cell or cytosol of a target cell by contacting the cell with a membrane-
destabilizing
polymer and a conjugate of the nucleic acid vaccine composition, a targeting
ligand and
- 108 -
Date Recue/Date Received 2021-04-23
an optional linker. Non-limiting examples of membrane-destabilizing polymers
are taught
in International PCT publication No. W02020093061, the contents of which are
herein
incorporated by reference in its entirety, such as, but not limited to, the
membrane-
destabilizing polymers of formula XX therein.
Excipients
[0337] In some embodiments, pharmaceutical formulations may additionally
comprise
a pharmaceutically acceptable excipient, which, as used herein, includes, but
are not
limited to, any and all solvents, dispersion media, diluents, or other liquid
vehicles,
dispersion or suspension aids, surface active agents, isotonic agents,
thickening or
emulsifying agents, preservatives, solid binders, lubricants, flavoring
agents, stabilizers,
anti-oxidants, osmolality adjusting agents, pH adjusting agents and the like,
as suited to
the particular dosage form desired. Various excipients for formulating
pharmaceutical
compositions and techniques for preparing the composition are known in the art
(see
Remington: The Science and Practice of Pharmacy, 21" Edition, A. R. Gennaro
(Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by
reference
in its entirety). The use of a conventional excipient medium may be
contemplated within
the scope of the present disclosure, except insofar as any conventional
excipient medium
is incompatible with a substance or its derivatives, such as by producing any
undesirable
biological effect or otherwise interacting in a deleterious manner with any
other
component(s) of the pharmaceutical composition, its use is contemplated to be
within the
scope of this disclosure.
[0338] In some embodiments, a pharmaceutically acceptable excipient may be
at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In
some
embodiments, an excipient is approved for use for humans and for veterinary
use. In
some embodiments, an excipient may be approved by United States Food and Drug
Administration. In some embodiments, an excipient may be of pharmaceutical
grade. In
some embodiments, an excipient may meet the standards of the United States
Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British
Pharmacopoeia,
and/or the International Pharmacopoeia.
- 109 -
Date Recue/Date Received 2021-04-23
[0339] Pharmaceutically acceptable excipients used in the manufacture of
pharmaceutical compositions include, but are not limited to, inert diluents,
dispersing
and/or granulating agents, surface active agents and/or emulsifiers,
disintegrating agents,
binding agents, preservatives, buffering agents, lubricating agents, and/or
oils. Such
excipients may optionally be included in pharmaceutical compositions. The
composition
may also include excipients such as cocoa butter and suppository waxes,
coloring agents,
coating agents, sweetening, flavoring, and/or perfuming agents.
[0340] Exemplary diluents include, but are not limited to, 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,
powdered
sugar, etc., and/or combinations thereof.
[0341] Exemplary granulating and/or dispersing agents include, but are not
limited to,
potato starch, corn starch, tapioca starch, sodium starch glycolate, clays,
alginic acid,
guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural
sponge,
cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-
linked
polyvinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium
starch
glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl
cellulose
(croscarmellose), methylcellulose, pregelatinized starch (starch 1500),
microcrystalline
starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium
aluminum
silicate (VEEGUMO), sodium lauryl sulfate, quaternary ammonium compounds,
etc.,
and/or combinations thereof.
[0342] Exemplary surface active agents and/or emulsifiers include, but are
not limited
to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate,
tragacanth, chon-
drux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat,
cholesterol, wax,
and lecithin), colloidal clays (e.g. bentonite (aluminum silicate) and VEEGUMO
(magnesium aluminum silicate)), long chain amino acid derivatives, high
molecular
weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin
monostearate,
ethylene glycol distearate, glyceryl monostearate, and propylene glycol
monostearate,
- 110 -
Date Recue/Date Received 2021-04-23
polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid,
acrylic acid
polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.
carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose,
hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose),
sorbitan fatty
acid esters (e.g. polyoxyethylene sorbitan monolaurate (TWEEN020),
polyoxyethylene
sorbitan (TWEEN060), polyoxyethylene sorbitan monooleate (TWEEN080), sorbitan
monopalmitate (SPAN040), sorbitan monostearate (SPAN060), sorbitan tristearate
(SPAN065), glyceryl monooleate, sorbitan monooleate (SPAN080)),
polyoxyethylene
esters (e.g. polyoxyethylene monostearate (MYRJ045), polyoxyethylene
hydrogenated
castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and
SOLUTOLO),
sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g.
CREMOPHORO),
polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether (BRIJ030)), poly
(vinyl-
pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium
oleate,
potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl
sulfate,
PLUORINCOF 68, POLOXAMERO 188, cetrimonium bromide, cetylpyridinium
chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations
thereof.
[0343] Exemplary binding agents include, but are not limited to, starch
(e.g.
cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,
dextrose, dextrin,
molasses, lactose, lactitol, mannitol); amino acids (e.g., glycine); natural
and synthetic
gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti
gum,
mucilage of isapol husks, carboxymethylcellulose, methylcellulose,
ethylcellulose,
hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,
microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone),
magnesium
aluminum silicate (VEEGUMO), and larch arabogalactan); alginates; polyethylene
oxide;
polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates;
waxes;
water; alcohol; etc.; and combinations thereof.
[0344] Exemplary preservatives may include, but are not limited to,
antioxidants,
chelating agents, antimicrobial preservatives, antifungal preservatives,
alcohol
preservatives, acidic preservatives, and/or other preservatives. Oxidation is
a potential
- 111 -
Date Recue/Date Received 2021-04-23
degradation pathway for mRNA, especially for liquid mRNA formulations. In
order to
prevent oxidation, antioxidants can be added to the formulation. Exemplary
antioxidants
include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl
palmitate, benzyl
alcohol, butylated hydroxyanisole, EDTA, m-cresol, methionine, butylated
hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid,
propyl
gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite,
thioglycerol and/or
sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic
acid
(EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic
acid,
fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid,
and/or trisodium
edetate. Exemplary antimicrobial preservatives include, but are not limited
to,
benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol,
cetrimide,
cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol,
chloroxylenol,
cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,
phenylethyl
alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal.
Exemplary
antifungal preservatives include, but are not limited to, butyl paraben,
methyl paraben,
ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium
benzoate,
potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
Exemplary
alcohol preservatives include, but are not limited to, ethanol, polyethylene
glycol, phenol,
phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenyl
ethyl
alcohol. Exemplary acidic preservatives include, but are not limited to,
vitamin A,
vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic
acid, ascorbic
acid, sorbic acid, and/or phytic acid. Other preservatives include, but are
not limited to,
tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated
hydroxyanisol
(BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate
(SLS),
sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite,
potassium
sulfite, potassium metabisulfite, GLYDANT PLUS , PHENONIPO, methylparaben,
GERMALL0115, GERMABENO!!, NEOLONETM, KATHONTm, and/or EUXYLO.
[0345] In some embodiments, the pH of the pharmaceutical solutions are
maintained
between pH 5 and pH 8 to improve stability. Exemplary buffers to control pH
may
- 112 -
Date Recue/Date Received 2021-04-23
include, but are not limited to sodium phosphate, sodium citrate, sodium
succinate,
histidine (or histidine-HC1), sodium carbonate, and/or sodium malate. In
another
embodiment, the exemplary buffers listed above may be used with additional
monovalent
counterions (including, but not limited to potassium). Divalent cations may
also be used
as buffer counterions; however, these are not preferred due to complex
formation and/or
mRNA degradation.
[0346] Exemplary buffering agents may also include, but are not limited to,
citrate
buffer solutions, acetate buffer solutions, phosphate buffer solutions,
ammonium
chloride, calcium carbonate, calcium chloride, calcium citrate, calcium
glubionate,
calcium gluceptate, calcium gluconate, D-gluconic acid, calcium
glycerophosphate,
calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic
calcium
phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide
phosphate,
potassium acetate, potassium chloride, potassium gluconate, potassium
mixtures, dibasic
potassium phosphate, monobasic potassium phosphate, potassium phosphate
mixtures,
sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium
lactate,
dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate
mixtures,
tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-
free
water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and/or
combinations thereof.
[0347] Exemplary lubricating agents include, but are not limited to,
magnesium
stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl
behanate, hydrogenated
vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium
chloride,
leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and
combinations thereof.
[0348] Exemplary oils include, but are not limited to, almond, apricot
kernel, avocado,
babassu, bergamot, black current seed, borage, cade, camomile, canola,
caraway,
carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn,
cotton seed,
emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape
seed, hazel nut,
hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,
litsea cubeba,
macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive,
orange,
orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin
seed,
- 113 -
Date Recue/Date Received 2021-04-23
rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea
buckthorn,
sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,
vetiver,
walnut, and wheat germ oils. Exemplary oils include, but are not limited to,
butyl
stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl
sebacate,
dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl
alcohol, silicone
oil, and/or combinations thereof.
[0349] Excipients such as cocoa butter and suppository waxes, coloring
agents,
coating agents, sweetening, flavoring, and/ or perfuming agents can be present
in the
composition, according to the judgment of the formulator.
[0350] Exemplary additives include physiologically biocompatible buffers
(e.g.,
trimethylamine hydrochloride), addition of chelants (such as, for example,
DTPA or
DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA,
CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for
example,
calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). In
addition,
antioxidants and suspending agents can be used.
Adjuvants
[0351] Adjuvants may also be administered with or in combination with one
or more
of the nucleic acid vaccines described herein. Adjuvants may be used to
enhance the
immunogenicity of the nucleic acid vaccine, modify the immune response, reduce
the
amount of nucleic acid vaccine needed for immunization, reduced the frequency
of
additional or "booster" immunizations needed or to create an improved immune
response
in those with weakened or immunocompromised immune systems or the elderly. The
adjuvants may be a component of the formulation containing the nucleic acid
vaccine or
they may be co-administered with the nucleic acid vaccines. Co-administration
of the
adjuvant may be any method known in the art or described herein such as, but
not limited
to, intravenous (IV), intramuscular (IM), subcutaneous (SC) or intradermal
(ID).
[0352] In some embodiments, the adjuvant is natural or synthetic. The
adjuvants may
also be organic or inorganic.
- 114 -
Date Recue/Date Received 2021-04-23
[0353] In some embodiments, the adjuvant used with the nucleic acid vaccine
is from
a class of adjuvants such as, but not limited to carbohydrates,
microorganisms, mineral
salts (e.g., aluminum hydroxide, aluminum phosphate gel, or calcium phosphate
gel),
emulsions (e.g., oil emulsion, surfactant based emulsion, purified saponin,
and oil-in
water emulsion), inert vehicles, particulate adjuvants (e.g., unilamellar
liposomal vehicles
such as virosomes or a structured complex of saponions and lipids such as
polylactide co-
glycolide (PLG)), microbial derivatives, endogenous human immunomodulators,
and
tensoactive compounds. Listings of adjuvants which may be used with the
nucleic acid
vaccines described herein may be found on the web-based vaccine adjuvant
database
Vaxjo (see e.g., violinet.org/vaxjo or Sayers S, Ulysse G, Xiang Z, and He Y.
Vaxjo.
Journal of Biomedicine and Biotechnology. 2012;2012:831486. Epub 2012 Mar 13.
PMID: 22505817; the contents of which is herein incorporated by reference in
its
entirety).
[0354] Adjuvants may be selected for use with the nucleic acid vaccines by
one of
ordinary skill in the art. Adjuvants may be interferons, TNF-alpha, TNF-beta,
chemokines (e.g., CCL21, eotaxin, HMGB1, SA100-8a1pha, GCSF, GMCSF,
granulysin,
lactoferrin, ovalbumin, CD4OL, CD28 agonists, PD1, soluble PD1, PDL1, PDL2) or
interleukins (e.g., ILL IL2, IL4, IL6, IL7, IL10, IL12, IL13, IL15, IL17,
IL18, IL21, and
IL23) Non-limiting examples of adjuvants include Abisco-100 vaccine adjuvant,
Adamantylamide Dipeptide Vaccine Adjuvant, AdjumerTM, AF03, Albumin-heparin
microparticles vaccine adjuvant, Algal Glucan, Algammulin, alhydrogel,
aluminum
hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum
potassium
sulfate adjuvant, Aluminum vaccine adjuvant, amorphous aluminum
hydroxyphosphate
sulfate adjuvant, Arlacel A, ASO, A504, A503, AS-2 vaccine adjuvant,
Avridine0, B7-2
vaccine adjuvant, Bay R1005, Bordetella pertussis component Vaccine Adjuvant,
Bupivacaine vaccine adjuvant, Calcium Phosphate Gel, Calcium phosphate vaccine
adjuvant, Cationic Liposomal Vaccine Adjuvant, cationic liposome-DNA complex
JVRS- 100, Cholera toxin, Cholera toxin B subunit, Corynebacterium-derived P40
Vaccine Adjuvant, CpG DNA Vaccine Adjuvant, CRL1005, CTAl-DD gene fusion
- 115 -
Date Recue/Date Received 2021-04-23
protein, DDA Adjuvant, DHEA vaccine adjuvant, DL-PGL (Polyester poly (DL-
lactide-
co-glycolide)) vaccine adjuvant, DOC/Alum Complex, E. coli heat-labile toxin,
Etx B
subunit Adjuvant, Flagellin, Freund's Complete Adjuvant, Freund's Incomplete
Adjuvant, Gamma Inulin, Gerbu Adjuvant, GM-CSF, GMDP, Imiquimod,
Immunoliposomes Containing Antibodies to Costimulatory Molecules, ISCOM(s)Tm,
ISCOMA-TRIXO, Killed Corynebacterium parvum Vaccine Adjuvant,
Lipopolysaccharide, Liposomes, Loxoribine, LTK63 Vaccine Mutant Adjuvant,
LTK72
vaccine adjuvant, LTR192G Vaccine Adjuvant, Matrix-S, MF59, Montanide
Incomplete
Seppic Adjuvant, Montanide ISA 51, Montanide ISA 720 Adjuvant, MPL-SE vaccine
adjuvant, MPLTM Adjuvant, MTP-PE Liposomes, Murametide, Muramyl Dipeptide
Adjuvant, Murapalmitine, D-Murapalmitine, NAGO, nanoemulsion vaccine adjuvant,
Non-Ionic Surfactant Vesicles, non-toxic mutant El 12K of Cholera Toxin mCT-
E112K,
PMMA, Poly(LC), Polygen Vaccine Adjuvant, Protein Cochleates, QS-21, Quil-A
vaccine adjuvant, RC529 vaccine adjuvant, Recombinant h1FN-gamma/Interferon-g,
Rehydragel EV, Rehydragel HPA, Resiquimod, Ribi Vaccine Adjuvant, SAF-1,
Saponin
Vaccine Adjuvant, Sclavo peptide, Sendai Proteoliposomes, Sendai-containing
Lipid
Matrices, Specol, SPT (Antigen Formulation), Squalene-based Adjuvants, Stearyl
Tyrosine, Theramide0, Threonyl muramyl dipeptide (TMDP), Titer-Max Gold
Adjuvant,
Ty Particles vaccine adjuvant, and VSA-3 Adjuvant.
[0355] In some embodiments, the nucleic acid vaccines described herein may
be used
as a vaccine and may further comprise an adjuvant which may enable the vaccine
to elicit
a higher immune response. As a non-limiting example, the adjuvant could be a
sub-
micron oil-in-water emulsion which can elicit a higher immune response in
human
pediatric populations (see e.g., the adjuvanted vaccines described in US
Patent
Publication No. U520120027813 and U.S. Pat. No. 8,506,966, the contents of
each of
which are herein incorporated by reference in its entirety).
Dosing and Administration
[0356] The present disclosure encompasses the delivery of nucleic acid
vaccine
compositions including, for example, nucleic acid vaccine for COVID-19 for any
- 116 -
Date Recue/Date Received 2021-04-23
therapeutic, prophylactic, pharmaceutical, diagnostic or imaging use by any
appropriate
route taking into consideration likely advances in the sciences of drug
delivery. Delivery
may be naked or formulated.
[0357] The nucleic acid vaccine compositions of the present disclosure may
be
delivered to a cell naked. As used herein in, "naked" refers to delivering
nucleic acid
vaccine compositions free from agents which promote transfection. For example,
the
nucleic acid vaccine compositions delivered to the cell may contain no
modifications.
The naked nucleic acid vaccine compositions may be delivered to the cell using
routes of
administration known in the art and described herein.
[0358] The nucleic acid vaccine compositions of the present disclosure may
be
formulated, using the methods described herein. The formulations may contain
nucleic
acid vaccine compositions which may be modified and/or unmodified. The
formulations
may further include, but are not limited to, cell penetration agents, a
pharmaceutically
acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer,
a solvent,
and a sustained-release delivery depot. The formulated nucleic acid vaccine
compositions
may be delivered to the cell using routes of administration known in the art
and described
herein.
[0359] The compositions may also be formulated for direct delivery to an
organ or
tissue in any of several ways in the art including, but not limited to, direct
soaking or
bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions,
and/or drops, by
using substrates such as fabric or biodegradable materials coated or
impregnated with the
compositions, and the like. The nucleic acid vaccine compositions of the
present
disclosure may also be cloned into a retroviral replicating vector (RRV) and
transduced
to cells.
Dosing
[0360] Provided herein are methods comprising administering the nucleic
acid
vaccines described herein to a subject in need thereof. The exact amount
required will
vary from subject to subject, depending on the species, age, and general
condition of the
subject, the severity of the disease, the particular composition, its mode of
administration,
- 117 -
Date Recue/Date Received 2021-04-23
its mode of activity, and the like. Compositions are typically formulated in
dosage unit
form for ease of administration and uniformity of dosage. It will be
understood, however,
that the total daily usage of the compositions may be decided by the attending
physician
within the scope of sound medical judgment. The specific therapeutically
effective,
prophylactically effective, or appropriate imaging dose level for any
particular patient
will depend upon a variety of factors including the disorder being treated and
the severity
of the disorder; the activity of the specific compound employed; the specific
composition
employed; the age, body weight, general health, sex and diet of the patient;
the time of
administration, route of administration, and rate of excretion of the specific
compound
employed; the duration of the treatment; drugs used in combination or
coincidental with
the specific compound employed; and like factors well known in the medical
arts.
[0361] The present disclosure contemplates dosage levels of between about
0.001 and
about 100 mg nucleic acid vaccine (e.g., nucleic acid vaccine for COVID-19)/kg
body
weight per day, preferably between about 0.005 and about 50 mg/kg, 0.01 and
about 10
mg/kg, 0.05 and about 5 mg/kg, 0.1 and about 1 mg/kg body weight. Other
embodiments
contemplate a dosage of between about 0.001-0.010, 0.010-0.050, 0.050-0.100,
0.1-0.5,
0.5-1.0, 1.0-5.0, 5.0-10, or 10-50 mg/kg body weight. The dosages may be
administered
about hourly, multiple times per day, daily, every other day, weekly, every
other week,
monthly, or on an as-needed basis.
