FORM 2
THE PATENTS ACT, 1970
[39 of 1970]5
&
THE PATENTS RULES, 2003
10
COMPLETE SPECIFICATION
15
[See Section 10 and Rule 13]
TITLE:20
Multitarget Vaccines and Therapeutics
APPLICANT:
Complete Name: Popvax Private Limited
Address: 8c Lohtse Chs, Millitary Road, Ruia Park, Mumbai, Maharashtra - 400049, India
Citizenship: Indian25
The following specification particularly describes the nature of the invention and the
manner in which it is to be performed.
30
2
RELATED APPLICATIONS
This application claims the benefit of priority to U.S. Provisional Patent Application
serial number 63/435,977, filed December 29, 2022, the contents of which are hereby
incorporated herein by reference in their entirety.5
FIELD OF THE INVENTION
The present disclosure relates to multitarget nucleic acid sequences, multitarget
peptides, and polypeptide nanoparticle and their compositions for vaccine and therapeutic10
purpose.
BACKGROUND
Variety of diseases are caused by infectious agents, such as bacteria, viruses, fungi,15
protozoans, and worms. Despite the advances in vaccination and other therapeutic
interventions, the economic burden of eight major diseases (HIV/AIDS, malaria, measles,
hepatitis, dengue fever, rabies, tuberculosis, and yellow fever) alone has been estimated to
be up to US$8 trillion, with more than 156 million life years lost for the year 2016 alone
(Armitage, Catherine Nature (2021) 598: S9).20
Many pathogens such as influenza virus, HIV, human papillomavirus, SARS-CoV-
2, Streptococcus pneumoniae, Neisseria meningitidis, Neisseria gonorrhoeae, Trypanosoma
brucei etc., have been ever evolving and adapting to escape the currently available vaccines
and therapeutics. Such evolutionary changes leading to antigenic drift or serotype shift
renders the current interventions ineffective. Targeting multiple facets of a disease or25
multiple pathogens through a single drug or therapeutic has always been a challenge.
Combination vaccines have been traditionally used to target multiple pathogens or
variations within a pathogen (Skibinski, David AG et al. Journal of Global Infectious
Diseases (2011) 3:63-72; Alderson, Mark R. et al. Microorganisms (2021) 9: 771).
Displaying antigens or epitopes over virus like particles or nanoparticles have been30
tried to increase the depth and breadth of immune response against emerging strains or
variants of a pathogen (Tretyakova, Irina et al. Virology (2013) 442: 67-73; Schellenbacher,
Christina et al. Journal of Virology (2009) 83: 10085-10095; Liu, Xingjian et al. (available
on the World Wide Web at doi.org/10.1101/2021.02.05.428685; Tumban, Ebenezer et al.
PLoS ONE (2012) 7: e49715). These methods used conventional approach of expressing the35
3
particles recombinantly in bacterial, insect or mammalian expression systems and purifying
the particles to be used for immunization.
More recently, mRNA based vaccines have emerged as promising alternative to
classical approaches to vaccine development. However, current mRNA based vaccines are
still inadequate to address the emerging strains or variants of a pathogen. For example, the5
antibody titres elicited by SARS-CoV-2 vaccine against the emerging and antigenically
divergent SARS-CoV-2 variants have been found to be lower, and wane over time
suggesting decreased effectiveness of the vaccines targeted against a single variant/strain of
the virus. Bivalent vaccines encapsulating different mRNAs, each targeting a specific variant
of SARS-CoV-2, are undergoing clinical trials to increase the breadth of coverage (Chalkias,10
Spyros et al. New England Journal of Medicine (2022) 387: 1279-1291; Chalkias, Spyros et
al. Nature Medicine (2022) available on the World Wide Web at doi.org/10.1038/s41591-
022-02031-7). Multivalent vaccines containing as many as eight different mRNAs have been
co-encapsulated in lipid nanoparticle for delivery (Chivukula, Sudha et al. NPJ Vaccines
(2021) 6:153; WO2022264109). Such strategies require multiple in vitro transcription (IVT)15
process to be performed complicating the manufacturing process and quick deployment of
vaccines during pandemics. Strategies that can simplify the manufacturing process and
induce more potent, durable, and/or broader immune response are necessary to combat
pandemics and evolving pathogens. Thus, it would be advantageous to develop vaccines and
therapeutics that provide protection against infection over a broad range of diseases,20
including multiple facets of a single disease or different strains and/or variations of a
pathogen.
SUMMARY
25
Accordingly, the present disclosure relates to a nucleic acid comprising a plurality of
polynucleotide sequences, wherein some or all polynucleotide sequence of the plurality
comprises either a target sequence, a linker sequence, and a self-assembling sequence or a
linker sequence, a target sequence, a linker sequence and a self-assembling sequence or a
combination thereof. In some embodiments, each polynucleotide sequence of the plurality30
is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence.
In some embodiments, the nucleic acid further comprises a signal sequence upstream of one
or more of the polynucleotide sequences of the plurality. In some embodiments, the target
4
sequence, the linker sequence, and the self-assembling sequence or the linker sequence, the
target sequence, the linker sequence and the self-assembling sequence are in 5′ to 3′ order.