[0362] In some embodiments, compositions of the nucleic acid vaccines may
be
administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to
about 100
mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to
about
0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg
to
about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg
to
about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg
to
about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg
to
about 25 mg/kg, of subject body weight per day, one or more times a day, to
obtain the
desired therapeutic, diagnostic, prophylactic, or imaging effect. The desired
dosage may
be delivered three times a day, two times a day, once a day, every other day,
every third
- 118 -
Date Recue/Date Received 2021-04-23
day, every week, every two weeks, every three weeks, or every four weeks. In
certain
embodiments, the desired dosage may be delivered using multiple
administrations (e.g.,
two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,
thirteen, fourteen, or
more administrations). When multiple administrations are employed, split
dosing
regimens such as those described herein may be used.
[0363] In some embodiments, the nucleic acid vaccines may be administered
in split-
dose regimens. As used herein, a "split dose" is the division of single unit
dose or total
daily dose into two or more doses, e.g., two or more administrations of the
single unit
dose. As used herein, a "single unit dose" is a dose of any therapeutic
administered in one
dose/at one time/single route/single point of contact, i.e., single
administration event. As
used herein, a "total daily dose" is an amount given or prescribed in 24 hour
period. It
may be administered as a single unit dose. In some embodiments, the nucleic
acid
vaccines described herein are administered to a subject in split doses. The
nucleic acid
vaccines may be formulated in buffer only or in a formulation described
herein.
[0364] Such administration can be used as a chronic or acute therapy. The
amount of
drug that may be combined with the carrier to produce a single dosage form
will vary
depending upon the host treated and the particular mode of administration. A
typical
preparation will contain from about 5% to about 95% active compound (w/w).
Preferably, such preparations contain from about 20% to about 80%, 30% to
about 70%,
40% to about 60%, or about 50% active compound. In other embodiments, the
preparations used in the present disclosure will be about 5-10%, 10-20%, 20-
30%, 30-
40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, or greater than 99% of
the
active ingredient.
[0365] Upon improvement of a patient's condition, a maintenance dose of a
compound, composition or combination of the present disclosure may be
administered, if
necessary. Subsequently, the dosage or frequency of administration, or both,
may be
reduced, as a function of the symptoms, to a level at which the improved
condition is
retained when the symptoms have been alleviated to the desired level,
treatment should
- 119 -
Date Recue/Date Received 2021-04-23
cease. Patients may, however, require intermittent treatment on a long-term
basis upon
any recurrence of disease symptoms.
[0366] As the skilled artisan will appreciate, lower or higher doses than
those recited
above may be required. Specific dosage and treatment regimens for any
particular patient
will depend upon a variety of factors, including the activity of the specific
compound
employed, the age, body weight, general health status, gender, diet, time of
administration, rate of excretion, drug combination, the severity and course
of an
infection, the patient's disposition to the infection and the judgment of the
treating
physician.
Delivery
[0367] In some embodiments, the delivery of the nucleic acid vaccines may
be naked
or formulated.
[0368] In some embodiments, the nucleic acid vaccines described herein may
be
delivered to a cell naked. As used herein in, "naked" refers to delivering
nucleic acid
vaccines free from agents which promote transfection. For example, the nucleic
acid
vaccines delivered to the cell may contain no modifications. The naked nucleic
acid
vaccines may be delivered to the cell using routes of administration known in
the art and
described herein.
[0369] In some embodiments, the nucleic acid vaccines described herein may
be
formulated, using the methods described herein. The formulations may further
include,
but are not limited to, cell penetration agents, a pharmaceutically acceptable
carrier, a
delivery agent, a bioerodible or biocompatible polymer, a solvent, and a
sustained-release
delivery depot. The formulated nucleic acid vaccines may be delivered to the
cell using
routes of administration known in the art and described herein.
[0370] The compositions may also be formulated for direct delivery to an
organ or
tissue in any of several ways in the art including, but not limited to, direct
soaking or
bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions,
and/or drops, by
using substrates such as fabric or biodegradable materials coated or
impregnated with the
compositions, and the like.
- 120 -
Date Recue/Date Received 2021-04-23
Administration
[0371] In some embodiments, the nucleic acid vaccine compositions of the
present
disclosure may be administered by any route which results in a therapeutically
effective
outcome. These include, but are not limited to enteral (into the intestine),
gastroenteral,
epidural (into the dura matter), oral (by way of the mouth), transdermal,
peridural,
intracerebral (into the cerebrum), intracerebroventricular (into the cerebral
ventricles),
epicutaneous (application onto the skin), intradermal, (into the skin itself),
subcutaneous
(under the skin), nasal administration (through the nose), intravenous (into a
vein),
intravenous bolus, intravenous drip, intraarterial (into an artery),
intramuscular (into a
muscle), intracardiac (into the heart), intraosseous infusion (into the bone
marrow),
intrathecal (into the spinal canal), intraperitoneal, (infusion or injection
into the
peritoneum), intravesical infusion, intravitreal, (through the eye),
intracavernous injection
(into a pathologic cavity) intracavitary (into the base of the penis),
intravaginal
administration, intrauterine, extra-amniotic administration, transdermal
(diffusion
through the intact skin for systemic distribution), transmucosal (diffusion
through a
mucous membrane), transvaginal, insufflation (snorting), sublingual,
sublabial, enema,
eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the
ear), buccal
(directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or
teeth),
electroosmosis, endoceryical, endosinusial, endotracheal, extracorporeal,
hemodialysis,
infiltration, interstitial, intraabdominal, intra-amniotic, intra-articular,
intrabiliary,
intrabronchial, intrabursal, intracartilaginous (within a cartilage),
intracaudal (within the
cauda equine), intracisternal (within the cisterna magna cerebellomedularis),
intracorneal
(within the cornea), dental intracornal, intracoronary (within the coronary
arteries),
intracorporus cavernosum (within the dilatable spaces of the corporus
cavernosa of the
penis), intradiscal (within a disc), intraductal (within a duct of a gland),
intraduodenal
(within the duodenum), intradural (within or beneath the dura), intraepidermal
(to the
epidermis), intraesophageal (to the esophagus), intragastric (within the
stomach),
intragingival (within the gingivae), intraileal (within the distal portion of
the small
intestine), intralesional (within or introduced directly to a localized
lesion), intraluminal
- 121 -
Date Recue/Date Received 2021-04-23
(within a lumen of a tube), intralymphatic (within the lymph), intramedullary
(within the
marrow cavity of a bone), intrameningeal (within the meninges), intraocular
(within the
eye), intraovarian (within the ovary), intrapericardial (within the
pericardium),
intrapleural (within the pleura), intraprostatic (within the prostate gland),
intrapulmonary
(within the lungs or its bronchi), intrasinal (within the nasal or periorbital
sinuses),
intraspinal (within the vertebral column), intrasynovial (within the synovial
cavity of a
joint), intratendinous (within a tendon), intratesticular (within the
testicle), intrathecal
(within the cerebrospinal fluid at any level of the cerebrospinal axis),
intrathoracic
(within the thorax), intratubular (within the tubules of an organ), intratumor
(within a
tumor), intratympanic (within the aurus media), intravascular (within a vessel
or vessels),
intraventricular (within a ventricle), iontophoresis (by means of electric
current where
ions of soluble salts migrate into the tissues of the body), irrigation (to
bathe or flush
open wounds or body cavities), laryngeal (directly upon the larynx),
nasogastric (through
the nose and into the stomach), occlusive dressing technique, ophthalmic (to
the external
eye), oropharyngeal (directly to the mouth and pharynx), parenteral,
percutaneous,
periarticular, peridural, perineural, periodontal, rectal, respiratory (within
the respiratory
tract by inhaling orally or nasally for local or systemic effect), retrobulbar
(behind the
pons or behind the eyeball), intramyocardial (entering the myocardium), soft
tissue,
subarachnoid, subconjunctival, submucosal, transplacental (through or across
the
placenta), transtracheal (through the wall of the trachea), transtympanic
(across or
through the tympanic cavity), ureteral (to the ureter), urethral (to the
urethra), vaginal,
caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion,
photopheresis
or spinal. In specific embodiments, compositions may be administered in a way
which
allows them to cross the blood-brain barrier, vascular barrier, or other
epithelial barrier.
[0372]
Delivery of modified therapeutic compounds described herein to a subject over
prolonged periods of time, for example, for periods of one week to one year,
may be
accomplished by a single administration of a controlled release system
containing
sufficient active ingredient for the desired release period. Various
controlled release
systems, such as monolithic or reservoir-type microcapsules, depot implants,
polymeric
- 122 -
Date Recue/Date Received 2021-04-23
hydrogels, osmotic pumps, vesicles, micelles, liposomes, transdermal patches,
iontophoretic devices and alternative injectable dosage forms may be utilized
for this
purpose. Localization at the site to which delivery of the active ingredient
is desired is an
additional feature of some controlled release devices, which may prove
beneficial in the
treatment of certain disorders.
[0373] In some embodiments, the nucleic acid vaccines described herein may
be
administered intranasally similar to the administration of live vaccines. In
another aspect
the polynucleotide may be administered intramuscularly or intradermally
similarly to the
administration of inactivated vaccines known in the art.
[0374] In certain embodiments for transdermal administration, delivery
across the
barrier of the skin would be enhanced using electrodes (e.g. iontophoresis),
electroporation, or the application of short, high-voltage electrical pulses
to the skin,
radiofrequencies, ultrasound (e.g. sonophoresis), microprojections (e.g.
microneedles), jet
injectors, thermal ablation, magnetophoresis, lasers, velocity, or
photomechanical waves.
The drug can be included in single-layer drug-in-adhesive, multi-layer drug-in-
adhesive,
reservoir, matrix, or vapor style patches, or could utilize patchless
technology. Delivery
across the barrier of the skin could also be enhanced using encapsulation, a
skin lipid
fluidizer, or a hollow or solid microstructured transdermal system (MTS, such
as that
manufactured by 3M), jet injectors. Additives to the formulation to aid in the
passage of
therapeutic compounds through the skin include prodrugs, chemicals,
surfactants, cell
penetrating peptides, permeation enhancers, encapsulation technologies,
enzymes,
enzyme inhibitors, gels, nanoparticles and peptide or protein chaperones.
[0375] One form of controlled-release formulation contains the therapeutic
compound
or its salt dispersed or encapsulated in a slowly degrading, non-toxic, non-
antigenic
polymer such as copoly(lactic/glycolic) acid, as described in the pioneering
work of Kent
et al., US Patent No. 4,675,189, incorporated by reference herein. The
compounds, or
their salts, may also be formulated in cholesterol or other lipid matrix
pellets, or
silastomer matrix implants. Additional slow release, depot implant or
injectable
formulations will be apparent to the skilled artisan. See, for example,
Sustained and
- 123 -
Date Recue/Date Received 2021-04-23
Controlled Release Drug Delivery Systems, JR Robinson ed., Marcel Dekker Inc.,
New
York, 1978; and Controlled Release of Biologically Active Agents, RW Baker,
John
Wiley & Sons, New York, 1987. The foregoing are incorporated by reference in
their
entirety.
[0376] An additional form of controlled-release formulation comprises a
solution of
biodegradable polymer, such as copoly(lactic/glycolic acid) or block
copolymers of lactic
acid and PEG, is a bioacceptable solvent, which is injected subcutaneously or
intramuscularly to achieve a depot formulation. Mixing of the therapeutic
compounds
described herein with such a polymeric formulation is suitable to achieve very
long
duration of action formulations.
[0377] When formulated for nasal administration, the absorption across the
nasal
mucous membrane may be further enhanced by surfactants, such as, for example,
glycocholic acid, cholic acid, taurocholic acid, ethocholic acid, deoxycholic
acid,
chenodeoxycholic acid, dehdryocholic acid, glycodeoxycholic acid,
cycledextrins and the
like in an amount in the range of between about 0.1 and 15 weight percent,
between about
0.5 and 4 weight percent, or about 2 weight percent. An additional class of
absorption
enhancers reported to exhibit greater efficacy with decreased irritation is
the class of
alkyl maltosides, such as tetradecylmaltoside (Arnold, JJ et al., 2004, J
Pharm Sci 93:
2205-13; Ahsan, F et al., 2001, Pharm Res 18:1742-46) and references therein,
all of
which are hereby incorporated by reference.
[0378] The pharmaceutical compositions may be in the form of a sterile
injectable
preparation, for example, as a sterile injectable aqueous or oleaginous
suspension. This
suspension may be formulated according to techniques known in the art using
suitable
dispersing or wetting agents (such as, for example, Tween 80) and suspending
agents.
The sterile injectable preparation may also be a sterile injectable solution
or suspension in
a non-toxic parenterally-acceptable diluent or solvent, for example, as a
solution in 1,3-
butanediol. Among the acceptable vehicles and solvents that may be employed
are
mannitol, water, Ringer's solution and isotonic sodium chloride solution. In
addition,
sterile, fixed oils are conventionally employed as a solvent or suspending
medium. For
- 124 -
Date Recue/Date Received 2021-04-23
this purpose, any bland fixed oil may be employed including synthetic mono- or
diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives
are useful in the
preparation of injectables, as are natural pharmaceutically-acceptable oils,
such as olive
oil or castor oil, especially in their polyoxyethylated versions. These oil
solutions or
suspensions may also contain a long-chain alcohol diluent or dispersant such
as Ph. Hely
or a similar alcohol.
[0379] The pharmaceutical compositions of the present disclosure may be
orally
administered in any orally acceptable dosage form including, but not limited
to, capsules,
tablets, and aqueous suspensions and solutions. In the case of tablets for
oral use, carriers
that are commonly used include lactose and corn starch. Lubricating agents,
such as
magnesium stearate, are also typically added. For oral administration in a
capsule form,
useful diluents include lactose and dried corn starch. When aqueous
suspensions are
administered orally, the active ingredient is combined with emulsifying and
suspending
agents. If desired, certain sweetening and/or flavoring and/or coloring agents
may be
added.
[0380] The pharmaceutical compositions of present disclosure may also be
administered in the form of suppositories for rectal administration. These
compositions
can be prepared by mixing a compound of the present disclosure with a suitable
non-
irritating excipient that is solid at room temperature but liquid at the
rectal temperature
and therefore will melt in the rectum to release the active components. Such
materials
include, but are not limited to, cocoa butter, beeswax and polyethylene
glycols.
[0381] Topical administration of the pharmaceutical compositions of the
present
disclosure is especially useful when the desired treatment involves areas or
organs readily
accessible by topical application. For application topically to the skin, the
pharmaceutical composition should be formulated with a suitable ointment
containing the
active components suspended or dissolved in a carrier. Carriers for topical
administration
of the compounds of the present disclosure include, but are not limited to,
mineral oil,
liquid petroleum, white petroleum, propylene glycol, polyoxyethylene
polyoxypropylene
compound, emulsifying wax and water. Alternatively, the pharmaceutical
composition
- 125 -
Date Recue/Date Received 2021-04-23
can be formulated with a suitable lotion or cream containing the active
compound
suspended or dissolved in a carrier. Suitable carriers include, but are not
limited to,
mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl
alcohol, 2-
octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of
the
present disclosure may also be topically applied to the lower intestinal tract
by rectal
suppository formulation or in a suitable enema formulation. Topical
transdermal patches
are also included in the present disclosure.
[0382] The pharmaceutical compositions of the present disclosure may be
administered by nasal aerosol or inhalation. Such compositions are prepared
according to
techniques well-known in the art of pharmaceutical formulation and may be
prepared as
solutions in saline, employing benzyl alcohol or other suitable preservatives,
absorption
promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing
or
dispersing agents known in the art.
[0383] When formulated for delivery by inhalation, a number of formulations
offer
advantages. Adsorption of the therapeutic compound to readily dispersed solids
such as
diketopiperazines (for example, Technosphere particles (Pfutzner, A and Forst,
T, 2005,
Expert Opin Drug Deliv 2:1097-1106) or similar structures gives a formulation
that
results in rapid initial uptake of the therapeutic compound. Lyophilized
powders,
especially glassy particles, containing the therapeutic compound and an
excipient are
useful for delivery to the lung with good bioavailability, for example, see
Exubera
(inhaled insulin, Pfizer, Inc. and Aventis Pharmaceuticals Inc.) and Afrezza
(inhaled
insulin, Mannkind, Corp.).
Dosage Forms
[0384] A pharmaceutical composition described herein can be formulated into
a
dosage form described herein, such as a topical, intranasal, intratracheal, or
injectable
(e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac,
intraperitoneal,
subcutaneous).
- 126 -
Date Recue/Date Received 2021-04-23
Liquid Dosage Forms
[0385] Liquid dosage forms for parenteral administration include, but are
not limited
to, pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions,
syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms
may comprise
inert diluents commonly used in the art including, but not limited to, 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 mixtures thereof. In certain
embodiments for
parenteral administration, compositions may be mixed with solubilizing agents
such as
CREMO- PHORO, alcohols, oils, modified oils, glycols, polysorbates,
cyclodextrins,
polymers, and/or combinations thereof.
Injectable
[0386] Injectable preparations, for example, sterile injectable aqueous or
oleaginous
suspensions may be formulated according to the known art and may include
suitable
dispersing agents, wetting agents, and/or suspending agents. Sterile
injectable
preparations may be sterile injectable solutions, suspensions, and/or
emulsions in
nontoxic parenterally acceptable diluents and/or solvents, for example, a
solution in 1,3-
butanediol. Among the acceptable vehicles and solvents that may be employed
include,
but are not limited to, water, Ringer's solution, U.S.P., and isotonic sodium
chloride
solution. Sterile, fixed oils are conventionally employed as a solvent or
suspending
medium. For this purpose, any bland fixed oil can be employed including
synthetic
mono- or diglycerides. Fatty acids such as oleic acid can be used in the
preparation of
injectables.
[0387] Injectable formulations can be sterilized, for example, by
filtration through a
bacterial-retaining filter, and/or by incorporating sterilizing agents in the
form of sterile
solid compositions which can be dissolved or dispersed in sterile water or
other sterile
injectable medium prior to use.
- 127 -
Date Recue/Date Received 2021-04-23
[0388] In order to prolong the effect of an active ingredient, it may be
desirable to
slow the absorption of the active ingredient from subcutaneous or
intramuscular injection.
This may be accomplished by the use of a liquid suspension of crystalline or
amorphous
material with poor water solubility. The rate of absorption of the nucleic
acid vaccine
then depends upon its rate of dissolution which, in turn, may depend upon
crystal size
and crystalline form. Alternatively, delayed absorption of a parenterally
administered
nucleic acid vaccine may be accomplished by dissolving or suspending the
nucleic acid
vaccine in an oil vehicle. Injectable depot forms are made by forming
microencapsule
matrices of the nucleic acid vaccine in biodegradable polymers such as
polylactide-
polyglycolide. Depending upon the ratio of nucleic acid vaccine to polymer and
the
nature of the particular polymer employed, the rate of polynucleotides release
can be
controlled. Examples of other biodegradable polymers include, but are not
limited to,
poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be
prepared
by entrapping the nucleic acid vaccine in liposomes or microemulsions which
are
compatible with body tissues.
Pulmonary
[0389] Formulations described herein as being useful for pulmonary delivery
may also
be used for intranasal delivery of a pharmaceutical composition. Another
formulation
suitable for intranasal administration may be a coarse powder comprising the
active
ingredient and having an average particle from about 0.2 pm to 500 pm. Such a
formulation may be administered in the manner in which snuff is taken, i.e. by
rapid
inhalation through the nasal passage from a container of the powder held close
to the
nose.
[0390] Formulations suitable for nasal administration may, for example,
comprise
from about as little as 0.1% (w/w) and as much as 100% (w/w) of active
ingredient, and
may comprise one or more of the additional ingredients described herein. A
pharmaceutical composition may be prepared, packaged, and/or sold in a
formulation
suitable for buccal administration. Such formulations may, for example, be in
the form of
tablets and/or lozenges made using conventional methods, and may, for example,
contain
- 128 -
Date Recue/Date Received 2021-04-23
about 0.1% to 20% (w/w) active ingredient, where the balance may comprise an
orally
dissolvable and/or degradable composition and, optionally, one or more of the
additional
ingredients described herein. Alternately, formulations suitable for buccal
administration
may comprise a powder and/or an aerosolized and/or atomized solution and/or
suspension comprising active ingredient. Such powdered, aerosolized, and/or
aerosolized
formulations, when dispersed, may have an average particle and/or droplet size
in the
range from about 0.1 nm to about 200 nm, and may further comprise one or more
of any
additional ingredients described herein.
[0391] 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.
Solid Dosage Forms: Coatings or Shells
[0392] Solid dosage forms of tablets, dragees, capsules, pills, and
granules can be
prepared with coatings and shells such as enteric coatings and other coatings
well known
in the pharmaceutical formulating art. They may optionally comprise pacifying
agents
and can be of a composition that they release the active ingredient(s) only,
or
preferentially, in a certain part of the intestinal tract, optionally, in a
delayed manner.
Examples of embedding compositions which can be used include polymeric
substances
and waxes. Solid compositions of a similar type may be employed as fillers in
soft and
hard-filled gelatin capsules using such excipients as lactose or milk sugar as
well as high
molecular weight polyethylene glycols and the like.
Properties of the Pharmaceutical Compositions
[0393] The nucleic acid vaccine pharmaceutical compositions described
herein may
be characterized using one or more of bioavailability, therapeutic window,
volume of
distribution, biological effect and detection of polynucleotides by mass
spectrometry.
Bioavailability
[0394] The nucleic acid vaccines, when formulated into a composition with
a delivery
agent as described herein, can exhibit an increase in bioavailability as
compared to a
composition lacking a delivery agent as described herein. As used herein, the
term
- 129 -
Date Recue/Date Received 2021-04-23
"bioavailability" refers to the systemic availability of a given amount of
nucleic acid
vaccines administered to a mammal. Bioavailability can be assessed by
measuring the
area under the curve (AUC) or the maximum serum or plasma concentration of the
unchanged form of a compound following administration of the compound to a
mammal.
AUC is a determination of the area under the curve plotting the serum or
plasma
concentration of a compound along the ordinate (Y-axis) against time along the
abscissa
(X-axis). Generally, the AUC for a particular compound can be calculated using
methods
known to those of ordinary skill in the art and as described in G. S. Banker,
Modem
Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72, Marcel Dekker,
N.Y, Inc.,
1996, herein incorporated by reference in its entirety.
[0395] The Cmax value is the maximum concentration of the compound achieved in
the serum or plasma of a mammal following administration of the compound to
the
mammal. The Cmax value of a particular compound can be measured using methods
known to those of ordinary skill in the art. The phrases "increasing
bioavailability" or
"improving the pharmacokinetics," as used herein mean that the systemic
availability of a
first nucleic acid vaccine, measured as AUC, Cmax, or Cmin, in a mammal is
greater,
when co-administered with a delivery agent as described herein, than when such
co-
administration does not take place. In some embodiments, the bioavailability
of the
nucleic acid vaccines can increase by at least about 2%, at least about 5%, 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
55%, at least about 60%, at least about 65%, at least about 70%, at least
about 75%, at
least about 80%, at least about 85%, at least about 90%, at least about 95%,
or about
100%.
[0396] In some embodiments, liquid formulations of nucleic acid vaccines
may have
varying in vivo half-life, requiring modulation of doses to yield a
therapeutic effect. To
address this, in some embodiments, nucleic acid vaccine formulations may be
designed to
improve bioavailability and/or therapeutic effect during repeat
administrations. Such
formulations may enable sustained release of nucleic acid vaccines and/or
reduce nucleic
- 130 -
Date Recue/Date Received 2021-04-23
acid vaccine degradation rates by nucleases. In some embodiments, suspension
formulations are provided comprising nucleic acid vaccines, water immiscible
oil depots,
surfactants and/or co-surfactants and/or co-solvents. Combinations of oils and
surfactants
may enable suspension formulation with nucleic acid vaccines. Delivery of
nucleic acid
vaccines in a water immiscible depot may be used to improve bioavailability
through
sustained release of polynucleotides from the depot to the surrounding
physiologic
environment and/or prevent polynucleotide degradation by nucleases.
[0397] In some embodiments, cationic nanoparticles comprising combinations
of
divalent and monovalent cations may be formulated with nucleic acid vaccines.
Such
nanoparticles may form spontaneously in solution over a given period (e.g.
hours, days,
etc.). Such nanoparticles do not form in the presence of divalent cations
alone or in the
presence of monovalent cations alone. The delivery of nucleic acid vaccines in
cationic
nanoparticles or in one or more depot comprising cationic nanoparticles may
improve
nucleic acid vaccine bioavailability by acting as a long-acting depot and/or
reducing the
rate of degradation by nucleases.
Therapeutic Window
[0398] The nucleic acid vaccines, when formulated into a composition with a
delivery
agent as described herein, can exhibit an increase in the therapeutic window
of the
administered nucleic acid vaccine composition as compared to the therapeutic
window of
the administered nucleic acid vaccine composition lacking a delivery agent as
described
herein. As used herein "therapeutic window" refers to the range of plasma
concentrations,
or the range of levels of therapeutically active substance at the site of
action, with a high
probability of eliciting a therapeutic effect. In some embodiments, the
therapeutic
window of the nucleic acid vaccines when co-administered with a delivery agent
as
described herein can increase by at least about 2%, at least about 5%, 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 55%,
at least about 60%, at least about 65%, at least about 70%, at least about
75%, at least
about 80%, at least about 85%, at least about 90%, at least about 95%, or
about 100%.
- 131 -
Date Recue/Date Received 2021-04-23
Volume ofDistribution
[0399] The nucleic acid vaccines, when formulated into a composition with a
delivery
agent as described herein, can exhibit an improved volume of distribution
(Vdist), e.g.,
reduced or targeted, relative to a composition lacking a delivery agent as
described
herein. The volume of distribution (Vdist) relates the amount of the drug in
the body to
the concentration of the drug in the blood or plasma. As used herein, the term
"volume of
distribution" refers to the fluid volume that would be required to contain the
total amount
of the drug in the body at the same concentration as in the blood or plasma:
Vdist equals
the amount of drug in the body/concentration of drug in blood or plasma. For
example,
for a 10 mg dose and a plasma concentration of 10 mg/L, the volume of
distribution
would be 1 liter. The volume of distribution reflects the extent to which the
drug is
present in the extravascular tissue. A large volume of distribution reflects
the tendency of
a compound to bind to the tissue components compared with plasma protein
binding. In a
clinical setting, Vdist can be used to determine a loading dose to achieve a
steady state
concentration. In some embodiments, the volume of distribution of the nucleic
acid
vaccines when co-administered with a delivery agent as described herein can
decrease at
least about 2%, at least about 5%, 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 55%, at least about 60%,
at least about
65%, at least about 70%.
Biological Effect
[0400] In some embodiments, the biological effect of the nucleic acid
vaccine
delivered to the animals may be categorized by analyzing the protein
expression in the
animals. The protein expression may be determined from analyzing a biological
sample
collected from a mammal administered the nucleic acid vaccine described
herein.
Detection of Polynucleotides by Mass Spectrometry
[0401] Mass spectrometry (MS) is an analytical technique that can provide
structural
and molecular mass/concentration information on molecules after their
conversion to
ions. The molecules are first ionized to acquire positive or negative charges
and then they
- 132 -
Date Recue/Date Received 2021-04-23
travel through the mass analyzer to arrive at different areas of the detector
according to
their mass/charge (m/z) ratio.
[0402] Mass spectrometry is performed using a mass spectrometer which
includes an
ion source for ionizing the fractionated sample and creating charged molecules
for further
analysis. For example ionization of the sample may be performed by
electrospray
ionization (ESI), atmospheric pressure chemical ionization (APCI),
photoionization,
electron ionization, fast atom bombardment (FAB)/liquid secondary ionization
(LSIMS),
matrix assisted laser desorption/ ionization (MALDI), field ionization, field
desorption,
thermospray/plasmaspray ionization, and particle beam ionization. The skilled
artisan
will understand that the choice of ionization method can be determined based
on the
analyte to be measured, type of sample, the type of detector, the choice of
positive versus
negative mode, etc.
[0403] After the sample has been ionized, the positively charged or
negatively
charged ions thereby created may be analyzed to determine a mass-to-charge
ratio (i.e.,
m/z). Suitable analyzers for determining mass-to-charge ratios include
quadropole
analyzers, ion traps analyzers, and time-of-flight analyzers. The ions may be
detected
using several detection modes. For example, selected ions may be detected
(i.e., using a
selective ion monitoring mode (SIM)), or alternatively, ions may be detected
using a
scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction
monitoring (SRM).
[0404] Liquid chromatography-multiple reaction monitoring (LC-MS/MRM) coupled
with stable isotope labeled dilution of peptide standards has been shown to be
an
effective method for protein verification (e.g., Keshishian et al., Mol Cell
Proteomics
2009 8: 2339-2349; Kuhn et al., Clin Chem 2009 55:1108-1117; Lopez et al.,
Clin Chem
2010 56:281- 290; each of which are herein incorporated by reference in its
entirety).
Unlike untargeted mass spectrometry frequently used in biomarker discovery
studies,
targeted MS methods are peptide sequence-based modes of MS that focus the full
analytical capacity of the instrument on tens to hundreds of selected peptides
in a
complex mixture. By restricting detection and fragmentation to only those
peptides
- 133 -
Date Recue/Date Received 2021-04-23
derived from proteins of interest, sensitivity and reproducibility are
improved
dramatically compared to discovery-mode MS methods. This method of mass
spectrometry based multiple reaction monitoring (MRM) quantitation of proteins
can
dramatically impact the discovery and quantitation of biomarkers via rapid,
targeted,
multiplexed protein expression profiling of clinical samples.
[0405] In some embodiments, the biological sample, once obtained from the
subject,
may be subjected to enzyme digestion. As used herein, the term "digest" means
to break
apart into shorter peptides. As used herein, the phrase "treating a sample to
digest
proteins" means manipulating a sample in such a way as to break down proteins
in a
sample. These enzymes include, but are not limited to, trypsin, endoproteinase
Glu-C and
chymotrypsin.
[0406] In some embodiments, a biological sample may be analyzed for protein
using
electrospray ionization. Electrospray ionization (ESI) mass spectrometry
(ESIMS) uses
electrical energy to aid in the transfer of ions from the solution to the
gaseous phase
before they are analyzed by mass spectrometry. Samples may be analyzed using
methods
known in the art (e.g., Ho et al., Clin Biochem Rev. 2003 24(1):3-12; herein
incorporated
by reference in its entirety). The ionic species contained in solution may be
transferred
into the gas phase by dispersing a fine spray of charge droplets, evaporating
the solvent
and ejecting the ions from the charged droplets to generate a mist of highly
charged
droplets. The mist of highly charged droplets may be analyzed using at least
1, at least 2,
at least 3 or at least 4 mass analyzers such as, but not limited to, a
quadropole mass
analyzer. Further, the mass spectrometry method may include a purification
step. As a
non-limiting example, the first quadrapole may be set to select a single m/z
ratio so it
may filter out other molecular ions having a different m/z ratio which may
eliminate
complicated and time-consuming sample purification procedures prior to MS
analysis.
[0407] In some embodiments, a biological sample may be analyzed for protein
in a
tandem ESIMS system (e.g., MS/MS). As non-limiting examples, the droplets may
be
analyzed using a product scan (or daughter scan) a precursor scan (parent
scan) a neutral
loss or a multiple reaction monitoring.
- 134 -
Date Recue/Date Received 2021-04-23
[0408] In some embodiments, a biological sample may be analyzed using
matrix-
assisted laser desorption/ionization (MALDI) mass spectrometry (MALDIMS).
MALDI
provides for the nondestructive vaporization and ionization of both large and
small
molecules, such as proteins. In MALDI analysis, the analyte is first co-
crystallized with a
large molar excess of a matrix compound, which may also include, but is not
limited to,
an ultraviolet absorbing weak organic acid. Non-limiting examples of matrices
used in
MALDI are a-cyano-4-hy- droxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic
acid
and 2,5-dihydroxybenzoic acid. Laser radiation of the analyte-matrix mixture
may result
in the vaporization of the matrix and the analyte. The laser induced
desorption provides
high ion yields of the intact analyte and allows for measurement of compounds
with high
accuracy. Samples may be analyzed using methods known in the art (e.g., Lewis,
Wei
and Siuzdak, Encyclopedia of Analytical Chemistry 2000:5880-5894; herein
incorporated
by reference in its entirety). As non-limiting examples, mass analyzers used
in the
MALDI analysis may include a linear time-of-flight (TOF), a TOF reflectron or
a Fourier
transform mass analyzer.
Expression Systems
[0409] In some embodiments, nucleic acid vaccines described herein may be
operably
linked to one or more regulatory nucleotide sequences and encoded in an
expression
construct. Regulatory nucleotide sequences will generally be appropriate for a
host cell
used for expression. Numerous types of appropriate expression vectors and
suitable
regulatory sequences are known in the art for a variety of host cells.
Typically, the one or
more regulatory nucleotide sequences may include, but are not limited to,
promoter
sequences, leader or signal sequences, transcriptional start and termination
sequences,
and enhancer or activator sequences. Constitutive or inducible promoters as
known in the
art are also contemplated. The promoters may be either naturally occurring
promoters, or
hybrid promoters that combine elements of more than one promoter. An
expression
construct may be present in a cell on an episome, such as a plasmid, or the
expression
construct may be inserted in a chromosome. In a specific embodiment, the
expression
vector includes a selectable marker gene to allow the selection of transformed
host cells.
- 135 -
Date Recue/Date Received 2021-04-23
Certain embodiments include an expression vector encoding a nucleic acid
vaccine for
COVID-19 sequence operably linked to at least one regulatory sequence.
Regulatory
sequences for use herein include promoters, enhancers, and other expression
control
elements. In certain embodiments, an expression vector is designed considering
the
choice of the host cell to be transformed, the particular nucleic acid vaccine
sequence to
be expressed, the vector's copy number, the ability to control that copy
number, or the
expression of other proteins encoded by the vector, such as antibiotic
markers.
[0410] In some embodiments, the gene products of the combinatorial
libraries
generated by the combinatorial mutagenesis of the nucleic acids described
herein may be
screened. Such screening methods include, for example, cloning the gene
library into
replicable expression vectors, transforming appropriate cells with the
resulting library of
vectors, and expressing the combinatorial genes under conditions to form such
library.
The screening methods optionally further comprise detecting a desired activity
and
isolating a product detected. Each of the illustrative assays described below
are amenable
to high-throughput analysis as necessary to screen large numbers of degenerate
sequences
created by combinatorial mutagenesis techniques.
[0411] In some embodiments, the nucleic acids described herein may be
expressed in
microorganisms. As a non-limiting example, the nucleic acid may be expressed
in a
bacterial system, for example, in Bacillus brevis , Bacillus megaterium,
Bacillus subtilis,
Caulobacter crescentus, Escherichia coli and their derivatives. Exemplary
promoters
include the 1-arabinose inducible araBAD promoter (PBAD), the lac promoter,
the 1-
rhamnose inducible rhaP BAD promoter, the T7 RNA polymerase promoter, the trc
and
tac promoter, the lambda phage promoter Pl, and the anhydrotetracycline-
inducible tetA
promoter/operator.
[0412] In some embodiments, the nucleic acids described herein may be
expressed in
a yeast expression system. Non-limiting examples of promoters which may be
used in
yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et
al., I
Biol. Chem. 255:2073 (1980)); other glycolytic enzymes (Hess et al., J Adv.
Enzyme Res.
7:149 (1968); Holland et al., Biochemistry 17:4900 (1978). Others promoters
are from,
- 136 -
Date Recue/Date Received 2021-04-23
e.g., enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-
phosphoglycerate
mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase,
glucokinase alcohol oxidase I (A0X1), alcohol dehydrogenase 2, isocytochrome
C, acid
phosphatase, degradative enzymes associated with nitrogen metabolism, and the
aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes
responsible
for maltose and galactose utilization. Any plasmid vector containing a yeast-
compatible
promoter and termination sequences, with or without an origin of replication,
is suitable.
Certain yeast expression systems are commercially available, for example, from
Clontech
Laboratories, Inc. (Palo Alto, Calif., e.g. Pyex 4T family of vectors for S.
cerevisiae),
Invitrogen (Carlsbad, Calif., e.g. Ppicz series Easy Select Pichia Expression
Kit) and
Stratagene (La Jolla, Calif., e.g. ESP.TM. Yeast Protein Expression and
Purification
System for S. pombe and Pesc vectors for S. cerevisiae).
[0413] In some embodiments, the nucleic acids described herein may be
expressed in
mammalian expression systems. Non-limiting examples of mammalian promoters
include, for example, promoters from the following genes: ubiquitin/527a
promoter of
the hamster (WO 97/15664), Simian vacuolating virus 40 (5V40) early promoter,
adenovirus major late promoter, mouse metallothionein-I promoter, the long
terminal
repeat region of Rous Sarcoma Virus (RSV), mouse mammary tumor virus promoter
(MMTV), Moloney murine leukemia virus Long Terminal repeat region, and the
early
promoter of human Cytomegalovirus (CMV). Examples of other heterologous
mammalian promoters are the actin, immunoglobulin or heat shock promoter(s).
In a
specific embodiment, a yeast alcohol oxidase promoter is used.
[0414] In some embodiments, promoters for use in mammalian host cells can
be
obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK
2,211,504 published 5 Jul. 1989), bovine papilloma virus, avian sarcoma virus,
cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (5V40).
In further
embodiments, heterologous mammalian promoters are used. Examples include the
actin
promoter, an immunoglobulin promoter, and heat-shock promoters. The early and
late
- 137 -
Date Recue/Date Received 2021-04-23
promoters of SV40 are conveniently obtained as an SV40 restriction fragment
which also
contains the SV40 viral origin of replication. Fiers et al., Nature 273: 113-
120 (1978).
The immediate early promoter of the human cytomegalovirus is conveniently
obtained as
a HindIII E restriction fragment. Greenaway, P. J. et al., Gene 18: 355-360
(1982). The
foregoing references are incorporated by reference in their entirety.
[0415] In some embodiments, the nucleic acids described herein may be
expressed in
insect cell expression systems. Eukaryotic expression systems employing insect
cell
hosts may rely on either plasmid or baculoviral expression systems. Typical
insect host
cells are derived from the fall army worm (Spodoptera frugiperda). For
expression of a
foreign protein these cells are infected with a recombinant form of the
baculovirus
Autographa californica nuclear polyhedrosis virus which has the gene of
interest
expressed under the control of the viral polyhedron promoter. Other insects
infected by
this virus include a cell line known commercially as "High 5" (Invitrogen)
which is
derived from the cabbage looper (Trichoplusia ni). Another baculovirus
sometimes used
is the Bombyx mori nuclear polyhedorsis virus which infect the silkworm
(Bombyx
mori). Numerous baculovirus expression systems are commercially available, for
example, from Thermo Fisher (Bac-N- BlueTMk or BAC-TO-BACTm Systems), Clontech
(BacPAKTM Baculovirus Expression System), Novagen (Bac Vector SystemTm), or
others
from Pharmingen or Quantum Biotechnologies. Another insect cell host is the
common
fruit fly, Drosophila melanogaster, for which a transient or stable plasmid
based
transfection kit is offered commercially by Thermo Fisher (The DESTM System).