In another aspect, provided herein is a nucleic acid comprising a plurality of
polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises
a target sequence, a linker sequence, and a self-assembling sequence. In some embodiments,5
each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide
sequence of the plurality by a cleavage sequence. In some embodiments, the nucleic acid
further comprises a signal sequence upstream of one or more of the polynucleotide
sequences of the plurality. In some embodiments, the target sequence, the linker sequence,
and the self-assembling sequence are in 5′ to 3′ order.10
In another aspect, provided herein is a nucleic acid comprising a plurality of
polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises
a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence. In
some embodiments, each polynucleotide sequence of the plurality is connected to an
adjacent polynucleotide sequence of the plurality by a cleavage sequence. In some15
embodiments, the nucleic acid further comprises a signal sequence upstream of one or more
of the polynucleotide sequences of the plurality. In some embodiments, the linker sequence,
the target sequence, the linker sequence and the self-assembling sequence are in 5′ to 3′
order.
In another aspect, provided herein is a nucleic acid encoding a plurality of20
polypeptides, wherein some or all polypeptides of the plurality comprises either a target
peptide, a linker peptide, and a self-assembling peptide or a linker peptide, a target peptide,
a linker peptide and a self-assembling peptide or a combination thereof. In some
embodiments, each polypeptide of the plurality is connected to an adjacent polypeptide of
the plurality by a cleavage peptide. In some embodiments, the nucleic acid further encodes25
a signal peptide on the amino-terminus of one or more of the polypeptides of the plurality.
In some embodiments, the target peptide, the linker peptide, and the self-assembling peptide
or the linker peptide, the target peptide, the linker peptide and the self-assembling peptide
are in N-terminus to C- terminus order.
In another aspect, provided herein is a nucleic acid encoding a plurality of30
polypeptides, wherein each polypeptide of the plurality comprises a target peptide, a linker
peptide, and a self-assembling peptide. In some embodiments, each polypeptide of the
plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide. In
5
some embodiments, the nucleic acid further encodes a signal peptide on the amino-terminus
of one or more of the polypeptides of the plurality. In some embodiments, the target peptide,
the linker peptide, and the self-assembling peptide are in N-terminus to C- terminus order.
In another aspect, provided herein is a nucleic acid encoding a plurality of
polypeptides, wherein each polypeptide of the plurality comprises a linker peptide, a target5
peptide, a linker peptide, and a self-assembling peptide. In some embodiments, each
polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a
cleavage peptide. In some embodiments, the nucleic acid further encodes a signal peptide
on the amino-terminus of one or more of the polypeptides of the plurality. In some
embodiments, the linker peptide, the target peptide, the linker peptide and the self-10
assembling peptide are in N-terminus to C- terminus order.
In some embodiments, total number of the polynucleotide sequences is not more than
100. In some embodiments, total number of the polynucleotide sequences is between 2-10,
10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-99. In some embodiments,
the nucleic acid is a DNA or an RNA. In some embodiments, the RNA is an mRNA. In some15
embodiments, the linker sequence encodes a linker peptide. In some embodiments, the linker
peptide is an amino acid linker, foldon, scaffold or a combination thereof. In some
embodiments, the amino acid linker comprises 2 to 49 amino acids. In some embodiments,
the amino acid linker is glycine serine linker, glycine proline linker, glycine threonine linker,
alanine serine linker, any combination of two amino acids, or a combination thereof.20
In some embodiments, the linker peptide has an amino acid sequence of any one of
SEQ ID NOs: 262-299, 330, and 350. In some embodiments, the self-assembling sequence
encodes a self-assembling peptide. In some embodiments, the self-assembling peptide is
lumazine synthase from Aquifex species, hepatitis B surface antigen (HBsAg) from
Hepatitis B Virus, hepatitis B core antigen (HBcAg) from Hepatitis B virus, human25
papillomavirus L1 (HPV L1) protein, matrix protein M1 from influenza A virus, ferritin,
riboflavin synthase, or a combination thereof, including their codon optimized nucleic acid
sequences, fragments, mutants, or variants thereof. In some embodiments, the ferritin
comprises of ferritin subunit or ferritin peptide. In some embodiments, the ferritin peptide
is derived from Helicobacter pylori ferritin. In some embodiments, the self-assembling30
peptide has an amino acid sequence of any one of SEQ ID NOs: 254-261, 331 and 333.
In some embodiments, the cleavage sequence encodes one or more cleavage peptide.
In some embodiments, the one or more cleavage peptides are optionally connected to each
6
other by a linker. In some embodiments, the cleavage peptide is a golgi specific cleavage
peptide or self-cleaving peptide. In some embodiments, the cleavage peptide has an amino
acid sequence of any one of SEQ ID NOs: 300-311 and 347-349.
In some embodiments, the signal sequence encodes a signal peptide. In some
embodiments, the signal peptide is present on the amino-terminus of the first polypeptide.5
In some embodiments, the nucleic acid further encodes a second signal peptide on the
amino-terminus of all or some polypeptides. In some embodiments, the signal peptide has
an amino acid sequence of any one of SEQ ID NOs: 312-329.