[0416] In some embodiments, cells are transformed with vectors that express
a nucleic
acid described herein. Transformation techniques for inserting new genetic
material into
eukaryotic cells, including animal and plant cells, are well known. Viral
vectors may be
used for inserting expression cassettes into host cell genomes. Alternatively,
the vectors
may be transfected into the host cells. Transfection may be accomplished by
methods as
described in the art such as, but not limited to, calcium phosphate
precipitation,
electroporation, optical transfection, protoplast fusion, impalefection, and
hydrodynamic
delivery.
- 138 -
Date Recue/Date Received 2021-04-23
IV. METHODS OF USE
[0417] One aspect of the present disclosure provides methods of using
nucleic acid
vaccines of the present disclosure and pharmaceutical compositions comprising
the
nucleic acid vaccines and at least one pharmaceutically acceptable carrier.
Provided
herein are compositions, methods, kits, and reagents for diagnosis, treatment
or
prevention of a disease or condition in humans or other mammals where the
active agent
is the nucleic acid vaccine, cells containing the nucleic acid vaccine or
polypeptides
translated from nucleic acid vaccine polynucleotides.
[0418] In some embodiments, the methods of use can be assessed using any
endpoint
indicating a benefit to the subject, including, without limitation, (1)
inhibition, to some
extent, of disease progression, including stabilization, slowing down and
complete arrest;
(2) reduction in the number of disease episodes and/or symptoms; (3)
inhibition (i.e.,
reduction, slowing down or complete stopping) of a disease cell infiltration
into adjacent
peripheral organs and/or tissues; (4) inhibition (i.e. reduction, slowing down
or complete
stopping) of disease spread; (5) decrease of an autoimmune condition; (6)
favorable
change in the expression of a biomarker associated with the disorder; (7)
relief, to some
extent, of one or more symptoms associated with a disorder; (8) increase in
the length of
disease-free presentation following treatment; or (9) decreased mortality at a
given point
of time following treatment.
Therapeutic or Prophylactic Uses
[0419] The nucleic acid vaccines described herein may be used to protect,
treat or cure
infection arising from contact with an infectious agent such as, but not
limited to, viruses,
bacteria, fungi, parasites and protozoa. As a non-limiting example, the
infectious agent is
a virus and the virus is SARS-CoV-2.
[0420] The nucleic acid vaccines described herein may be used as
therapeutic or
prophylactic agents where the nucleic acid vaccines are administered to a
subject, and
wherein the nucleic acid vaccine polynucleotide is translated in vivo to
produce one or
more proteins, peptides, fragments or variants thereof of SARS-CoV-2 for the
treatment
and/or prevention of COVID-19.
- 139 -
Date Recue/Date Received 2021-04-23
[0421] In some embodiments, provided are methods for treating or preventing
a viral
infection and/or a disease, disorder, or condition associate with a viral
infection or a
symptom thereof, in a subject, by administering a nucleic acid vaccine
comprising one or
more polynucleotides encoding a viral polypeptide. The administration may be
in
combination with an anti-viral or anti-bacterial agent or a small molecule
compound
described herein or known in the art.
[0422] In some embodiments, the nucleic acid vaccines described herein may
be used
to protect against and/or prevent the transmission of an emerging or
engineered threat
which may be known or unknown.
[0423] In some embodiments, provided herein are methods of inducing
translation of
a polypeptide (e.g., one or more proteins, peptides, fragments or variants
thereof of
SARS-CoV-2 for the treatment and/or prevention of COVID-19) in a cell, tissue
or
organism using the nucleic acid polynucleotides described herein. Such
translation can be
in vitro, in vivo, ex vivo, or in culture. The cell, tissue or organism may be
contacted with
an effective amount of a composition or pharmaceutical composition containing
the
nucleic acid vaccine which includes a polynucleotide with at least one region
encoding
the polypeptide of interest (e.g., one or more proteins, peptides, fragments
or variants
thereof of SARS-CoV-2 for the treatment and/or prevention of COVID-19).
[0424] In some embodiments, the effective amount of the nucleic acid
vaccine
described herein provided to a cell, a tissue or a subject may be enough for
immune
prophylaxis.
[0425] An "effective amount" of the composition of the nucleic acid vaccine
is
provided based, at least in part, on the target tissue, target cell type,
means of
administration, physical characteristics of the polynucleotide (e.g., size,
and the number
of unmodified and modified nucleosides) and other components of the nucleic
acid
vaccine. An effective amount of the composition containing the nucleic acid
vaccine
described herein is one that provides an induced or boosted immune response as
a
function of production in the cell of one or more proteins, peptides,
fragments or variants
thereof of SARS-CoV-2 as compared to an untreated cell. Increased production
may be
- 140 -
Date Recue/Date Received 2021-04-23
demonstrated by increased cell transfection (i.e., the percentage of cells
transfected with
the nucleic acid vaccine), increased protein translation from the
polynucleotide or altered
innate immune response of the host cell.
[0426] Provided herein are directed to methods of inducing in vivo
translation of one
or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 in a
mammalian subject in need thereof. An effective amount of a nucleic acid
vaccine
composition containing a polynucleotide that has at least one translatable
region encoding
the polypeptide (e.g., one or more proteins, peptides, fragments or variants
thereof of
SARS-CoV-2) is administered to the subject using the delivery methods
described herein.
The polynucleotide is provided in an amount and under other conditions such
that the
polynucleotide is translated in the cell. The cell in which the polynucleotide
is localized,
or the tissue in which the cell is present, may be targeted with one or more
rounds of
nucleic acid vaccine administration.
[0427] In certain embodiments, the administered nucleic acid vaccine
comprising
polynucleotides directs production of one or more polypeptides that provide a
functional
immune system-related activity which is substantially absent in the cell,
tissue or
organism in which the polypeptide is translated. For example, the missing
functional
activity may be enzymatic, structural, or gene regulatory in nature. In
related
embodiments, the administered polynucleotides directs production of one or
more
polypeptides that increases a functional activity related to the immune system
which is
present but substantially deficient in the cell in which the polypeptide is
translated.
[0428] Additionally, the polypeptide translated from the nucleic acid
vaccine may
antagonize, directly or indirectly, the activity of a biological moiety
present in, on the
surface of, or secreted from the cell. Non-limiting examples of biological
moieties that
may be antagonized include a nucleic acid, a carbohydrate, a protein toxin
such as shiga
and tetanus toxins, lipids (e.g., cholesterol), a lipoprotein (e.g., low
density lipoprotein),
or a small molecule toxin (e.g., cholera, botulinum, and diphtheria toxins).
In some
embodiments, the biological molecule which may be antagonized may be an
endogenous
protein that may have an undesirable activity such as, but not limited to,
cytotoxic or
- 141 -
Date Recue/Date Received 2021-04-23
cytostatic activity. The proteins described herein may be engineered for
localization
within the cell, potentially within a specific compaiiment such as the
cytoplasm or
nucleus, or are engineered for secretion from the cell or translocation to the
plasma
membrane of the cell.
[0429] In some embodiments, nucleic acid vaccine polynucleotides and their
encoded
polypeptides may be used for treatment of any of a variety of diseases,
disorders, and/or
conditions, including but not limited to viral infections (e.g., infections
caused by SARS-
CoV-2).
[0430] The subject to whom the nucleic acid vaccine may be administered
suffers
from or may be at risk of developing a disease, disorder, or deleterious
condition.
Provided are methods of identifying, diagnosing, and classifying subjects on
these bases,
which may include clinical diagnosis, biomarker levels, genome-wide
association studies
(GWAS), and other methods known in the art.
[0431] The agents (e.g., compositions of nucleic acid vaccines and any
additional
moieties) can be administered simultaneously, for example in a combined unit
dose (e.g.,
providing simultaneous delivery of both agents). The agents can also be
administered at a
specified time interval, such as, but not limited to, an interval of minutes,
hours, days or
weeks. Generally, the agents may be concurrently bioavailable, e.g.,
detectable, in the
subject. In some embodiments, the agents may be administered essentially
simultaneously, for example two unit dosages administered at the same time, or
a
combined unit dosage of the two agents. In other embodiments, the agents may
be
delivered in separate unit dosages. The agents may be administered in any
order, or as
one or more preparations that includes two or more agents. In a preferred
embodiment, at
least one administration of one of the agents, e.g., the first agent, may be
made within
minutes, one, two, three, or four hours, or even within one or two days of the
other agent,
e.g., the second agent. In some embodiments, combinations can achieve
synergistic
results, e.g., greater than additive results, e.g., at least 25, 50, 75, 100,
200, 300, 400, or
500% greater than additive results.
- 142 -
Date Recue/Date Received 2021-04-23
[0432] In some embodiments, the nucleic acid vaccine described herein may
be
administrated with other prophylactic or therapeutic compounds. As a non-
limiting
example, the prophylactic or therapeutic compound may be an adjuvant or a
booster. As
used herein, when referring to a prophylactic composition, such as a vaccine,
the term
"booster" refers to an extra administration of the prophylactic composition. A
booster (or
booster vaccine) may be given after an earlier administration of the
prophylactic
composition. The time of administration between the initial administration of
the
prophylactic composition and the booster may be, but is not limited to, 1
minute, 2
minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9
minutes, 10
minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50
minutes, 55
minutes, 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, 19
hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3
days, 4 days, 5
days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months,
4 months,
months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year,
18
months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9
years, 10 years, 11
years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years,
19 years, 20
years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years,
60 years, 65
years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more than
99 years.
[0433] In some embodiments, the nucleic acid vaccines may be formulated by
the
methods described herein. In one aspect, the formulation may comprise a
nucleic acid
vaccine or polynucleotide which can have a therapeutic and/or prophylactic
effect on
more than one disease, disorder or condition. As a non-limiting example, the
formulation
may comprise polynucleotides encoding one or more proteins, peptides,
fragments or
variants thereof of SARS-CoV-2 for the treatment and/or prevention of COVID-
19.
[0434] In some embodiments, the nucleic acid vaccines described herein may
be used
for research in many applications, such as, but not limited to, identifying
and locating
intracellular and extracellular proteins, protein interaction, signal pathways
and cell
biology.
- 143 -
Date Recue/Date Received 2021-04-23
Modulation of the Immune Response
[0435] In some embodiments, the nucleic acid vaccines comprising the
polynucleotides described herein may act as a single composition as a vaccine.
As used
herein, a "vaccine" refers to a composition, a substance or preparation that
stimulates,
induces, causes or improves immunity in an organism, e.g., an animal organism,
for
example, a mammalian organism (e.g., a human). Preferably, a vaccine provides
immunity against one or more diseases or disorders in the organism, including
prophylactic and/or therapeutic immunity. Exemplary vaccines includes one or
more
agents that resembles an infectious agent, e.g., a disease-causing
microorganism, and can
be made, for example, from live, attenuated, modified, weakened or killed
forms of
disease-causing microorganisms, or antigens derived therefrom, including
combinations
of antigenic components. In exemplary embodiments, a vaccine stimulates,
induces
causes or improves immunity in an organism or causes or mimics infection in
the
organism without inducing any disease or disorder. A vaccine introduces an
antigen into
the tissues, extracellular space or cells of a subject and elicits an immune
response,
thereby protecting the subject from a particular disease or pathogen
infection. The nucleic
acid vaccines described herein may encode an antigen and when the
polynucleotides are
expressed in cells, a desired immune response is achieved. As a non-limiting
example,
the nucleic acid vaccines described herein may encode one or more proteins,
peptides,
fragments or variants thereof of SARS-CoV-2 and when the polynucleotides are
expressed in cells, a desired immune response against SARS-CoV-2 is achieved
to treat
and/or prevent COVID-19.
[0436] Nucleic acid vaccines may be administered prophylactically or
therapeutically
as part of an active immunization scheme to healthy individuals or early in
infection
during the incubation phase or during active infection after onset of
symptoms.
[0437] The nucleic acid vaccines described herein may also be administered
as a
second line treatment after the standard first line treatments such as
antibiotics and
antivirals have failed to induce passive immunity. In this regard, the nucleic
acid vaccines
- 144 -
Date Recue/Date Received 2021-04-23
described herein are useful in settings where resistance to first line
treatments has
developed and disease persists and induces chronic disease.
[0438] Nucleic acid vaccines may be administered as part of a treatment
regimen for
latent viral infections, such as SARS-CoV-2 infections. In this embodiment,
one or more
polynucleotides are administered which ultimately produce proteins which
result a
desired immune response against SARS-CoV-2 is achieved to treat and/or prevent
COVID-19.
[0439] The use of RNA in or as a vaccine overcomes the disadvantages of
conventional genetic vaccination involving incorporating DNA into cells in
terms of
safeness, feasibility, applicability, and effectiveness to generate immune
responses. RNA
molecules are considered to be significantly safer than DNA vaccines, as RNAs
are more
easily degraded. They are cleared quickly out of the organism and cannot
integrate into
the genome and influence the cell's gene expression in an uncontrollable
manner. It is
also less likely for RNA vaccines to cause severe side effects like the
generation of
autoimmune disease or anti-DNA antibodies (Bringmann A. et al., Journal of
Biomedicine and Biotechnology (2010), vol. 2010, article ID623687).
Transfection with
RNA requires only insertion into the cell's cytoplasm, which is easier to
achieve than into
the nucleus. However, RNA is susceptible to RNase degradation and other
natural
decomposition in the cytoplasm of cells.
[0440] Various attempts to increase the stability and shelf life of RNA
vaccines. US
2005/0032730 to Von Der Mulbe et al. discloses improving the stability of mRNA
vaccine compositions by increasing G(guanosine)/C(cytosine) content of the
mRNA
molecules. U.S. Pat. No. 5,580,859 to Feigner et al. teaches incorporating
polynucleotide
sequences coding for regulatory proteins that binds to and regulates the
stabilities of
mRNA. While not wishing to be bound by theory, it is believed that the nucleic
acid
vaccines described herein may result in improved stability and therapeutic
efficacy due at
least in part to the specificity, purity and selectivity of the construct
designs.
Additionally, modified nucleosides, or combinations thereof, may be introduced
into the
nucleic acid vaccines described herein to activate the innate immune response.
Such
- 145 -
Date Recue/Date Received 2021-04-23
activating molecules are useful as adjuvants when combined with polypeptides
and/or
other vaccines. In certain embodiments, the activating molecules contain a
translatable
region which encodes for a polypeptide sequence useful as a vaccine, thus
providing the
ability to be a self-adjuvant.
[0441] In some embodiments, the nucleic acid vaccines described herein may
be used
in the prevention, treatment and diagnosis of diseases and physical
disturbances caused
by infectious agents such as, but not limited to, SARS-CoV-2. The nucleic acid
vaccines
described herein may encode at least one polypeptide of interest (e.g., one or
more
proteins, peptides, fragments or variants thereof of SARS-CoV-2) and may be
provided
to an individual in order to stimulate the immune system to protect against
the disease-
causing agents. As a non-limiting example, the biological activity and/or
effect from an
infectious agent may be inhibited and/or abolished by providing one or more
polynucleotides which have the ability to bind and neutralize the infectious
agent.
[0442] As a non-limiting example, the polynucleotides encoding an immunogen
may
be delivered to cells to trigger multiple innate response pathways (see
International Pub.
No. W02012006377 and US Patent Publication No. US20130177639; herein
incorporated by reference in its entirety). As another non-limiting example,
the nucleic
acid vaccines described herein may be delivered to a vertebrate in a dose
amount large
enough to be immunogenic to the vertebrate (see International Pub. No.
W02012006372
and W02012006369 and US Publication No. US20130149375 and US20130177640; the
contents of each of which are herein incorporated by reference in their
entirety).
[0443] As a non-limiting example, the nucleic acid vaccines described
herein may
treat and/or prevent infectious diseases including viral infectious diseases
such as
COVID-19 caused by SARS-CoV-2.
[0444] Nucleic acid vaccines described herein may be utilized in various
settings
depending on the prevalence of the infection or the degree or level of unmet
medical
need. As a non-limiting example, the nucleic acid vaccines described herein
may be
utilized to treat and/or prevent COVID-19 infection, including the diseases
and
- 146 -
Date Recue/Date Received 2021-04-23
conditions related to COVID-19 infection (including infection by the original
and
mutated versions of SARS-CoV-2).
[0445] Symptoms of COVID-19 infection are changing as more is learned
about the
disease but the current symptoms include fever or chills, cough, shortness of
breath or
difficulty breathing, fatigue, body aches, muscle aches, headaches, sore
throat, congestion
or runny nose, nausea and/or vomiting, diarrhea, and a new loss of taste or
smell.
[0446] In some embodiments, the nucleic acid vaccines described herein may
be
better designed, as compared to current anti-viral treatments, to produce the
appropriate
protein conformation on translation as the nucleic acid vaccines co-opt
natural cellular
machinery. Unlike traditional vaccines which are manufactured ex vivo and may
trigger
unwanted cellular responses, the nucleic acid vaccines are presented to the
cellular
system in a more native fashion. In some embodiments, the nucleic acid
vaccines
described herein are a tailored active vaccine for COVID-19 that not only can
prevent
infection by SARS-CoV-2 but can limit transmission of SARS-CoV-2.
[0447] In some embodiments, the nucleic acid vaccines described herein may
be used
to prevent pandemic COVID-19 by reacting to emerging new strains with the very
rapid
nucleic acid based vaccine production process.
[0448] In some embodiments, a single injection of a nucleic acid vaccine
may provide
protection for an entire season.
[0449] In some embodiments, the nucleic acid vaccines described herein may
be
immunostimulatory. The polynucleotide sequence of the nucleic acid vaccine may
further
comprise a sequence region encoding a cytokine that promotes the immune
response,
such as a monokine, lymphokine, interleukin or chemokine, such as IL-1, IL-2,
IL-3, IL-
4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-a, INF-y, GM-CFS, LT-a, or
growth
factors such as hGH.
Treatment and/or Prevention of COVID-19
[0450] In some embodiments, the nucleic acid vaccines described herein
encode one
or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 and
may be
used for the treatment and/or prevention of COVID-19
- 147 -
Date Recue/Date Received 2021-04-23
[0451] In some embodiments, the nucleic acid vaccines described herein can
produce
much higher antibody titers and they may produce responses early than
commercially
available anti-virals.
[0452] In some embodiments, the nucleic acid vaccines described herein co-
opt the
natural cellular machinery to produce the appropriate protein conformation on
translation.
Unlike traditional vaccines which are manufactured ex vivo and may trigger
unwanted
cellular responses, the nucleic acid vaccines described herein are introduced
to the
cellular system in a way that is closer to the native way or the way normal
cellular
processing occurs. Additionally, formulations may be used to shield or target
delivery of
the nucleic acid vaccines to specific cells or tissues in the subject.
[0453] In some embodiments, nucleic acid vaccines described herein
represent a
targeted active vaccine that not only can prevent infection but can limit
transmission of
COVID-19.
[0454] In some embodiments, the nucleic acid vaccines may be used to
prevent
pandemic SARS-CoV-2 infection or COVID-19 by reacting to emerging new strains
with
the very rapid vaccine production process.
[0455] In some embodiments a single injection of nucleic acid vaccines
encoding one
or more proteins, peptides, fragments or variants thereof of SARS-CoV-2 may
provide
protection for at least 1 year, at least 2 years, at least 3 years, at least 4
years, at least 5
years, at least 6 years, at least 7 years, at least 8 years, at least 9 years,
at least 10 years, at
least 11 years, at least 12 years, at least 13 years, at least 15 years or
more than 15 years.
[0456] The nucleic acid vaccines described herein may also be used to
maintain or
restore antigenic memory in a subject or population as part of a vaccination
plan for
COVID-19 or other diseases caused by SARS-CoV-2.
[0457] In some embodiments, nucleic acid vaccines compositions may be
created
which include polynucleotides that encode one or more proteins, peptides,
fragments or
variants thereof of SARS-CoV-2 which are showing prevalence increased
infection rates
for the year. The protein sequences of SARS-CoV-2 have been shown to change or
mutate over time, wherein some of the mutations have shown increased infection
rates.
- 148 -
Date Recue/Date Received 2021-04-23
As a non-limiting example, the nucleic acid vaccines compositions may be
created which
include polynucleotides that encode one or more proteins, peptides, fragments
or variants
thereof of SARS-CoV-2 which are showing prevalence increased infection rates
for the
year such as, but not limited to the D614G mutation.
[0458] In some embodiments, a vaccination scheme or plan is developed which
allows
for not only ongoing vaccination in the current year but memory booster
vaccinations
across years, strains, or groups thereof to establish and maintain memory in a
population.
In this manner, a population is less likely to succumb to any pandemic or
outbreak
involving recurrence of older strains. Any combination of a prior vaccine
component
strain can be utilized to create or design a memory booster vaccine.
[0459] In some embodiments, nucleic acid vaccines which are memory booster
vaccines are administered to boost memory across a time period of 2, 3, 4, 5,
6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 35, 40,45,
50 or more than 50 years.
[0460] In some embodiments, nucleic acid vaccines which are memory booster
vaccines are administered to boost memory for alternating historic years
including every
other year from the past vaccine component strains relative to a current year.
In some
embodiments the selection of the vaccine components can be from every 2nd,
3rd, 4th,
5th, 6th, 7th, 8th, 9th, 10th or more years.
[0461] In some embodiments, nucleic acid vaccines which are memory booster
vaccines are administered to boost memory over ten year periods.
[0462] In some embodiments, the nucleic acid booster vaccine may be used in
a
population either once or periodically to create herd immunity which means
greater than
30% of a population is protected.
[0463] In some embodiments, the nucleic acid booster vaccine may be used in
a
population either once or periodically to create herd immunity against COVID-
19 which
means greater than 30% of a population is protected.
[0464] In some embodiments, the nucleic acid vaccines are used to target at
risk
populations for COVID-19 such as those having pre-existing conditions
including, but
- 149 -
Date Recue/Date Received 2021-04-23
not limited to, cancer, chronic kidney disease, chronic obstructive pulmonary
disease
(COPD), immunocompromised state (weakened immune system) from solid organ
transplant, blood or bone marrow transplant, immune deficiencies, HIV, and use
of
corticosteroids or other immune weaking medicines, obesity (body mass index
(BMI) of
30 or higher), heart conditions such as heart failure, coronary artery
disease, or
cardiomyopathies, sickle cell disease, type 1 or type 2 diabetes mellitus,
asthma
(moderate-to-severe), cerebrovascular disease, cystic fibrosis, hypertension
or high blood
pressure, neurological conditions such as dementia, liver disease, pregnancy,
pulmonary
fibrosis, smoking, and thalassemia.
[0465] In some embodiments, the nucleic acid vaccines are used to treat
healthcare
workers who are at risk of being exposed to SARS-CoV-2.
V. KITS AND DEVICES
Kits
[0466] The disclosure provides a variety of kits for conveniently and/or
effectively
carrying out methods of the present disclosure. Typically, kits will comprise
sufficient
amounts and/or numbers of components to allow a user to perform multiple
treatments of
a subject(s) and/or to perform multiple experiments.
[0467] In some embodiments, the present disclosure provides kits for
modulating the
expression of genes in vitro or in vivo, comprising nucleic acid vaccine
compositions of
the present disclosure or a combination of nucleic acid vaccine compositions
of the
present disclosure, nucleic acid vaccine compositions modulating other genes,
siRNAs,
miRNAs or other oligonucleotide molecules.
[0468] The kit may further comprise packaging and instructions and/or a
delivery
agent to form a formulation, e.g., for administration to a subject in need of
treatment
using the nucleic acid vaccine compositions described herein. The delivery
agent may
comprise a saline, a buffered solution, a lipidoid, a dendrimer or any
suitable delivery
agent.
[0469] In one non-limiting example, the buffer solution may include sodium
chloride,
calcium chloride, phosphate and/or EDTA. In another non-limiting example, the
buffer
- 150 -
Date Recue/Date Received 2021-04-23
solution may include, but is not limited to, saline, saline with 2mM calcium,
5% sucrose,
5% sucrose with 2mM calcium, 5% Mannitol, 5% Mannitol with 2mM calcium,
Ringer's
lactate, sodium chloride, sodium chloride with 2mM calcium and mannose (See
U.S. Pub.
No. 20120258046; herein incorporated by reference in its entirety). In yet
another non-
limiting example, the buffer solutions may be precipitated or it may be
lyophilized. The
amount of each component may be varied to enable consistent, reproducible
higher
concentration saline or simple buffer formulations. The components may also be
varied
in order to increase the stability of nucleic acid vaccine compositions in the
buffer
solution over a period of time and/or under a variety of conditions.
Devices
[0470] The present disclosure provides for devices which may incorporate
nucleic
acid vaccine compositions of the present disclosure. These devices can contain
a stable
formulation available to be immediately delivered to a subject in need
thereof, such as a
human patient.
[0471] Non-limiting examples of the devices include a pump, a catheter, a
needle, a
transdermal patch, a pressurized olfactory delivery device, electroporation
devices,
iontophoresis devices, multi-layered microfluidic devices. The devices may be
employed
to deliver nucleic acid vaccine compositions of the present disclosure
according to single,
multi- or split-dosing regiments. The devices may be employed to deliver
nucleic acid
vaccine compositions of the present disclosure across biological tissue,
intradermal,
subcutaneously, or intramuscularly. More examples of devices suitable for
delivering
oligonucleotides are disclosed in International Publication WO 2013/090648,
the contents
of which are incorporated herein by reference in their entirety.
VI. DEFINITIONS
[0472] At various places in the present specification, substituents of
compounds of the
present disclosure are disclosed in groups or in ranges. It is specifically
intended that the
present disclosure include each and every individual subcombination of the
members of
such groups and ranges.
[0473] About: As used herein, the term "about" means +/-10% of the recited
value.
- 151 -
Date Recue/Date Received 2021-04-23
[0474] Administered in combination: As used herein, the term "administered
in
combination" or "combined administration" means that two or more agents are
administered to a subject at the same time or within an interval such that
there may be an
overlap of an effect of each agent on the patient. In some embodiments, they
are
administered within about 60, 30, 15, 10, 5, or 1 minute of one another. In
some
embodiments, the administrations of the agents are spaced sufficiently closely
together
such that a combinatorial (e.g., a synergistic) effect is achieved.
[0475] Adjuvant: As used herein, the term "adjuvant" means a substance that
enhances a subject's immune response to an antigen. The nucleic acid vaccines
described
herein may optionally comprise one or more adjuvants.
[0476] Animal: As used herein, the term "animal" refers to any member of
the animal
kingdom. In some embodiments, "animal" refers to humans at any stage of
development.
In some embodiments, "animal" refers to non-human animals at any stage of
development. In certain embodiments, the non-human animal is a mammal (e.g., a
rodent,
a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate,
or a pig). In
some embodiments, animals include, but are not limited to, mammals, birds,
reptiles,
amphibians, fish, and worms. In some embodiments, the animal is a transgenic
animal,
genetically-engineered animal, or a clone.
[0477] Antigen: As defined herein, the term "antigen" refers to a
composition, for
example, a substance or agent which causes an immune response in an organism,
e.g.,
causes the immune response of the organism to produce antibodies against the
substance
or agent in particular, which provokes an adaptive immune response in an
organism.
Antigens can be any immunogenic substance including, in particular, proteins,
polypeptides, polysaccharides, nucleic acids, lipids and the like. Exemplary
antigens are
derived from infectious agents. Such agents can include parts or subunits of
infectious
agents, for example, coats, coat components, e.g., coat protein or
polypeptides, surface
components, e.g., surface proteins or polypeptides, capsule components, cell
wall
components, flagella, fimbrae, and/or toxins or toxoids) of infectious agents,
for example,
bacteria, viruses, and other microorganisms. Certain antigens, for example,
lipids and/or
- 152 -
Date Recue/Date Received 2021-04-23
nucleic acids are antigenic, preferably, when combined with proteins and/or
polysaccharides.
[0478] Approximately: As used herein, the term "approximately" or "about,"
as
applied to one or more values of interest, refers to a value that is similar
to a stated
reference value. In certain embodiments, the term "approximately" or "about"
refers to a
range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,
12%,
11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction
(greater
than or less than) of the stated reference value unless otherwise stated or
otherwise
evident from the context (except where such number would exceed 100% of a
possible
value).
[0479] Associated with: As used herein, the terms "associated with,"
"conjugated,"
"linked," "attached," and "tethered," when used with respect to two or more
moieties,
means that the moieties are physically associated or connected with one
another, either
directly or via one or more additional moieties that serves as a linking
agent, to form a
structure that is sufficiently stable so that the moieties remain physically
associated under
the conditions in which the structure is used, e.g., physiological conditions.
An
"association" need not be strictly through direct covalent chemical bonding.
It may also
suggest ionic or hydrogen bonding or a hybridization based connectivity
sufficiently
stable such that the "associated" entities remain physically associated.
[0480] Bifunctional: As used herein, the term "bifunctional" refers to any
substance,
molecule or moiety which is capable of or maintains at least two functions.
The functions
may affect the same outcome or a different outcome. The structure that
produces the
function may be the same or different.
[0481] Biocompatible: As used herein, the term "biocompatible" means
compatible
with living cells, tissues, organs or systems posing little to no risk of
injury, toxicity or
rejection by the immune system.
[0482] Biodegradable: As used herein, the term "biodegradable" means
capable of
being broken down into innocuous products by the action of living things.
- 153 -
Date Recue/Date Received 2021-04-23
[0483] Biologically active: As used herein, the phrase "biologically
active" refers to a
characteristic of any substance that has activity in a biological system
and/or organism.
For instance, a substance that, when administered to an organism, has a
biological effect
on that organism, is considered to be biologically active. In particular
embodiments, a
polynucleotide described herein may be considered biologically active if even
a portion
of the polynucleotides is biologically active or mimics an activity considered
biologically
relevant.
[0484] Chimera: As used herein, "chimera" is an entity having two or more
incongruous or heterogeneous parts or regions.
[0485] Compound: As used herein, the term "compound," is meant to include
all
stereoisomers, geometric isomers, tautomers, and isotopes of the structures
depicted.
[0486] The compounds described herein can be asymmetric (e.g., having one
or more
stereocenters). All stereoisomers, such as enantiomers and diastereomers, are
intended
unless otherwise indicated. Compounds of the present disclosure that contain
asymmetrically substituted carbon atoms can be isolated in optically active or
racemic
forms. Methods on how to prepare optically active forms from optically active
starting
materials are known in the art, such as by resolution of racemic mixtures or
by
stereoselective synthesis. Many geometric isomers of olefins, C=N double
bonds, and the
like can also be present in the compounds described herein, and all such
stable isomers
are contemplated in the present disclosure. Cis and trans geometric isomers of
the
compounds of the present disclosure are described and may be isolated as a
mixture of
isomers or as separated isomeric forms.
[0487] Compounds of the present disclosure also include tautomeric forms.
Tautomeric forms result from the swapping of a single bond with an adjacent
double
bond and the concomitant migration of a proton. Tautomeric forms include
prototropic
tautomers which are isomeric protonation states having the same empirical
formula and
total charge.
[0488] Compounds of the present disclosure also include all of the isotopes
of the
atoms occurring in the intermediate or final compounds. "Isotopes" refers to
atoms
- 154 -
Date Recue/Date Received 2021-04-23
having the same atomic number but different mass numbers resulting from a
different
number of neutrons in the nuclei. For example, isotopes of hydrogen include
tritium and
deuterium.
[0489] The compounds and salts of the present disclosure can be prepared in
combination with solvent or water molecules to form solvates and hydrates by
routine
methods.
[0490] Conserved: As used herein, the term "conserved" refers to
nucleotides or
amino acid residues of a polynucleotide sequence or polypeptide sequence,
respectively,
that are those that occur unaltered in the same position of two or more
sequences being
compared. Nucleotides or amino acids that are relatively conserved are those
that are
conserved amongst more related sequences than nucleotides or amino acids
appearing
elsewhere in the sequences.
[0491] In some embodiments, two or more sequences are said to be
"completely
conserved" if they are 100% identical to one another. In some embodiments, two
or more
sequences are said to be "highly conserved" if they are at least 70%
identical, at least
80% identical, at least 90% identical, or at least 95% identical to one
another. In some
embodiments, two or more sequences are said to be "highly conserved" if they
are about
70% identical, about 80% identical, about 90% identical, about 95%, about 98%,
or about
99% identical to one another. In some embodiments, two or more sequences are
said to
be "conserved" if they are at least 30% identical, at least 40% identical, at
least 50%
identical, at least 60% identical, at least 70% identical, at least 80%
identical, at least
90% identical, or at least 95% identical to one another. In some embodiments,
two or
more sequences are said to be "conserved" if they are about 30% identical,
about 40%
identical, about 50% identical, about 60% identical, about 70% identical,
about 80%
identical, about 90% identical, about 95% identical, about 98% identical, or
about 99%
identical to one another. Conservation of sequence may apply to the entire
length of an
polynucleotide or polypeptide or may apply to a portion, region or feature
thereof.
- 155 -
Date Recue/Date Received 2021-04-23
[0492] Controlled Release: As used herein, the term "controlled release"
refers to a
pharmaceutical composition or compound release profile that conforms to a
particular
pattern of release to effect a therapeutic outcome.
[0493] Cytostatic: As used herein, "cytostatic" refers to inhibiting,
reducing,
suppressing the growth, division, or multiplication of a cell (e.g., a
mammalian cell (e.g.,
a human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or a
combination
thereof.
[0494] Cytotoxic: As used herein, "cytotoxic" refers to killing or causing
injurious,
toxic, or deadly effect on a cell (e.g., a mammalian cell (e.g., a human
cell)), bacterium,
virus, fungus, protozoan, parasite, prion, or a combination thereof.
[0495] Delivery: As used herein, "delivery" refers to the act or manner of
delivering a
compound, substance, entity, moiety, cargo or payload.
[0496] Delivery Agent: As used herein, "delivery agent" refers to any
substance
which facilitates, at least in part, the in vivo delivery of a polynucleotide
to targeted cells.
[0497] Destabilized: As used herein, the term "destable," "destabilize,"
or
"destabilizing region" means a region or molecule that is less stable than a
starting, wild-
type or native form of the same region or molecule.
[0498] Detectable label: As used herein, "detectable label" refers to one
or more
markers, signals, or moieties which are attached, incorporated or associated
with another
entity that is readily detected by methods known in the art including
radiography,
fluorescence, chemiluminescence, enzymatic activity, absorbance and the like.
Detectable
labels include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal
ions,
ligands such as biotin, avidin, streptavidin and haptens, quantum dots, and
the like.
Detectable labels may be located at any position in the peptides or proteins
disclosed
herein. They may be within the amino acids, the peptides, or proteins, or
located at the N-
or C-termini.
[0499] Digest: As used herein, the term "digest" means to break apart into
smaller
pieces or components. When referring to polypeptides or proteins, digestion
results in the
production of peptides.
- 156 -
Date Recue/Date Received 2021-04-23
[0500] Dosing regimen: As used herein, a "dosing regimen" is a schedule of
administration or physician determined regimen of treatment, prophylaxis, or
palliative
care.
[0501] Encapsulate: As used herein, the term "encapsulate" means to
enclose,
surround or encase.
[0502] Encoded protein cleavage signal: As used herein, "encoded protein
cleavage
signal" refers to the nucleotide sequence which encodes a protein cleavage
signal.
[0503] Engineered: As used herein, embodiments of the nucleic acid vaccines
are
"engineered" when they are designed to have a feature or property, whether
structural or
chemical, that varies from a starting point, wild type or native molecule.
[0504] Effective Amount: As used herein, the term "effective amount" of an
agent is
that amount sufficient to effect beneficial or desired results, for example,
clinical results,
and, as such, an "effective amount" depends upon the context in which it is
being applied.
For example, in the context of administering an agent that treats cancer, an
effective
amount of an agent is, for example, an amount sufficient to achieve treatment,
as defined
herein, of cancer, as compared to the response obtained without administration
of the
agent.
[0505] Exosome: As used herein, "exosome" is a vesicle secreted by
mammalian cells
or a complex involved in RNA degradation.
[0506] Expression: As used herein, "expression" of a nucleic acid sequence
refers to
one or more of the following events: (1) production of an RNA template from a
DNA
sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g.,
by splicing,
editing, 5' cap formation, and/or 3' end processing); (3) translation of an
RNA into a
polypeptide or protein; and (4) post-translational modification of a
polypeptide or protein.
[0507] Feature: As used herein, a "feature" refers to a characteristic, a
property, or a
distinctive element.
[0508] Formulation: As used herein, a "formulation" includes at least a
polynucleotide
of a nucleic acid vaccine and a delivery agent.
- 157 -
Date Recue/Date Received 2021-04-23
[0509] Fragment: A "fragment," as used herein, refers to a portion. For
example,
fragments of proteins may comprise polypeptides obtained by digesting full-
length
protein isolated from cultured cells.
[0510] Functional: As used herein, a "functional" biological molecule is a
biological
molecule in a form in which it exhibits a property and/or activity by which it
is
characterized.
[0511] Homology: As used herein, the term "homology" refers to the overall
relatedness between polymeric molecules, e.g. between nucleic acid molecules
(e.g. DNA
molecules and/or RNA molecules) and/or between polypeptide molecules. In some
embodiments, polymeric molecules are considered to be "homologous" to one
another if
their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term "homologous"
necessarily refers to a comparison between at least two sequences
(polynucleotide or
polypeptide sequences). Two polynucleotide sequences are considered to be
homologous
if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%,
95%, or
even 99% for at least one stretch of at least about 20 amino acids. In some
embodiments,
homologous polynucleotide sequences are characterized by the ability to encode
a stretch
of at least 4-5 uniquely specified amino acids. For polynucleotide sequences
less than 60
nucleotides in length, homology is determined by the ability to encode a
stretch of at least
4-5 uniquely specified amino acids. Two protein sequences are considered to be
homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90%
identical for
at least one stretch of at least about 20 amino acids.