In some embodiments, the target sequence encodes a target peptide. In some
embodiments, the target peptide is encoded by a codon optimized nucleic acid sequence, or10
fragments, mutants, or variants thereof. In some embodiments, the target peptide is obtained
from a prokaryote, a eukaryote, a unicellular organism, a multicellular organism, a virus, a
bacterium, a fungus, a protozoan, a worm, a mycoplasma, an animal, a human or a
combination thereof. In some embodiments, the virus is selected from the family comprising
picornaviride, calciviridae, astroviridae, togaviridae, flaviviridae, coronaviridae,15
arteriviridae, rhabndoviridae, filoviridae, paramyxoviridae, bornaviridae,
orthomyxoviridae, bunyaviridae, arenaviridae, reoviridae, retroviridae, polyomaviridae,
herpesviridae, poxviridae, papillomaviridae, hepadnaviridae, adenoviridae, parvoviridae,
hepeviridae, circoviridae or a combination thereof. In some embodiments, the bacterium is
selected from the genus comprising Bacillus, Bordetella, Borrelia, Brucella, Campylobacter,20
Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus,
Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria,
Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio,
Yersinia, or a combination thereof. In some embodiments, the virus is selected from the
family consisting of coronaviridae, herpesviridae, poxviridae, flaviviridae, togaviridae,25
retroviridae, paramyxoviridae, or a combination thereof. In some embodiments, the virus is
alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus, torovirus or a
combination thereof.
In some embodiments, the betacoronavirus is SARS-CoV-1, SARS-CoV-2, MERS-
CoV, OC43, HKU1, bat coronavirus, other betacoronavirus or a combination thereof. In30
some embodiments, the target peptide is a spike protein, a membrane protein, an envelope
protein or a nucleocapsid protein of coronaviruses. In some embodiments, the target peptide
is a receptor binding domain, fusion peptide, or stem helix of the spike protein, or a
7
combination thereof, including their codon optimized nucleic acid sequences, fragments,
mutants or variants thereof. In some embodiments, the target peptide is a receptor binding
domain obtained or derived from a betacoronavirus comprising SARS-CoV-1, SARS-CoV-
2, MERS-CoV, OC43, HKU1, bat coronavirus, other betacoronavirus, or a combination
thereof. In some embodiments, the target peptide is glycoprotein B, glycoprotein C,5
glycoprotein D, glycoprotein E, glycoprotein K, glycoprotein L, and glycoprotein M, of
herpes simplex virus 1 (HSV-1) or herpes simplex virus 2 (HSV-2), or a combination thereof,
including their codon optimized nucleic acid sequences, fragments, mutants or variants
thereof.
In some embodiments, the target peptide is glycoprotein B, glycoprotein H,10
glycoprotein L, glycoprotein M, or glycoprotein N of human cytomegalovirus (HCMV), or
a combination thereof, including their codon optimized nucleic acid sequences, fragments,
mutants, or variants thereof. In some embodiments, the target peptide is glycoprotein B,
glycoprotein C, glycoprotein H, or glycoprotein L of varicella-zoster virus (VZV), or a
combination thereof, including their codon optimized nucleic acid sequences, fragments,15
mutants, or variants thereof. In some embodiments, the target peptide is glycoprotein B,
glycoprotein H, glycoprotein L, glycoprotein M, glycoprotein N, glycoprotein 42, or
glycoprotein 350 of Epstein-Barr virus (EBV), or a combination thereof, including their
codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some
embodiments, the target peptide is F9 membrane protein of a poxvirus, H3L protein of a20
poxvirus, A4 protein of a poxvirus, A27 protein of poxvirus, A33 protein of a poxvirus, A56
protein of a poxvirus, B5 protein of a poxvirus, or L1 protein of a poxvirus, or a combination
thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or
variants thereof.
In some embodiments, the target peptide is a capsid protein, a membrane protein, an25
envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A,
NS4B, or NS5) of flaviviruses or hepaciviruses, or a combination thereof, including their
codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some
embodiments, the target peptide is capsid protein, membrane protein, envelope protein, or
non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of30
Japanese encephalitis virus, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the
target peptide is capsid protein, membrane protein, envelope protein, or non-structural
8
proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of zika virus, or a
combination thereof, including their codon optimized nucleic acid sequences, fragments,
mutants, or variants thereof. In some embodiments, the target peptide is capsid protein,
membrane protein, envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B,
NS3, NS4A, NS4B, or NS5) of yellow fever virus, or a combination thereof, including their5
codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some
embodiments, the target peptide is capsid protein, membrane protein, envelope protein, or
non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of west
nile virus, or a combination thereof, including their codon optimized nucleic acid sequences,
fragments, mutants, or variants thereof. In some embodiments, the target peptide is capsid10
protein, membrane protein, envelope protein, or non-structural proteins (such as NS1,
NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of hepatitis C virus, or a combination thereof,
including their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof. In some embodiments, the target peptide is capsid protein, membrane protein,
envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A,15
NS4B, or NS5) of dengue virus, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide is capsid protein, or envelope protein such
as E1, E2 and E3 protein of alphaviruses, or a combination thereof, including their codon
optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some20
embodiments, the target peptide is domain A, domain B, or domain C of the E2 protein of