[0512] Identity: As used herein, the term "identity" refers to the overall
relatedness
between polymeric molecules, e.g., between polynucleotide molecules (e.g. DNA
molecules and/or RNA molecules) and/or between polypeptide molecules.
[0513] Calculation of the percent identity of two polynucleotide sequences,
for
example, can be performed by aligning the two sequences for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first and a second
nucleic acid
sequences for optimal alignment and nonidentical sequences can be disregarded
for
- 158 -
Date Recue/Date Received 2021-04-23
comparison purposes). In certain embodiments, the length of a sequence aligned
for
comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%,
at least
70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the
reference
sequence. The nucleotides at corresponding nucleotide positions are then
compared.
When a position in the first sequence is occupied by the same nucleotide as
the
corresponding position in the second sequence, then the molecules are
identical at that
position. The percent identity between the two sequences is a function of the
number of
identical positions shared by the sequences, taking into account the number of
gaps, and
the length of each gap, which needs to be introduced for optimal alignment of
the two
sequences. The comparison of sequences and determination of percent identity
between
two sequences can be accomplished using a mathematical algorithm. For example,
the
percent identity between two nucleotide sequences can be determined using
methods
such as those described in Computational Molecular Biology, Lesk, A. M, ed.,
Oxford
University Press, N.Y., 1988; Biocomputing: Informatics and Genome Projects,
Smith,
D. W., ed., Academic Press, N.Y, 1993; Sequence Analysis in Molecular Biology,
von
Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I,
Griffin,
A. M, and Griffin, H. G., eds., Humana Press, N.J., 1994; and Sequence
Analysis Primer,
Gribskov, M. and Devereux, J., eds., M Stockton Press, N.Y, 1991; each of
which is
incorporated herein by reference. For example, the percent identity between
two
nucleotide sequences can be determined using the algorithm of Meyers and
Miller
(CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program
(version 2.0) using a PAM 120 weight residue table, a gap length penalty of 12
and a gap
penalty of 4. The percent identity between two nucleotide sequences can,
alternatively, be
determined using the GAP program in the GCG software package using an
NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity
between sequences include, but are not limited to those disclosed in Carillo,
H., and
Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by
reference.
Techniques for determining identity are codified in publicly available
computer
programs. Exemplary computer software to determine homology between two
sequences
- 159 -
Date Recue/Date Received 2021-04-23
include, but are not limited to, GCG program package, Devereux, J., et al.,
Nucleic Acids
Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al,
J.
Molec. Biol., 215, 403 (1990)).
[0514] Infectious Agent: As used herein, the phrase "infectious agent"
means an agent
capable of producing an infection in an organism, for example, in an animal.
An
infectious agent may refer to any microorganism, virus, infectious substance,
or
biological product that may be engineered as a result of biotechnology, or any
naturally
occurring or bioengineered component of any such microorganism, virus,
infectious
substance, or biological product, can cause emerging and contagious disease,
death or
other biological malfunction in a human, an animal, a plant or another living
organism.
[0515] In vitro: As used herein, the term "in vitro" refers to events that
occur in an
artificial environment, e.g., in a test tube or reaction vessel, in cell
culture, in a Petri dish,
etc., rather than within an organism (e.g., animal, plant, or microbe).
[0516] In vivo: As used herein, the term "in vivo" refers to events that
occur within an
organism (e.g., animal, plant, or microbe or cell or tissue thereof).
[0517] Isolated: As used herein, the term "isolated" refers to a substance
or entity that
has been separated from at least some of the components with which it was
associated
(whether in nature or in an experimental setting). Isolated substances may
have varying
levels of purity in reference to the substances from which they have been
associated.
Isolated substances and/or entities may be separated from at least about 10%,
about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,
or
more of the other components with which they were initially associated. In
some
embodiments, isolated agents are more than about 80%, about 85%, about 90%,
about
91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about
98%,
about 99%, or more than about 99% pure. As used herein, a substance is "pure"
if it is
substantially free of other components. Substantially isolated: By
"substantially isolated"
is meant that the compound is substantially separated from the environment in
which it
was formed or detected. Partial separation can include, for example, a
composition
enriched in the compound of the present disclosure. Substantial separation can
include
- 160 -
Date Recue/Date Received 2021-04-23
compositions containing at least about 50%, at least about 60%, at least about
70%, at
least about 80%, at least about 90%, at least about 95%, at least about 97%,
or at least
about 99% by weight of the compound of the present disclosure, or salt
thereof. Methods
for isolating compounds and their salts are routine in the art.
[0518] Linker: As used herein, a "linker" refers to a group of atoms, e.g.,
10-1,000
atoms, and can be comprised of the atoms or groups such as, but not limited
to, carbon,
amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine.
The linker
can be attached to a modified nucleoside or nucleotide on the nucleobase or
sugar moiety
at a first end, and to a payload, e.g., a detectable or therapeutic agent, at
a second end.
The linker may be of sufficient length as to not interfere with incorporation
into a nucleic
acid sequence.
[0519] Modified: As used herein "modified" refers to a changed state or
structure of a
molecule described herein. Molecules may be modified in many ways including
chemically, structurally, and functionally.
[0520] Mucus: As used herein, "mucus" refers to the natural substance that
is viscous
and comprises mucin glycoproteins.
[0521] Naturally occurring: As used herein, "naturally occurring" means
existing in
nature without artificial aid.
[0522] Neutralizing antibody: As used herein, a "neutralizing antibody"
refers to an
antibody which binds to its antigen and defends a cell from an antigen or
infectious agent
by neutralizing or abolishing any biological activity it has.
[0523] Non-human vertebrate: As used herein, a "non-human vertebrate"
includes all
vertebrates except Homo sapiens, including wild and domesticated species.
Examples of
non- human vertebrates include, but are not limited to, mammals, such as
alpaca,
banteng, bison, camel, cat, cattle, deer, dog, donkey, gayal, goat, guinea
pig, horse, llama,
mule, pig, rabbit, reindeer, sheep water buffalo, and yak.
[0524] Nucleic Acid Vaccine: As used herein, "nucleic acid vaccine" refers
to a
vaccine or vaccine composition which includes a nucleic acid or nucleic acid
molecule
(e.g., a polynucleotide) encoding an antigen (e.g., an antigenic protein or
polypeptide.) In
- 161 -
Date Recue/Date Received 2021-04-23
exemplary embodiments, a nucleic acid vaccine includes a ribonucleic ("RNA")
polynucleotide, ribonucleic acid ("RNA") or ribonucleic acid ("RNA") molecule.
Such
embodiments can be referred to as ribonucleic acid ("RNA") vaccines.
[0525] Off-target: As used herein, "off target" refers to any unintended
effect on any
one or more target, gene, or cellular transcript.
[0526] Open reading frame: As used herein, the term "open reading frame"
or "ORF"
refers to a continuous polynucleotide sequence, for example, a DNA sequence or
RNA
sequence (e.g., an mRNA sequence), comprising a start codon, a subsequent
region
comprising a plurality of amino acid-encoding codons, and a terminal stop
codon,
wherein the region comprising the plurality of amino acid-encoding codons
contains no
stop codons.
[0527] Operably linked: As used herein, the phrase "operably linked"
refers to a
functional connection between two or more molecules, constructs, transcripts,
entities,
moieties or the like.
[0528] Part: As used herein, a "part" or "region" of a polynucleotide is
defined as any
portion of the polynucleotide which is less than the entire length of the
polynucleotide.
[0529] Peptide: As used herein, "peptide" is less than or equal to 50
amino acids long,
e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
[0530] Paratope: As used herein, a "paratope" refers to the antigen-
binding site of an
antibody.
[0531] Patient: As used herein, "patient" refers to a subject who may seek
or be in
need of treatment, requires treatment, is receiving treatment, will receive
treatment, or a
subject who is under care by a trained professional for a particular disease
or condition.
[0532] Pharmaceutically acceptable: The phrase "pharmaceutically
acceptable" is
employed herein to refer to those compounds, materials, compositions, and/or
dosage
forms which are, within the scope of sound medical judgment, suitable for use
in contact
with the tissues of human beings and animals without excessive toxicity,
irritation,
allergic response, or other problem or complication, commensurate with a
reasonable
benefit/risk ratio.
- 162 -
Date Recue/Date Received 2021-04-23
[0533] Pharmaceutically acceptable excipients: The phrase "pharmaceutically
acceptable excipient," as used herein, refers any ingredient other than the
compounds
described herein (for example, a vehicle capable of suspending or dissolving
the active
compound) and having the properties of being substantially nontoxic and non-
inflammatory in a patient. Excipients may include, for example: antiadherents,
antioxidants, binders, coatings, compression aids, disintegrants, dyes
(colors), emollients,
emulsifiers, fillers (diluents), film formers or coatings, flavors,
fragrances, glidants (flow
enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or
dispersing
agents, sweeteners, and waters of hydration. Exemplary excipients include, but
are not
limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium
phosphate
(dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl
pyrrolidone, citric acid,
crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose,
hydroxypropyl
methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine,
methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene
glycol,
polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben,
retinyl palmitate,
shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate,
sodium starch
glycolate, sorbitol, starch (com), stearic acid, sucrose, talc, titanium
dioxide, vitamin A,
vitamin E, vitamin C, and xylitol.
[0534] Pharmaceutically acceptable salts: The present disclosure also
includes
pharmaceutically acceptable salts of the compounds described herein. As used
herein,
"pharmaceutically acceptable salts" refers to derivatives of the disclosed
compounds
wherein the parent compound is modified by converting an existing acid or base
moiety
to its salt form (e.g., by reacting the free base group with a suitable
organic acid).
Examples of pharmaceutically acceptable salts include, but are not limited to,
mineral or
organic acid salts of basic residues such as amines; alkali or organic salts
of acidic
residues such as carboxylic acids; and the like. Representative acid addition
salts include
acetate, acetic acid, adipate, alginate, ascorbate, aspartate,
benzenesulfonate, benzene
sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,
fumarate,
- 163 -
Date Recue/Date Received 2021-04-23
glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate,
hydrobromide,
hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate,
lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-
naphthalenesulfonate,
nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
persulfate, 3-
phenylpropionate, phosphate, picrate, pivalate, propionate, stearate,
succinate, sulfate,
tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the
like.
Representative alkali or alkaline earth metal salts include sodium, lithium,
potassium,
calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary
ammonium, and amine cations, including, but not limited to ammonium,
tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,
trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically
acceptable
salts of the present disclosure include the conventional non-toxic salts of
the parent
compound formed, for example, from non-toxic inorganic or organic acids. The
pharmaceutically acceptable salts of the present disclosure can be synthesized
from the
parent compound which contains a basic or acidic moiety by conventional
chemical
methods. Generally, such salts can be prepared by reacting the free acid or
base forms of
these compounds with a stoichiometric amount of the appropriate base or acid
in water or
in an organic solvent, or in a mixture of the two; generally, nonaqueous media
like ether,
ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of
suitable salts are
found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing
Company,
Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and
Use, P. H.
Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Beige et al., Journal of
Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein
by
reference in its entirety.
[0535] Pharmaceutically acceptable solvate: The term "pharmaceutically
acceptable
solvate," as used herein, means a compound described herein wherein molecules
of a
suitable solvent are incorporated in the crystal lattice. A suitable solvent
is
physiologically tolerable at the dosage administered. For example, solvates
may be
prepared by crystallization, recrystallization, or precipitation from a
solution that includes
- 164 -
Date Recue/Date Received 2021-04-23
organic solvents, water, or a mixture thereof. Examples of suitable solvents
are ethanol,
water (for example, mono-, di-, and tri-hydrates), N-methylpyrrolidi- none
(NMP),
dimethyl sulfoxide (DMSO), N,N'-dimethyl- formamide (DiVif ), N,N'-
dimethylacetamide
(DMAC), 1,3- dimethy1-2-imidazolidinone (DMEU),1,3-dimethy1-3,4,5, 6-
tetrahydro-2-
(111)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl
acetate, benzyl
alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the
solvent, the
solvate is referred to as a "hydrate."
[0536] Pharmacokinetic: As used herein, "pharmacokinetic" refers to any one
or more
properties of a molecule or compound as it relates to the determination of the
fate of
substances administered to a living organism. Pharmacokinetics is divided into
several
areas including the extent and rate of absorption, distribution, metabolism
and excretion.
This is commonly referred to as ADME where: (A) Absorption is the process of a
substance entering the blood circulation; (D) Distribution is the dispersion
or
dissemination of substances throughout the fluids and tissues of the body; (M)
Metabolism (or Biotransformation) is the irreversible transformation of parent
compounds into daughter metabolites; and (E) Excretion (or Elimination) refers
to the
elimination of the substances from the body. In rare cases, some drugs
irreversibly
accumulate in body tissue.
[0537] Physicochemical: As used herein, "physicochemical" means of or
relating to a
physical and/or chemical property.
[0538] Polypeptide per unit drug (PUD): As used herein, a PUD or product
per unit
drug, is defined as a subdivided portion of total daily dose, usually 1 mg,
pg, kg, etc., of a
product (such as a polypeptide) as measured in body fluid or tissue, usually
defined in
concentration such as pmol/mL, mmol/ mL, etc. divided by the measure in the
body fluid.
[0539] Preventing: As used herein, the term "preventing" refers to
partially or
completely delaying onset of an infection, disease, disorder and/or condition;
partially or
completely delaying onset of one or more symptoms, features, or clinical
manifestations
of a particular infection, disease, disorder, and/or condition; partially or
completely
delaying onset of one or more symptoms, features, or manifestations of a
particular
- 165 -
Date Recue/Date Received 2021-04-23
infection, disease, disorder, and/or condition; partially or completely
delaying
progression from an infection, a particular disease, disorder and/or
condition; and/or
decreasing the risk of developing pathology associated with the infection, the
disease,
disorder, and/or condition.
[0540] Proliferate: As used herein, the term "proliferate" means to grow,
expand or
increase or cause to grow, expand or increase rapidly. "Proliferative" means
having the
ability to proliferate. "Anti-proliferative" means having properties counter
to or
inapposite to proliferative properties.
[0541] Prophylactic: As used herein, "prophylactic" refers to a therapeutic
or course
of action used to prevent the spread of disease.
[0542] Prophylaxis: As used herein, a "prophylaxis" refers to a measure
taken to
maintain health and prevent the spread of disease. An "immune prophylaxis"
refers to a
measure to produce active or passive immunity to prevent the spread of
disease.
[0543] Protein cleavage site: As used herein, "protein cleavage site"
refers to a site
where controlled cleavage of the amino acid chain can be accomplished by
chemical,
enzymatic or photochemical means.
[0544] Protein cleavage signal: As used herein "protein cleavage signal"
refers to at
least one amino acid that flags or marks a polypeptide for cleavage.
[0545] Protein of interest: As used herein, the terms "proteins of
interest" or "desired
proteins" include those provided herein and fragments, mutants, variants, and
alterations
thereof.
[0546] Purified: As used herein, "purify," "purified," "purification" means
to make
substantially pure or clear from unwanted components, material defilement,
admixture or
imperfection.
[0547] Repeated transfection: As used herein, the term "repeated
transfection" refers
to transfection of the same cell culture with a polynucleotide a plurality of
times. The cell
culture can be transfected at least twice, at least 3 times, at least 4 times,
at least 5 times,
at least 6 times, at least 7 times, at least 8 times, at least 9 times, at
least 10 times, at least
11 times, at least 12 times, at least 13 times, at least 14 times, at least 15
times, at least 16
- 166 -
Date Recue/Date Received 2021-04-23
times, at least 17 times at least 18 times, at least 19 times, at least 20
times, at least 25
times, at least 30 times, at least 35 times, at least 40 times, at least 45
times, at least 50
times or more.
[0548] Sample: As used herein, the term "sample" or "biological sample"
refers to a
subset of its tissues, cells or component parts (e.g. body fluids, including
but not limited
to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva,
amniotic
fluid, amniotic cord blood, urine, vaginal fluid and semen). A sample further
may include
a homogenate, lysate or extract prepared from a whole organism or a subset of
its tissues,
cells or component parts, or a fraction or portion thereof, including but not
limited to, for
example, plasma, serum, spinal fluid, lymph fluid, the external sections of
the skin,
respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood
cells, tumors,
organs. A sample further refers to a medium, such as a nutrient broth or gel,
which may
contain cellular components, such as proteins or nucleic acid molecule.
[0549] Signal Sequences: As used herein, the phrase "signal sequences"
refers to a
sequence which can direct the transport or localization of a protein.
[0550] Single unit dose: As used herein, a "single unit dose" is a dose of
any
therapeutic administered in one dose/at one time/single route/single point of
contact, i.e.,
single administration event.
[0551] Similarity: As used herein, the term "similarity" refers to the
overall
relatedness between polymeric molecules, e.g. between polynucleotide molecules
(e.g.
DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
Calculation of percent similarity of polymeric molecules to one another can be
performed
in the same manner as a calculation of percent identity, except that
calculation of percent
similarity takes into account conservative substitutions as is understood in
the art.
[0552] Split dose: As used herein, a "split dose" is the division of
single unit dose or
total daily dose into two or more doses.
[0553] Stable: As used herein "stable" refers to a compound that is
sufficiently robust
to survive isolation to a useful degree of purity from a reaction mixture, and
preferably
capable of formulation into an efficacious therapeutic agent.
- 167 -
Date Recue/Date Received 2021-04-23
[0554] Stabilized: As used herein, the term "stabilize", "stabilized,"
"stabilized
region" means to make or become stable.
[0555] Subject: As used herein, the term "subject" or "patient" refers to
any organism
to which a composition may be administered, e.g., for experimental,
diagnostic,
prophylactic, and/or therapeutic purposes. Typical subjects include animals
(e.g.,
mammals such as mice, rats, rabbits, non-human primates, and humans).
[0556] Substantially: As used herein, the term "substantially" refers to
the qualitative
condition of exhibiting total or near-total extent or degree of a
characteristic or property
of interest. One of ordinary skill in the biological arts will understand that
biological and
chemical phenomena rarely, if ever, go to completion and/or proceed to
completeness or
achieve or avoid an absolute result. The term "substantially" is therefore
used herein to
capture the potential lack of completeness inherent in many biological and
chemical
phenomena.
[0557] Substantially equal: As used herein as it relates to time
differences between
doses, the term means plus/minus 2%.
[0558] Substantially simultaneously: As used herein and as it relates to
plurality of
doses, the term means within 2 seconds.
[0559] Suffering from: An individual who is "suffering from" a disease,
disorder,
and/or condition has been diagnosed with or displays one or more symptoms of a
disease,
disorder, and/or condition.
[0560] Susceptible to: An individual who is "susceptible to" a disease,
disorder,
and/or condition has not been diagnosed with and/or may not exhibit symptoms
of the
disease, disorder, and/or condition but harbors a propensity to develop a
disease or its
symptoms. In some embodiments, an individual who is susceptible to a disease,
disorder,
and/or condition (for example, cancer) may be characterized by one or more of
the
following: (1) a genetic mutation associated with development of the disease,
disorder,
and/or condition; (2) a genetic polymorphism associated with development of
the disease,
disorder, and/or condition; (3) increased and/or decreased expression and/or
activity of a
protein and/or nucleic acid associated with the disease, disorder, and/or
condition; (4)
- 168 -
Date Recue/Date Received 2021-04-23
habits and/or lifestyles associated with development of the disease, disorder,
and/or
condition; (5) a family history of the disease, disorder, and/or condition;
and (6) exposure
to and/or infection with a microbe associated with development of the disease,
disorder,
and/or condition. In some embodiments, an individual who is susceptible to a
disease,
disorder, and/or condition will develop the disease, disorder, and/or
condition. In some
embodiments, an individual who is susceptible to a disease, disorder, and/or
condition
will not develop the disease, disorder, and/or condition.
[0561] Sustained release: As used herein, the term "sustained release"
refers to a
pharmaceutical composition or compound release profile that conforms to a
release rate
over a specific period of time.
[0562] Synthetic: The term "synthetic" means produced, prepared, and/or
manufactured by the hand of man. Synthesis of polynucleotides or polypeptides
or other
molecules described herein may be chemical or enzymatic.
[0563] Vaccine: As used herein, a vaccine is a compound or composition
which
comprises at least one polynucleotide encoding at least one antigen.
[0564] Targeted Cells: As used herein, "targeted cells" refers to any one
or more cells
of interest. The cells may be found in vitro, in vivo, in situ or in the
tissue or organ of an
organism. The organism may be an animal, preferably a mammal, more preferably
a
human and most preferably a patient.
[0565] Therapeutic Agent: The term "therapeutic agent" refers to any agent
that, when
administered to a subject, has a therapeutic, diagnostic, and/or prophylactic
effect and/or
elicits a desired biological and/or pharmacological effect.
[0566] Therapeutically effective amount: As used herein, the term
"therapeutically
effective amount" means an amount of an agent to be delivered (e.g., nucleic
acid, drug,
therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is
sufficient, when
administered to a subject suffering from or susceptible to an infection,
disease, disorder,
and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or
delay the
onset of the infection, disease, disorder, and/or condition.
- 169 -
Date Recue/Date Received 2021-04-23
[0567] Therapeutically effective outcome: As used herein, the term
"therapeutically
effective outcome" means an outcome that is sufficient in a subject suffering
from or
susceptible to an infection, disease, disorder, and/or condition, to treat,
improve
symptoms of, diagnose, prevent, and/or delay the onset of the infection,
disease, disorder,
and/or condition.
[0568] Total daily dose: As used herein, a "total daily dose" is an amount
given or
prescribed in 24 hr. period. It may be administered as a single unit dose.
[0569] Transfection: As used herein, the term "transfection" refers to
methods to
introduce exogenous nucleic acids into a cell. Methods of transfection
include, but are not
limited to, chemical methods, physical treatments and cationic lipids or
mixtures.
[0570] Translation: As used herein "translation" is the process by which a
polynucleotide molecule is processed by a ribosome or ribosomal-like
machinery, e.g.,
cellular or artificial, to produce a peptide or polypeptide.
[0571] Transcription: As used herein "transcription" is the process by
which a
polynucleotide molecule is processed by a polymerase or other enzyme to
produce a
polynucleotide, e.g., an RNA polynucleotide.
[0572] Treating: As used herein, the term "treating" refers to partially or
completely
alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting
progression
of, reducing severity of, and/or reducing incidence of one or more symptoms or
features
of a particular infection, disease, disorder, and/or condition. Treatment may
be
administered to a subject who does not exhibit signs of a disease, infection,
disorder,
and/or condition and/or to a subject who exhibits only early signs of a
disease, infection,
disorder, and/or condition for the purpose of decreasing the risk of
developing pathology
associated with the disease, infection, disorder, and/or condition.
[0573] Unmodified: As used herein, "unmodified" refers to any substance,
compound
or molecule prior to being changed in any way. Unmodified may, but does not
always,
refer to the wild type or native form of a biomolecule. Molecules may undergo
a series of
modifications whereby each modified molecule may serve as the "unmodified"
starting
molecule for a subsequent modification.
- 170 -
Date Recue/Date Received 2021-04-23
[0574] Vaccine: As used herein, the phrase "vaccine" refers to a biological
preparation that improves immunity in the context of a particular disease,
disorder or
condition.
[0575] Viral protein: As used herein, the phrase "viral protein" means any
protein
originating from a virus.
EXAMPLES
Example 1. In vivo Study of LNP Formulated mRNA
[0576] 5 groups of week 6 female C57b1/6 mice, 8 mice/group, are
administered
formulations as described in Table 6. On day 0 and 21 the mice are bled before
receiving
20 micrograms (" g") of one of the formulations in Table 6 via intramuscular
administration on day 1 and 22. On day 43 the mice were euthanized, and blood
was
collected by cardiac puncture, the spleen was harvested and splenocytes
isolated.
Table 6. Formulation Table
Group Description of Formulation
1 LNP formulated TdTomato mRNA negative control
2 LNP formulated S protein receptor binding domain (RBD) mRNA
3 LNP formulated full-length S protein mRNA ("PTX-B")
4 LNP formulated full-length S protein with mutated furin site mRNA
Dulbecco's Phosphate-Buffered Saline (DPBS) control
[0577] Clinical isolate virus neutralization assay was performed on the
pooled
samples from the live bleed from day 21. At termination (Day 43), sera
antibody binding
to SARS-CoV-2 RBD and S protein, pseudovirus neutralization, clinical isolate
virus
neutralization and T cell response (determined by enzyme-linked immunospot
(ELIspot)
and flow cytometry) were measured.
[0578] There were no apparent adverse reactions from the mice. Data from
the initial
preliminary bleed showed that all three constructs did have neutralizing
activity. Group 3
formulation (full-length S protein mRNA) was the best, followed closely by the
Group 4
formulation (full-length S protein with mutated furin site); the Group 2
formulation (S
protein RBD domain) was the lowest performer; and no activity was seen in
either
negative control treated groups.
- 171 -
Date Recue/Date Received 2021-04-23
[0579] Splenocytes were stimulated with SARS-CoV antigens (RBD peptide
pool
plus S protein), and antigen-specific T cell responses were measured by
counting IFN-y
secreting T cells in ELIspot, or Thl cytokine (IFN-y/TNF-a/1L-2) and Th2
cytokine (IL-
4/IL-5) producing T cells in flow cytometry. Thl cytokine (IFN-y/TNF-ailL-2)
and Th2
cytokine (IL-4/IL-5) were also measured in the supernatant of the SARS-CoV
antigen-
stimulated T cells by a multiplex mouse cytokine assay.
ELIspot Assay
[0580] The ELIspot assay demonstrated that the splenocytes from the mice
treated
with Group 2, Group 3 and Group 4 formulations produced T cells that were
secreting
IFN- y in response to a peptide pool which contained overlapping peptides
within the
RBD and conserved S2 regions of the S protein. The responses in Group 2, Group
3, and
Group 4 were similar and neither of the two control groups showed a
significant response
to the peptide pool. Flow cytometry assay did not detect a significant Thl or
Th2
responses in the three groups as compared to the two control groups.
[0581] A direct binding ELISA was used to determine if any antibodies were
elicited
to bind to the S protein. Either the RBD domain or the full-length S protein
was bound to
plates and different dilutions of sera from the treated mice were incubated in
the plates
before being washed and detected by an anti-mouse antibody. Sera from Group 2,
Group
3 and Group 4 were positive for antibodies that could bind both the RBD and
the full-
length S protein and the controls were negative for binding.
SARS-CoV2 Neutralization Assay
[0582] For determining whether the antibodies elicited from the Group 2,
Group 3 or
Group 4 formulations were neutralizing, two different assays were used. The
first assay
used a SARS-CoV-2 virus that was isolated from one of the first COVID-19
patients in
Ontario and the readout for this assay is a microscopic reading on the health
of Vero2 E6
cells that have been incubated with live virus and different dilutions of the
sera from the
treated mice. This assay has been used to characterize sera from convalescent
patients
where the ID50 values have been mostly in the range of 1:80 to 1:320,
therefore the
dilution series chosen for the test of these mouse sera was between 1:20 and
1:2560. The
- 172 -
Date Recue/Date Received 2021-04-23
sera from Group 1 mice had minimal detectable neutralization activity. Group 2
showed
some moderate activity with 6 of the 8 samples with 'DSOs between 1:20 and a
1:80.
Group 3 and Group 4 showed strong neutralizing activity with most sera samples
retaining 100% neutralization activity even at the highest dilution of 1:2560.
Results are
provided in FIG. 1.
Pseudovirus Neutralization Assay
[0583] The second neutralization assay that was performed was a pseudovirus
assay.
This uses a SARS-CoV-2 S protein pseudotype lentivirus that encodes a
luciferase gene
and can infect HEK293T cells made to express hACE2 and TMPRSS2 for better
transduction efficiency. This assay has been characterized by determining the
titer of sera
from ¨50 convalescent patients where the sera had an average ID50 of ¨1:500
with a
range of 1:1 to ¨1:10,000, but for this study, a range of dilutions from 1:40
to 1:24,400
was used. The sera from both of the control groups (Group 1 and Group 5) had
minimal
to no activity. Group 2 had significant but low neutralization activity. The
activity of
Group 3 and Group 4 was above the quantitative range of the assay. Values were
extrapolated and the average ID50 values were ¨1:50,000 (Group 3) and
¨1:45,000
(Group 4). Results are provided in FIG. 2.
Conclusion
[0584] This study showed our LNP formulated mRNA vaccines when injected into
mice intramuscularly twice over a three-week period were able to elicit T cell
responses
and antibodies that can bind the S protein of SARS-CoV-2. The treated mice
produced
antibodies that could neutralize a clinical isolate of SARS-CoV-2 as well as a
SARS-
CoV-2 pseudotyped lentivirus. This was particularly true of Group 3 and Group
4
formulations which resulted in titers above the quantitative range in each
assay.
Example 2. Neutralizing Antibody Study in Mice
[0585] The lead vaccine candidate, LNP formulated vaccine encoding full-
length S
protein (vaccine formulation applied to Group 3 in Table 6, referred to
hereafter as
"PTX-B"), was chosen as the candidate for further study. The ability of PTX-B
to
- 173 -
Date Recue/Date Received 2021-04-23
produce neutralizing antibodies and T cell response in mice was evaluated.
Three groups
of female C57BL/6 mice (10 mice/group) were vaccinated on Days 1 and 22 as
follows:
Group 1: 10 g LNP formulated tdTomato mRNA (negative control)
Group 2: 1 tg PTX-B
Group 3: 10 g PTX-B
[0586] Parameters evaluated in this study included clinical isolate virus
neutralization
assay and pseudovirus neutralization assay on pooled samples from live bleed
three
weeks (Day 22) after the first immunization; and the following assessments at
termination (Day 43) after the second immunization: sera antibody binding to S
protein
from SARS-CoV-2, pseudovirus neutralization, clinical isolate virus
neutralization,
splenocyte T cell responses by ELISpot and flow cytometry, and cytokine
secretion.
[0587] The live phase of the experiment showed no apparent adverse
reactions in the
mice. Data from the initial preliminary bleed on Day 22 showed that the 10 tg
dose level
produced neutralizing antibodies while the 1 tg dose level was only marginally
different
from the negative control group.
[0588] At termination, three weeks after the second immunization (booster),
the 1 tg
and 10 tg dose groups showed approximately equal T cell responses in the
ELISpot
assay, but the 10 tg dose level group performed much better in the antibody-
based assays
with evidence of high levels of IgG isotypes (total IgG, IgGl, IgG2b and
IgG2c). The
levels of IgM were higher in the mice dosed with 1 tg than those dosed with 10
g,
perhaps due to an early class switching due to a stronger stimulus in the 10
tg group.
There was evidence of IgGA, especially at the 10 tg dose, but this isotype was
not
induced to as high a level as the IgG isotypes. Both 1 and 10 pg PTX-B
elicited very
strong S-specific IgG, IgGl, IgG2b, IgG2c (end-point titers for 1 and 10 [tg
PTX-B are,
respectively: 2.7 0.9E6, 3.0 0.5E7 for IgG; 1.1 0.2E6, 2.8 0.8E6 for IgGl; 9.4
2.0E5,
9.7 3.4E6 for IgG2b; 3.5 1.8E7, 1.95 0.0E8 for IgG2c). Both 1 and 10 [tg PTX-B
also
elicited strong S-specific IgA (end-point titer for 1 and 10 pg PTX-B is,
respectively:
3.3 3.1E4, 1.7 0.6E7), although the titers were lower than those of the IgG.
The dose of
[tg PTX-B usually induced higher S-specific binding antibody than the dose of
1 pg.
- 174 -
Date Recue/Date Received 2021-04-23
The preponderance of the Thl antibody (IgG2b and IgG2c) over the Th2 antibody
(IgG1)
also indicated that PTX-B induced a Thl-biased antibody response. Very low or
little 5-
specific binding antibodies were detected in the sera of the control mice
receiving the
tdTomato mRNA.
[0589] As in Example 1, the first neutralization assay used a SARS-CoV-2
virus that
was isolated from one of the first COVID-19 patients in Ontario and the second
assay
was a pseudovirus neutralization assay using a SARS-CoV-2 S protein
pseudotyped
lentivirus. In both antibody neutralization assays the 10 jig dose group
greatly
outperformed the 1 jig dose group, though this dose group did show
considerable
neutralizing activity (comparable to that seen with sera from convalescent
patients). FIG.
3 shows the ID50 (dilution at which 50% inhibition of infectivity is seen) for
both the
SARS-CoV-2 clinical isolate and pseudovirus neutralization assays. The sera
from the
negative control group showed no activity in either assay. There was a dose-
responsive
effect with sera from the 10 jig group demonstrating considerably more
neutralization
activity, especially in the SARS-CoV-2 clinical isolate assay. Statistics were
performed
by Kruskal-Wallis test using multiple comparisons; in FIG. 3, **=13<0.01,
***=13<0.001
****=P<0.0001. There was no significant activity in the negative control
group,
moderate activity in the 1 jig dose group and very strong neutralizing
activity in the 10
jig group with ID50s up to 1:90,000 in the pseudovirus assay.
[0590] IFN-y analysis by ELISpot was performed to determine the T cell
response to
immunization with the vaccine. Splenocytes from mice were stimulated with
peptide
pools of SARS-CoV-2 S protein (315 15mer peptides with 1 lmer overlap). IFN-y
producing T cells were measured by ELISpot analysis. A higher frequency of T
cells
from PTX-B-immunized mice produced IFN-y compared with those from mice
vaccinated with the negative control (FIG. 4). Mice were vaccinated with a
prime and
booster of PTX-B at Days 1 and 22. Mice were sacrificed at Day 43 and
splenocytes were
stimulated in the presence of SARS-CoV-2 peptide pool overnight on a 96 well
ELISpot
plate precoated with anti-IFN-y antibodies. Following incubation, the plates
were washed
- 175 -
Date Recue/Date Received 2021-04-23
stained and treated with an anti-IFN-y EIRP antibody and read on an ELISpot
reader.
Statistics were performed using Kruskal-Wallis test with multiple comparison
analysis.
[0591] Cytokine profiling by Luminex showed that mice immunized with PTX-B
produced in a dose dependent manner high levels of IL-2, IFN-y, and GM-CSF but
low
levels of IL-4 and IL-10 (FIG. 5). Mice were vaccinated with a prime and
booster of
PTX-B at Days 1 and 22. Mice were sacrificed at Day 43 and splenocytes were
stimulated in the presence of SARS-CoV-2 peptide pool overnight. Supernatants
were
analyzed by Luminex for the presence of IL-2, IFN-y, GM-CSF, IL-4, IL-5, and
IL-10.
Statistics were performed by Kruskal-Wallis test by multiple comparisons. The
levels of
TNF-a were not detectable in the assay for mice immunized with the PTX-B or
control.
Interestingly, levels of IL-5 were detectable in PTX-B-immunized mice but did
not
increase with vaccination.
Cellular Immune Response
[0592] PTX-B also elicited a strong cellular immune response. Mouse
splenocytes
were prepared at 3 weeks after the boost vaccination, stimulated with a S
peptide pool,
and the S-specific cellular responses were measured by IFN-y/lL-4 ELISPOT,
flow
cytometry analysis of cytokine production by CD4+ and CD8+ T cells, and a
multiplex
immunoassay to detect the cytokines in the supernatant of the stimulated
splenocytes.
These assays showed that both 1 [ig and 10 [ig PTX-B induced robust S-specific
cellular
immune responses, which is Thl-biased as indicated by the predominant Thl
cytokine
(IFN- y/TNF-a/IL-2) production over Th2 cytokine (IL-4/IL-5) from CD4+ T
cells. Of
note, significant amount of S-specific CD8+ T cells were induced by PTX-B. In
contrast
to the humoral response, especially the nAb response, the cellular responses
elicited by 1
[ig and 10 [ig PTX-B were usually comparable. Cytokine profiling by flow
cytometry
showed significant proportions of CD4+ (FIG. 6A) and CD8+ (FIG. 6B) cells
producing
IL-2 and IFN-y detected in PTX-B-immunized mice, especially CD8+ IFN-y
producing
cells. In contrast, IL-4 and IL-5 producing cells were not significantly
different in
immunized mice compared to the control mice. Mice were vaccinated with a prime
and
booster of PTX-B at Days 1 and 22. Mice were sacrificed at Day 43 and
splenocytes were
- 176 -
Date Recue/Date Received 2021-04-23
stimulated in the presence of SARS-CoV-2 peptide pool overnight. Following
overnight
stimulation, cells were surface stained for anti-CD3, anti-CD4 and anti-CD8
antibodies.
Cells were then fixed and permeabilized and stained for IL-2, IFN-y, TNF-a, IL-
4 and IL-
5. Cells were evaluated using flow cytometry. FIG. 6A and FIG. 6B show that
TNF-a
producing cells were slightly higher than control mice but not consistently
high in a dose
dependent manner. These results demonstrate that vaccination with PTX-B
induced an S
protein specific Thl response.
[0593] In conclusion, immunizations with either 1 i.ig or 10 i.ig PTX-B led
to similar T
cell response, both well above the background in the negative control group.
For the
antibody-based assays (antibody levels and neutralization ability), the 10
i.ig dose greatly
outperformed the 1 jig dose.
Example 3. Mouse AAV6-hACE2 Challenge Model
[0594] A non-GLP challenge study was conducted in AAV6-hACE2 (receptor for
SARS-CoV-2) transfected C57BL/6 mice to investigate the protective efficacy of
PTX-B.
Four groups of female C57BL/6 mice (12 mice/group) were vaccinated with PTX-B
on
Days 1 and 22 as follows:
Group 1: Formulation buffer (negative control)
Group 2: 20 g PTX-B
Group 3: 4 g PTX-B
Group 4: 1 g PTX-B
[0595] On Day 29, the animals were transduced with lx10" vector genome
copies of
AAV-hACE2 per mouse and then challenged intranasally with 2.5x104 TCID50 with
SARS-CoV-2 per mouse on Day 38. Study termination was on Day 42. The
parameters
evaluated in this study included infectivity of lung homogenates, viral RNA
levels in the
lung, and lung histopathology. Mice were euthanized and one lung was taken for
histology while the second lung was split in half for homogenization in media
for
infectivity test and homogenization in RNA extracting buffer for viral load
determination.
[0596] The live phase of the experiment showed no apparent adverse
reactions in the
mice. Body weights were measured on Day 38, at the time of challenge and then
again on
- 177 -
Date Recue/Date Received 2021-04-23
Day 42, immediately prior to sacrifice. There was a statistically significant
weight loss
observed in the 1 lig PTX-B vaccination group (20.39 vs 18.54, 9.07% p=0.0016)
(FIG.