alphaviruses, or a combination thereof, including their codon optimized nucleic acid
sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide
is capsid protein, or envelope protein such as E1, E2 and E3 protein of chikungunya virus,
or a combination thereof, including their codon optimized nucleic acid sequences,25
fragments, mutants, or variants thereof. In some embodiments, the target peptide is gag, pol
and env proteins of retroviruses, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide is p17Gag, p24Gag, p7Gag, p6Gag, gp12Env,
gp41Env, or pol proteins from lentiviruses, or a combination thereof, including their codon30
optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some
embodiments, the target peptide is p17Gag, p24Gag, p7Gag, p6Gag, gp12Env, gp41Env, or pol
9
proteins from human immunodeficiency virus (HIV), or a combination thereof, including
their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide is nucleocapsid protein, P protein, V
protein, W protein, D protein, I protein, C protein, L protein, M protein, H (hemagglutinin)
protein, HN (hemagglutinin-neuraminidase) protein, G protein or F protein of Mumps virus5
(MuV), Parainfluenza virus type 5 (PIV5), Human parainfluenza virus type 2, types 4a and
4b (HPIV2/4a/4b), Newcastle disease virus (NDV), Human parainfluenza virus type 1 and
type 3 (HPIV1/3), Nipah virus (NiV), Measles virus (MeV), Human respiratory syncytial
virus A2, B1, S2, (HRSV), or Human metapneumovirus (HMPV), or a combination thereof,
including their codon optimized nucleic acid sequences, fragments, mutants, or variants10
thereof. In some embodiments, the target peptide is nucleocapsid protein, P protein, V
protein, W protein, D protein, I protein, C protein, L protein, M protein, H (hemagglutinin)
protein, HN (hemagglutinin-neuraminidase) protein, G protein or F protein of human
respiratory syncytial virus A2, B1, S2 (HRSV), or a combination thereof, including their
codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some15
embodiments, the target peptide is early protein for example (E1, E2, E4, E5, E6, or E7), or
late protein L1 (major capsid protein) or L2 (minor capsid protein) of human papillomavirus,
or a combination thereof, including their codon optimized nucleic acid sequences,
fragments, mutants, or variants, thereof. In some embodiments, the target peptide is a major
capsid protein (L1) of a human papillomavirus that has lost the ability to self-assemble,20
including their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof. In some embodiments, the target peptide is a minor capsid protein (L2) of a human
papillomavirus, including their codon optimized nucleic acid sequences, fragments, mutants,
or variants thereof.
In some embodiments, the target peptide has an amino acid sequence of any one of25
SEQ ID NOs: 1-253, 334-337, 338-346, and 353-388.
In another aspect, provided herein is a lipid nanoparticle composition comprising
cationic lipid, a phospholipid, a sterol, a PEG-lipid, and the nucleic acid according to any of
the preceding embodiments or paragraphs. In some embodiments, the cationic lipid
comprises an ionizable lipid. In some embodiments, the ionizable lipid is present in an30
amount from 25 mol percent to 70 mol percent. In some embodiments, the phospholipid is
present in an amount from 2 mol percent to about 30 mol percent. In some embodiments,
the sterol is present in an amount from 30 mol percent to about 65 mol percent. In some
10
embodiments, the PEG-lipid is present in an amount from 0.2 mol percent to about 2.0 mol
percent. In some embodiments, the lipid nanoparticle composition additionally comprises
an ionizable polymer. In some embodiments, the ionizable polymer is present in an amount
from 1 mol percent to 25 mol percent. In some embodiments, the ionizable polymer is
selected from the group comprising a chitosan, a cellulose derivative, a poly-L-lysine, a5
poly-L-glutamic acid, and/or their derivatives or a combination thereof.
In another aspect, provided herein is a method of treating or preventing a disease,
comprising administrating to a subject in need thereof the nucleic acid disclosed herein.
In another aspect, provided herein is a method of treating or preventing a disease,
comprising administrating to a subject in need thereof the multitarget peptide disclosed10
herein.
In another aspect, provided herein is a method of treating or preventing a disease,
comprising administrating to a subject in need thereof the lipid nanoparticle composition
disclosed herein.
In another aspect, provided herein is use of the nucleic acid sequence disclosed15
herein, in the manufacture of a medicament for the treatment or prevention of a disease in a
subject.
In another aspect, provided herein is use of a lipid nanoparticle composition
disclosed herein in the manufacture of a medicament for the treatment or prevention of a
disease in a subject.20
In another aspect, provided herein is a multitarget peptide encoded by the nucleic
acid disclosed herein.
In another aspect, provided herein is a multitarget peptide comprising two or more
polypeptides, wherein some or all polypeptides comprises either a target peptide, a linker
peptide, and a self-assembling peptide, or a linker peptide, a target peptide, a linker peptide,25
and a self-assembling peptide, or a combination thereof, wherein one polypeptide is
connected to another polypeptide by a cleavage peptide, wherein the multitarget peptide
includes a signal peptide upstream (amino-terminus) of one or more of the polypeptides. In
some embodiments, the signal peptide may be present on the amino-terminus of the first
polypeptide. In some embodiments, the signal peptide may be present on the amino-terminus30
of some or each of the polypeptides.
In another aspect, provided herein is a polypeptide nanoparticle comprising at least
2 or up to 100 polypeptides disclosed herein. In some embodiments, the polypeptides are
11
homologous polypeptides, heterologous polypeptides, oligomeric complex or combination
thereof. In some embodiments, the polypeptide nanoparticle is icosahedral, helical,
spherical, rod-like or combination thereof.
In another aspect, provided herein is a nucleic acid sequence comprising a
multitarget nucleic acid sequence comprising two or more polynucleotide sequences,5
wherein some or all polynucleotide sequences comprises either a target sequence, a linker
sequence, and a self-assembling sequence, or a linker sequence, a target sequence, a linker
sequence, and a self-assembling sequence, or a combination thereof, wherein one
polynucleotide sequence is connected to another polynucleotide sequence by a cleavage
sequence, wherein the multitarget nucleic acid sequence includes a signal sequence10
upstream of one or more of the polynucleotide sequences. In some embodiments, the linker
sequence connects signal sequence with the first polynucleotide sequence. In some
embodiments, the signal sequence may be present upstream of all or some of the
polynucleotide sequences. In some embodiments, signal sequence is present upstream of the
first polynucleotide sequence. In some embodiments, signal sequence is present upstream15
of all polynucleotide sequences. In some embodiments, linker sequence connects target
sequence with the self-assembling sequence in a polynucleotide sequence. In some
embodiments, one linker sequence connects cleavage sequence with the target sequence and
another linker sequence connects the target sequence with the self-assembling sequence in
a polynucleotide sequence. In some embodiments, the multitarget nucleic acid sequence is20
a DNA or an RNA. In some embodiments, the multitarget nucleic acid sequence is an
mRNA. In some embodiments, the multitarget nucleic acid sequence encodes a multitarget
peptide. In some embodiments, the multitarget nucleic acid is encapsulated in a lipid
nanoparticle composition. In some embodiments, the multitarget nucleic acid sequence is
synthesized through a single in vitro transcription (IVT) process.25
In some embodiments, the disclosure relates to a nucleic acid sequence encoding a
multitarget peptide described herein.
In some embodiments, the disclosure relates to a nucleic acid sequence encoding a
multitarget peptide comprising two or more polypeptides, wherein some or all polypeptides
comprises either a target peptide, a linker peptide, and a self-assembling peptide, or a linker30
peptide, a target peptide, a linker peptide, and a self-assembling peptide, or a combination
thereof, wherein one polypeptide is connected to another polypeptide by a cleavage peptide,
wherein the multitarget peptide includes a signal peptide upstream of one or more of the
12
polypeptides. In some embodiments, signal peptide may be present upstream of all or some
of the polypeptides. In some embodiments, signal peptide may be present upstream of the
first polypeptide. In some embodiments, signal peptide may be present upstream of all
polypeptides.
In some embodiments, the disclosure also relates to a multitarget peptide comprising5
two or more polypeptides, wherein some or all polypeptides comprises either a target
peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a target peptide,
a linker peptide, and a self-assembling peptide, or a combination thereof, wherein one
polypeptide is connected to another polypeptide by a cleavage peptide, wherein the
multitarget peptide includes a signal peptide upstream (amino-terminus) of one or more of10
the polypeptides. In some embodiments, signal peptide may be present upstream (amino-
terminus) of all or some of the polypeptides. In some embodiments, signal peptide may be
present upstream (amino-terminus) of the first polypeptide. In some embodiments, signal
peptide may be present upstream (amino-terminus) of all the polypeptides.
In some embodiments, the linker peptide connects signal peptide with the first15
polypeptide in a multitarget peptide. In some embodiments, the linker peptide connects
target peptide with the self-assembling peptide in a polypeptide. In some embodiments, one
linker peptide connects cleavage peptide with the target peptide and another linker peptide
connects the target peptide with the self-assembling peptide in a polypeptide. In some
embodiments, the multitarget peptide comprise homologous polypeptides. In some other20
embodiments, the multitarget peptide comprise heterologous polypeptides. In some
embodiments, multitarget peptide comprise homologous polypeptides, or heterologous
polypeptides. In some embodiments, the disclosure relates to a polypeptide nanoparticle
comprising one or more homologous polypeptides, one or more heterologous polypeptides,
one or more oligomeric complex, or a combination thereof. In some embodiments, the25
homologous polypeptides, heterologous polypeptides, oligomeric complex may comprise
either a target peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a
target peptide, a linker peptide, and a self-assembling peptide, or a combination thereof. In
some embodiments, the polypeptides in a polypeptide nanoparticle may also have some
residues of cleavage peptide.30
In some embodiments, the disclosure relates to a polypeptide nanoparticle formed
from the self-assembly of two or more polypeptides, wherein some or all polypeptides
comprises either a target peptide, a linker peptide, and a self-assembling peptide, or a linker
13
peptide, a target peptide, a linker peptide, and a self-assembling peptide or a combination
thereof. In some embodiments, the polypeptides in the polypeptide nanoparticle may also
have some residues of cleavage peptide. In some embodiments, the polypeptide nanoparticle
comprises of homologous polypeptides, heterologous polypeptides, oligomeric complexes,
or a combination thereof.5
In some aspects, provided herein is a multitarget nucleic acid sequences described
herein, encapsulated in a lipid nanoparticle composition. In some aspects, the lipid
nanoparticle composition comprises a cationic lipid, a phospholipid, a sterol, a PEG lipid
and a multitarget nucleic acid sequence described herein.