7). C57BL/6 mice were immunized with PTX-B prime-booster and transduced with
AAV6-hACE2. Body weights were measured at time of challenge (Day 38) and
immediately before sacrifice (Day 42). Analysis performed by 2-way ANOVA with
multiple comparisons. In FIG. 7, ** p<0.01. No significant weight loss was
observed in
the 20 lig or 4 lig groups, or in the formulation control group.
[0597] As shown in FIG. 8, PTX-B provided protective efficacy at all three
dose
levels tested. No infective virus was found in the mice immunized with 20 or 4
lig of
vaccine (TCID50 = 0) and 10 of 12 mice immunized with 1 lig were also free of
infective
virus (mean TCID50 = 1.25 2.93) while 11 of the 12 mice in the formulation
buffer
negative control group had easily detectable infectious SARS-CoV-2. In FIG. 8,
TCID50
means tissue culture 50% infectious dose. As shown, PTX-B neutralizes SARS-CoV-
2.
TCID50 were measured in AAV6-hACE2 transduced C57BL/6 mice that were
immunized by prime-booster with PTX-B at 3 different doses or a formulation
buffer
negative control. Mice were transduced with AAV6-hACE2 at 7 days post booster
and
challenged 9 days later. All mice were sacrificed at 4 days post challenge
with SARS-
CoV-2 and virus measured from lung homogenates (n=12 per group) (****
p<0.0001).
[0598] Additionally, detection of viral RNA in the lungs by PCR
demonstrated a
dose-responsive reduction with more than 100-fold difference between the
averages of
the high dose and negative control groups. Sections of one lung were graded
for lung
histopathology. All mice demonstrated significant histopathology. It is not
clear how
much of the pathology was due to SARS-CoV-2 and how much was due to the AAV6
virus used to express hACE2; however, there was a trend to lower
histopathology scores
in the groups of mice treated with the two higher dose levels of PTX-B (FIG.
9). To
summarize, mice were immunized with the indicated amount of PTX-B, transduced
with
AAV6-hACE2 and nine days later challenged with SARS-CoV-2. Four days after
challenge, mice were immunized and the left lung was fixed in formalin,
processed for
histology and examined under the microscope by a certified pathologist who was
blinded
- 178 -
Date Recue/Date Received 2021-04-23
to the treatment conditions. Each sample was assigned a histology score from 1-
5 with
the lowest being normal. A trend to lower pathology was seen with increasing
doses of
the vaccine.
[0599] In conclusion, administration of PTX-B (1, 4 and 20 jig) conferred
protection
against SARS-CoV-2 infection using the AAV6-hACE2 transduction mouse model.
There was also a reduced total amount of SARS-CoV-2 mRNA in the lungs at
euthanasia. A post-challenge weight loss was observed in the low dose (1 jig)
vaccination
group.
Example 4. Hamster Challenge Model
[0600] A challenge study was performed in 6-8 week old male Syrian Golden
hamsters challenged with SARS-CoV-2 to determine if the vaccine protected from
infection.
[0601] The Syrian golden hamster is susceptible to SARS-CoV-2 infection and
has
demonstrated utility for evaluating candidate vaccines.
[0602] Group 1 hamsters received 20 jig LNP formulated full-length S
protein mRNA
(PTX-B).
[0603] Group 2 hamsters received 4 jig LNP formulated full-length S protein
mRNA
(PTX-B).
[0604] Group 3 hamsters received formulation buffer (PBS sham/negative
vaccine
control group).
[0605] Group 4 hamsters received 1 jig LNP formulated full-length S protein
mRNA
(PTX-B).
[0606] On Day 0, all hamsters were pre-bled for baseline analysis. On Day 1
all
hamsters received the first intramuscular injection (vaccine or control,
according to
Group 1 ¨ Group 4). Animals were allowed to acclimatize for 7 days prior to
receiving
the first vaccine dose. On Day 21 all hamsters were subjected to live
bleeding. On Day 22
all hamsters received the second (booster) vaccination according to Group 1 ¨
Group 4.
On Day 29 all hamsters were received AAV6-hACE2 (see, e.g., Example 3)
intranasally
to facilitate SARS-CoV-2 infection. On Day 38, all hamsters were infected with
SARS-
- 179 -
Date Recue/Date Received 2021-04-23
CoV-2 via intranasal infection. All animals received a total dose of 7.5 x
10A5 TCID50 as
determined by back-titration. Following this, animals were monitored daily for
weight
loss and signs of disease or distress. Additionally, viral shedding was
monitored by
collecting oral swabs on every second day. Hamsters were euthanized on Day 42
for
endpoint analysis: (i) infectivity of lung homogenates; and (ii) viral RNA
level in lung;
(iii) lung histopathology. Animals were monitored during the study for any
observable
clinical signs during the vaccination phase. There were no apparent adverse
reactions
observed. Full experimental design is illustrated in Table 7.
Table 7. Experimental Overview
Route of
Hamsters were vaccinated at two sites intramuscularly in the hind
vaccination leg,
with 200 uL (100u1 per site) total using 23-25 gauge, 3/8-1-
inch needles.
Time of vaccination 2 doses on days 1 and 22, challenge on day 43
Challenge virus SARS-CoV-2
Dose 7.5 x 105 TCID50
Route Intra nasal
[0607] At 4 days and 8 days post-infection (dpi), four animals from each
group were
selected at random and euthanized. Tissues were collected for viral load by
qRT-PCR and
infectious virus titre levels as well as for histopathology. At the terminal
point of the
experiment (8 dpi), blood was also collected from animals in both groups to
evaluate
titers of neutralizing antibodies.
[0608]
Animals in the vaccinated groups showed on average no weight loss during the
course of the experiment. By contrast, hamsters in the sham-vaccine group
showed
moderate average weight loss beginning at 3 dpi. Overall, the average weight
loss was
11% by terminal point of the experiment. No other significant clinical signs
of disease
were reported for either group.
[0609]
Following euthanization of the animals at 4 dpi and 8 dpi, half of the lung
was
placed into formalin for tissue fixation. Tissues subsequently underwent H&E
staining
and were evaluated by a pathologist who was blinded to the groups. Pathology
scores
were significantly higher at both timepoints in the control group (sham
vaccination)
- 180 -
Date Recue/Date Received 2021-04-23
compared to the vaccinated group. This suggested more severe disease in the
unvaccinated group.
[0610] Collection of oral swabs over the course of the experiment was used
to
evaluate viral shedding. Interestingly, while viral RNA was detected in both
groups
throughout the experiment, the levels of actual infectious virus was
significantly lower in
vaccinated animals (ranging from 2-3 log reduction, as illustrated in FIG.
14). This
suggests that viral shedding was lower in the vaccinated group throughout the
course of
the experiment.
[0611] Examination of the viral burden in nasal turbinates demonstrated
significantly
lower quantities of infectious virus at 4 dpi in the vaccinated group and
undetectable
levels of infectious virus at 8 dpi. A similar trend was seen in the lungs,
although a
notable difference was that infectious virus was not detected at either
timepoint in the
lungs of vaccinated animals. Viral RNA was detected in lungs in both
vaccinated and
unvaccinated groups at both timepoints.
[0612] These data indicate that vaccination with PTX-B conferred
protection against
an intranasal challenge with SARS-CoV2-2 in the hamster model of infection.
Example 5. Immunogenicity and Local Tolerance Study in Mice
[0613] The goal of this 2-dose immunogenicity and tolerability study was
to obtain
basic safety data pertinent to mRNA vaccines in addition to immunological data
in a
different strain of mice than used for the other preclinical experiments. PTX-
B was
administered to groups of BALB/c mice by IM injection on Days 1 and 22 at dose
levels
of 0, 4, or 20 jig as outlined in Table 8. Main study animals were evaluated
for clinical
signs, body weight changes, and dermal observations by modified Draize
scoring. The
hematology cohort of animals was sacrificed on Day 24 (two days after second
dose),
blood was drawn for hematology and organ weights were recorded, gross
pathology
evaluated, and liver, spleen, and injection site tissues were examined
microscopically.
The cytokine cohort was sacrificed on Day 22 for determination of serum
cytokine
concentrations. The main study animals were terminated on Day 43 (three weeks
after the
- 181 -
Date Recue/Date Received 2021-04-23
second dose) and assessed for immunogenicity end points, hematology, clinical
chemistry, liver function tests, gross pathology, and organ weights.
Table 8. Immunogenicity and Local Tolerance Study in Mice ¨ Study Design
No. of Animals
Dose Dose
Group Test Dose Main
Volume Concentration Hematology Cytokine
No. Material (jag) Study
( mL)
110 (jag /
Males Females Males Females Males Females
1 Control 0 50 0 10 10 5 5 5 5
2 PTX-B 4 50 0.08 10 10 5 5 5 5
3 PTX-B 20 50 0.4 10 10 5 5 5 5
Safety-Related Endpoints
[0614] Transient, slight body weight loss was observed in both sexes after
the second
dose of 20 jig PTX-B; however, no difference in the average body weights among
the
groups was apparent at the end of the study (data not shown).
[0615] Test material-related findings at the injection site were noted upon
clinical
observation, and gross and microscopic examination; all findings were
reversible. Based
on Draize scoring, occasional findings of redness and/or swelling were
observed at the
injection site at both 4 and 20 jig and erected fur was seen in a minority of
female mice
for one to two days after the first dose, but these were not considered
significant findings.
Upon termination two days after the second dose, histopathological findings at
the
injection site included minimal to moderate mixed cell inflammation at 4 and
20 jig in
both sexes; the finding was accompanied by edema and, in one female at 20 jig,
by
mineralized material. A dose relationship in the incidence and severity of the
finding was
noted in females. Inflammation correlated grossly with firm abnormal
consistency and
swelling. In addition, minimal to mild hemorrhage was noted in a few animals
at both
doses, correlating grossly with dark focus of the injection site or subcutis;
no dose
relationship was evident. At the end of the study, no macroscopic findings
were observed
at the injection site.
[0616] Serum cytokine analysis 6 hours after the second dose (Day 22) was
performed
to monitor for cytokine release syndrome, which is a known potential side
effect of LNP-
formulated mRNAs. IFN-y, IL-113, IL-6, IL-10, MCP-1, and TNF-a were analyzed
using
- 182 -
Date Recue/Date Received 2021-04-23
a validated immunoassay method. PTX-B-related increases in serum
concentrations of
IL-6 (up to 53 fold and 266 fold of control in males and females respectively)
and MCP-1
(up to 20 fold and 15 fold of control in males and females respectively) were
observed in
both sexes at the two dose levels. In general, the magnitude of the responses
was dose
related. For MCP-1, the response had no meaningful sex-related difference. For
IL-6, the
increase was greater in females than in males. Mild increases (up to 2.5 fold)
in serum
concentrations of IFN-y were observed in some animals of both sexes. No PTX-B-
related
changes in IL-113, IL-10, and TNF-a were apparent. The pattern of cytokine
changes
observed was not consistent with cytokine release syndrome.
[0617] Body weight was determined weekly during the study. A slight dip in
body
weight was seen in both males and females in the 20 i.ig group. The body
weight of each
group had recovered by the end of the study (data not shown.)
[0618] Hematological parameters were determined two days (Day 24) and three
weeks (Day 43) after the second vaccination. At the first time point, the only
changes that
changed dose-responsively in both sexes were leukocytes (males 264% and 420%
of
control at 4 and 20 i.ig respectively, females 329% and 514% of control at 4
and 20 i.ig
respectively) and reticulocytes (males 69% and 41% of control at 4 and 20 i.ig
respectively, females 53% and 27% of control at 4 and 20 i.ig respectively);
no effect on
red cell parameters was observed. All hematological parameters were within
normal
ranges on Day 43 (leukocytes: males 77% and 77% of control at 4 and 20 i.ig
respectively, females 67% and 75% of control at 4 and 20 i.ig respectively,
reticulocytes
(males 107% and 117% of control at 4 and 20 i.ig respectively, females 122%
and 129%
of control at 4 and 20 i.ig respectively).
[0619] At two days (Day 24) after the second dose, the liver of females at
20 i.ig
showed minimal hepatocellular cytoplasmic alteration, characterized by
accumulation of
glycogen-like material, was noted. The change correlated with increased
weights
(absolute and relative to brain weight, 24% to 27%) and pale discoloration.
However, at
Day 43, all liver function tests were within normal ranges and not
significantly different
from the control group.
- 183 -
Date Recue/Date Received 2021-04-23
[0620] On Day 24, increased spleen weights (absolute and relative to brain
weight,
32% to 49%) were noted in both sexes at 4 and 20 jig. This increase was
statistically
significant and correlated grossly with enlargement in females. No microscopic
correlate
could be established.
[0621] By the end of the study (Day 43, three weeks after the second
dose), no PTX-
B-related gross findings were noted. Although absolute spleen weights remained
increased (14% to 18%) at 20 jig, the magnitude of the increase was
substantially lower
than at two days after the second dose and all hematological parameters were
normal.
[0622] In summary, PTX-B administered to BALB/c mice by IM injection on Days 1
and 22 at dose levels of 4 and 20 jig was well tolerated. Findings observed
after the
second dose were limited primarily increases in serum concentrations of IL-6
and MCP-
1, dose-related increases in leukocytes and decreases in reticulocytes, dose-
related
injection site reactions, and increased spleen weight with no microscopic
correlate.
Additional findings noted only at 20 ug/dose included slight body weight loss
and
hepatocellular cytoplasmic alteration. All findings were fully or partially
reversible; by
Day 43, test material-related effects were limited to slight increase in
spleen weights at
20 ug/dose.
Immunogenicity-Related Endpoints
[0623] Splenocytes were collected at end of study for analysis by ELISpot
(FIG. 10,
IFN-y and IL-4 ELISpots of splenocytes from PTX-B immunized mice). Splenocytes
collected from PTX-B mice were stimulated in the presence of SARS-CoV-2 S
protein
peptide pools S158 and S157 (available, e.g., from JPT Peptide Technologies,
Berlin,
Germany) on IFN-y and IL-4 multiplexed ELISpot plate. Spots were counted after
overnight stimulation. Statistics performed by two-way ANOVA. As shown in FIG.
10, a
significant increase in expression of IFN-y was observed from both male and
female mice
stimulated with the S158 peptide pool. Similarly, the S157 peptide pool
induced
significant increase in IFN-y expression in male mice. IL-4 expression was not
significantly increased by stimulation with either peptide pool. This
combination of
results indicates a Thl skewed response.
- 184 -
Date Recue/Date Received 2021-04-23
[0624] In the SARS-CoV-2 neutralization assay, sera from both female and
male mice
treated with formulation buffer as a negative control provided no protection
from
infection with a SARS-CoV-2 clinical isolate at any dilution tested (FIG. 11).
Conversely, sera from female and male mice immunized with 4 i.ig of PTX-B gave
mean
ID50 titers of 1353 and 480, respectively. Sera from mice immunized with 20
i.ig of PTX-
B provided even greater protection with mean ID50 titers of 7645 (females) and
5118
(males) demonstrating a dose-responsive effect (FIG. 11).
[0625] To confirm the strong neutralizing effect of the sera from mice
treated with
PTX-B, a second independent pseudovirus neutralization assay was performed.
Sera from
the negative control group showed no neutralizing capacity (FIG. 12). Sera
from the
female and male mice treated with 4 i.ig of PTX-B provided protective activity
with ID50
values of 4048 and 1863, respectively. Sera from mice immunized with 20 i.ig
of PTX-B
showed mean ID50 values of 16390 (females) and 1414 (males).
[0626] Immunization with PTX-B, at both 4 and 20 lig, resulted in
production of a
strong neutralizing antibody response in BALB/c mice (FIG. 13A ¨ FIG. 13C).
Serial
dilutions of sera from PTX-B treated mice were performed and the anti-SARS-CoV-
2
Spike IgG, IgGl, IgG2a, IgG2b, IgM and IgA were measured using anti-isotype
HRP
antibodies. Median values are represented using box-plots with whiskers
representing the
Tukey analysis of the Q1 and Q3 of the interquartile range with statistical
outliers
represented with individual dots. The results were dose-responsive and
consistent with
what was demonstrated in C57BL/6 mice in previous experiments. The anti-SARS-
CoV-
2 anti-Spike protein antibody profile induced by prime-booster with PTX-B
showed that
this formulated vaccine promoted seroconversion against SARS-CoV-2 Spike
protein.
SARS-CoV-2 spike protein specific IgG (FIG. 13A, left panel), IgG1 (FIG. 13A,
right
panel), IgG2a (FIG. 13B, left panel), IgG2b (FIG. 13B, right panel), IgM (FIG.
13C, left
panel) and IgA (FIG. 13C, right panel) were induced at both dose levels
tested.
Equivalents and Scope
[0627] Those skilled in the art will recognize, or be able to ascertain
using no more
than routine experimentation, many equivalents to the specific embodiments
described
- 185 -
Date Recue/Date Received 2021-04-23
herein. The scope is not intended to be limited to the above Description, but
rather is as
set forth in the appended claims.
[0628] In the claims, articles such as "a," "an," and "the" may mean one or
more than
one unless indicated to the contrary or otherwise evident from the context.
Claims or
descriptions that include "or" between one or more members of a group are
considered
satisfied if one, more than one, or all of the group members are present in,
employed in,
or otherwise relevant to a given product or process unless indicated to the
contrary or
otherwise evident from the context. The present disclosure includes
embodiments in
which exactly one member of the group is present in, employed in, or otherwise
relevant
to a given product or process. The present disclosure includes embodiments in
which
more than one, or the entire group members are present in, employed in, or
otherwise
relevant to a given product or process.
[0629] It is also noted that the term "comprising" is intended to be open
and permits
but does not require the inclusion of additional elements or steps. When the
term
"comprising" is used herein, the term "consisting of" is thus also encompassed
and
disclosed.
[0630] Where ranges are given, endpoints are included. Furthermore, it is
to be
understood that unless otherwise indicated or otherwise evident from the
context and
understanding of one of ordinary skill in the art, values that are expressed
as ranges can
assume any specific value or subrange within the stated ranges in different
embodiments,
to the tenth of the unit of the lower limit of the range, unless the context
clearly dictates
otherwise.
[0631] In addition, it is to be understood that any particular embodiment
that falls
within the prior art may be explicitly excluded from any one or more of the
claims. Since
such embodiments are deemed to be known to one of ordinary skill in the art,
they may
be excluded even if the exclusion is not set forth explicitly herein. Any
particular
embodiment of the compositions described herein (e.g., any therapeutic or
active
ingredient; any method of production; any method of use; etc.) can be excluded
from any
one or more claims, for any reason, whether or not related to the existence of
prior art.
- 186 -
Date Recue/Date Received 2021-04-23
[0632] It is to be understood that the words which have been used are words
of
description rather than limitation, and that changes may be made within the
purview of
the appended claims without departing from the true scope and spirit of the
present
disclosure in its broader aspects.
[0633] While the present disclosure has been described at some length and
with some
particularity with respect to the several described embodiments, it is not
intended that it
should be limited to any such particulars or embodiments or any particular
embodiment,
but it is to be construed with references to the appended claims so as to
provide the
broadest possible interpretation of such claims in view of the prior art and,
therefore, to
effectively encompass the intended scope of the disclosure.
- 187 -
Date Recue/Date Received 2021-04-23