In some other aspects, the lipid nanoparticle composition comprises an ionizable10
polymer, a cationic lipid, a phospholipid, a sterol, a PEG-lipid and the multitarget nucleic
acid sequence described herein.
In some aspects, provided herein is a method of treating or preventing a disease,
comprising administering to a subject in need thereof the multitarget nucleic acid sequence
as described herein.15
In some aspects, provided herein is a method of treating or preventing a disease,
comprising administering to a subject in need thereof the lipid nanoparticle composition
comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid and the multitarget nucleic
acid sequence as described herein.
In some aspects, provided herein is a method of treating or preventing a disease,20
comprising administering to a subject in need thereof the lipid nanoparticle composition
comprising an ionizable polymer, a cationic lipid, a phospholipid, a sterol, a PEG-lipid and
the multitarget nucleic acid sequence as described herein.
In some aspects, the disclosure relates to use of a lipid nanoparticle composition
comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and the multitarget nucleic25
acid sequence as described herein in the manufacture of a medicament for the treatment or
prevention of a disease in a subject.
In some embodiments, the target sequence is obtained from a prokaryote or a
eukaryote, a unicellular organism or a multicellular organism, a virus, a bacterium, a fungus,
a protozoan, a worm, a mycoplasma, an animal, human, or a combination thereof, including30
the codon optimized sequences, fragments, variants, or mutants of such target sequences. In
some embodiments, the target sequence may be modified or unmodified. In some
embodiments, the target sequence is obtained from viruses belonging to the families, for
14
example, picornaviride, calciviridae, astroviridae, togaviridae, flaviviridae, coronoviridae,
arteriviridae, rhabndoviridae, filoviridae, paramyxoviridae, bornaviridae,
orthomyxoviridae, bunyaviridae, arenaviridae, reoviridae, retroviridae, polyomaviridae,
herpesviridae, poxviridae, papillomaviridae, hepadnaviridae, adenoviridae, parvoviridae,
hepeviridae, circoviridae, or a combination thereof. In some embodiments, the target5
sequence is obtained from bacteria belonging to genera, for example, Bacillus, Bordetella,
Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium,
Enterococcus, Escherichia, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria,
Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella,
Staphylococcus, Streptococcus, Vibrio, Yersinia, or a combination thereof. In some10
embodiments, the target sequence encodes a target peptide. In some embodiments, the target
peptide is an antigen, its fragment, or mutant thereof. In some embodiments, the target
sequence or target peptide may be modified or unmodified. In some embodiments, the target
peptide regulates or modulates cellular functions. In some embodiments, the target peptide
may have immunostimulatory or immunomodulatory effect.15
In some embodiments, the self-assembling sequence encodes a self-assembling
peptide. In some embodiments, the self-assembling peptide includes, but not limited to,
lumazine synthase from Aquifex species, hepatitis B surface antigen (HBsAg) from
Hepatitis B Virus, hepatitis B core antigen (HBcAg) from hepatitis B virus, human
papillomavirus L1 (HPV L1) protein from human papillomavirus, matrix protein (M1) from20
influenza A virus, ferritin peptide, or a combination thereof, including their fragments,
mutants, or variants thereof. In some embodiments, the ferritin peptide is a Helicobacter
pylori ferritin or its fragment, mutant, or variant thereof.
In some embodiments, the linker sequence encodes a linker peptide. In some
embodiments, the linker peptide connects the target peptide with the self-assembling peptide25
in a polypeptide. In some embodiments, the linker peptide connects the signal peptide with
the first polypeptide. In some embodiments, one linker peptide connects cleavage peptide
with the target peptide and another linker peptide connects target peptide with the self-
assembling peptide in a polypeptide. The linker peptide may be an amino acid linker, a
foldon, a scaffold or a combination thereof.30
In some embodiments, the cleavage sequence encodes a cleavage peptide. The
cleavage peptide connects one polypeptide with another polypeptide, for example, adjacent
polypeptide. The cleavage peptide carries a cleavage site. In some embodiments, the
15
cleavage peptide facilitates the action of cellular proteases to cleave the multitarget peptide
into individual polypeptides. In some embodiments, the cleavage peptide self cleaves into
individual polypeptides. In some embodiments, the cleavage peptide comprises two or more
cleavage peptides (for example, cleavage peptide-1, cleavage peptide-2 and so on),
optionally connected via a linker. In some embodiments, the cleavage peptide may self5
cleave into individual polypeptides or may be cleaved by the action of cellular proteases. In
some embodiments, the cleavage peptide is a substrate for golgi specific proteases.
In some embodiments, the signal sequence encodes a signal peptide. The signal
peptide is present upstream (amino-terminus) of one or more polypeptides in a multitarget
peptide. In some embodiments, the signal peptide is present upstream (amino-terminus) of10
the first polypeptide. In some embodiments, the signal peptide is present upstream (amino-
terminus) of some polypeptides. In some embodiments, the signal peptide is present
upstream (amino-terminus) of all polypeptides. In some embodiments, the signal peptide
transports the multitarget peptide to cell organelles. In some embodiments, the signal peptide
transports the multitarget peptide to golgi body or golgi apparatus. In some embodiments,15
the signal peptide is a golgi targeting signal peptide.
In some aspects, the present disclosure also includes a method of transforming a cell
with the multitarget nucleic acid sequence as described herein.
BRIEF DESCRITPITON OF THE DRAWINGS20
Figure 1 – shows representative schematic illustration of multitarget nucleic acid
sequence wherein each polynucleotide sequence (PS) comprises a target sequence (TS), a
linker sequence (LS), and a self-assembling sequence (SAS) or a linker sequence (LS), a
target sequence (TS), a linker sequence (LS), and a self-assembling sequence (SAS) .25
Multiple polynucleotide sequences are connected through a cleavage sequence (CS) such
that between any two polynucleotide sequences there is present a cleavage sequence. The
multitarget nucleic acid sequence has a signal sequence (SS) upstream of the first
polynucleotide sequence. The letter ‘n’ in figure 1 represents any number between 1 to 98.
The multitarget nucleic acid sequence may additionally have 5’ cap and 3’ poly(A) tail. This30
multitarget nucleic acid sequence encodes corresponding multitarget peptide depicted in
Figure 2.
Figure 2 – shows representative schematic illustration of multitarget peptide wherein
each polypeptide (PP) comprises a target peptide (TP), a linker peptide (LP), and a self-
16
assembling peptide (SAP) or a linker peptide (LP), a target peptide (TP), a linker peptide
(LP), and a self-assembling peptide (SAP). Multiple polypeptides are connected through a
cleavage peptide (CP) such that between any two polypeptides there is present a cleavage
peptide. The multitarget peptide has a signal peptide (SP) on the N-terminus of the first
polypeptide. The letter ‘n’ represents any number between 1 to 98.5
Figure 3a – shows a western blot of cell lysate (lanes 4 and 5) showing expression
of a multitarget peptide using polyclonal anti-spike antibody against SARS-CoV-2.
Figure 3b – shows a western blot of supernatant (lane 5) confirming the cleavage of
multitarget peptide using polyclonal anti-spike RBD antibody against SARS-CoV-2.
Figure 4 – shows estimation of polypeptide nanoparticles in cell lysate and10
supernatant by ELISA. There is a decrease in protein concentration in the cell lysate at 48 h
with a corresponding increase in protein concentration of supernatant at 48 h. These results
indicate increase in the amount of polypeptide nanoparticle with the progression of time in
the supernatant.
Figure 5 – shows TEM image showing the formation of polypeptide nanoparticle.15
Figure 6 – shows a western blot of cell lysate (lane 2) showing expression of target
peptides encoded by multitarget nucleic acid sequence - trivalent RBD construct of example
2 using polyclonal anti-spike antibody against SARS-CoV-2. Lanes 1 and 3 are protein
ladder and negative control respectively.
Figures 7a, 7b, and 7c – show ELISA data showing generation of antibodies (IgG)20
against the respective target peptides encoded by the multitarget nucleic acid sequence –
trivalent RBD construct of example 2.
Figures 8a, 8b, and 8c – show pseudovirus neutralization of the antibodies
generated against the respective target peptides encoded by the multitarget nucleic acid
sequence – trivalent RBD construct of example 2.25
Figure 9 – shows a western blot of cell lysate (lane 3) showing expression of target
peptides encoded by multitarget nucleic acid sequence - pentavalent RBD construct of
example 3 using polyclonal anti-spike antibody against SARS-CoV-2. Lanes 1 and 2 are
protein ladder and negative control respectively.
30
DESCRIPTION
17
Unless defined otherwise, technical, and scientific terms used herein have the same
meaning as commonly understood by one of person skill in the art. Some of the terms are
defined briefly here below; the definitions should not be construed in a limiting sense.
The singular forms “a”, “an” and “the” as used in the specification also include plural
aspects unless the context dictates otherwise. Similarly, any singular term used in the5
specification also mean plural or vice versa unless the context dictates otherwise. As used
herein in the claim(s), when used in conjunction with the word “comprising”, the words “a”
or “an” may mean one or more than one. As used herein “another” may mean at least a
second or more.
It must be noted that the words “comprising” or any of its form such as “comprise”10
or “comprises”, “having” or any of its forms such as “have” or “has”, “including” or any of
its forms such as “include” or “includes”, or “containing” or any of its forms such as
“contains” or “contains” are open-ended and do not exclude additional unrecited elements
or method steps.
Wherever any quantity or range is stated one skilled in the art will recognize that15
quantity or range within 10 or 20 percent of the stated values can also be expected to be
appropriate and reasonable and included within the scope of the invention.
Unless otherwise defined herein, scientific, and technical terms used in connection
with the present invention shall have the meanings that are commonly understood by those
of ordinary skilled in the art. Generally, nomenclatures used in connection with, and20
techniques of, cell and tissue culture, molecular biology, immunology, microbiology,
protein, adjuvant, pharmaceutical biotechnology, and biopharmaceutical manufacturing
described herein are those well known and commonly used in the art. The methods and
techniques of the present invention are generally performed according to conventional
methods well known in the art and as described in various general and more specific25
references that are cited and discussed throughout the present specification.
The term “composition” or “formulation” has been used interchangeably to mean a
lipid nanoparticle composition comprising a multitarget nucleic acid sequence, and lipid
components such as cationic lipid, phospholipid, sterol, and PEG-lipid. The composition
may optionally contain an ionizable polymer. The composition may additionally contain30
pharmaceutical carriers or excipients, such as but not limited to, buffering agents, stabilizers,
tonicity modifiers, surfactants, chelating agents, salts, anti-oxidants, diluents, and/or
preservatives or combinations thereof.
18
The term "therapeutic", “therapeutic agent”, “prophylactic”, “prophylactic agent’, or
drug has been used interchangeably to mean a compound (such as multitarget nucleic acid
sequence) or composition (such as a lipid nanoparticle composition described herein) having
a biological effect or a combination of biological effects that prevents, inhibits, eliminates
or prevents the progression of a disease or other aberrant biological processes in a subject,5
for example, an animal or human.
The term “preventing” is art-recognized, and when used in relation to a condition,
such as an infection is well understood in the art, and includes administration of a
composition, which reduces the frequency or severity, or delays the onset, of one or more
symptoms of the medical condition in a subject relative to a subject who does not receive10
the composition. Thus, the prevention of a condition, such as an infection, includes, for
example, the reduction of the frequency or severity of one or more symptoms of the medical
condition in a population of patients receiving a therapy relative to a control population that
did not receive the therapy, e.g., by a statistically and/or clinically significant amount.
Similarly, the prevention of an infection includes reducing the likelihood that a patient15
receiving a therapy will develop the infection or related symptoms, relative to a patient who
does not receive the therapy.
The term “molar ratio”, “mol ratio”, “molar percent”, “mol percent”, “molar %”, or
“mol %” have been used interchangeably to mean number of moles of a component
expressed as percentage relative to total moles of all lipid components (such as cationic lipid,20
phospholipid, sterol and PEG-lipid) and, if present, ionizable polymer component(s) present
in the lipid nanoparticle compositions described herein. For example, 50 mol % of cationic
lipid means, 50 mol % of cationic lipid is present in the lipid nanoparticle composition and
other lipids components constitute the remaining 50% such that the total amount of all the
lipid components together constitute 100 mol %. Alternatively, 50 mol % of cationic lipid25
also means, 50 mol % of cationic lipid present in the lipid nanoparticle composition and
other lipids components and ionizable polymer components together constitute the
remaining 50 mol % such that the total amount of all the lipid components and ionizable
polymer components constitute 100 mol %.
The terms “antibody” and “antibodies” have been used interchangeably herein and30
means any antibody or antibody fragment (whether produced naturally or recombinantly)
which retains antigen binding activity. This includes a monoclonal or polyclonal antibody, a
single chain antibody, a Fab fragment of a monoclonal or polyclonal antibody, a chimeric
19
antibody, a humanized antibody, a human antibody, a bispecific antibody, a multispecific
antibody, or a nanobody.
The term “buffer” as used herein means those agents that maintains the pH of a
solution in a desired range.
The term “cell” as used herein means a single cell or a population of cells or plurality5
of cells.
The term “biologically effective amount” or “therapeutically effective amount” as
used herein means an amount of an agent, for example, a therapeutic, drug, therapeutic
agent, prophylactic agent, diagnostic agent, composition, etc., that is sufficient, when
administered to a subject suffering from or susceptible to an infection, disease, disorder,10
and/or condition, to treat, prevent, diagnose, improve symptoms of, and/or delay the onset
of the infection, disease, disorder, and/or condition. A therapeutically effective amount
herein may vary according to factors such as the disease state, age, sex, and weight of the
patient.
As used herein, the term “treating” or “treatment” includes reducing, arresting, or15
reversing the symptoms, clinical signs, or underlying pathology of a condition to stabilize
or improve a subject's condition or to reduce the likelihood that the subject’s condition will
worsen as much as if the subject did not receive the treatment. Treatment may be
administered to a subject who does not exhibit signs of a disease and/or exhibits only early
signs of the disease for the purpose of decreasing the risk of developing pathology associated20
with the disease.
The term “subject” as used herein refers to a living mammal and may be
interchangeably used with the term “patient”. Examples of mammals include, but are not
limited to, any member of the mammalian class: humans, non-human primates such as
chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep,25
goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including
rodents, such as rats, mice, and guinea pigs, and the like. The term does not denote a
particular age or gender.
As used herein, an individual “at risk” of developing a particular disease, disorder,
or condition may or may not have detectable disease or symptoms of disease, and may or30
may not have displayed detectable disease or symptoms of disease prior to the treatment
methods described herein. “At risk” denotes that an individual has one or more risk factors,
which are measurable parameters that correlate with development of a particular disease,
20
disorder, or condition, as known in the art. An individual having one or more of these risk
factors has a higher probability of developing a particular disease, disorder, or condition than
an individual without one or more of these risk factors.
The term “disease” as used herein, means an interruption, cessation, or disorder of
body function, system, or organ. Non limiting examples of disease include malignant5
diseases, autoimmune diseases, inherited diseases, metabolic disorders, or infectious
diseases.
As used herein, administration “conjointly” with another compound or composition
includes simultaneous administration and/or administration at different times. Conjoint
administration also encompasses administration as a co-formulation or administration as10
separate compositions, including at different dosing frequencies or intervals, and using the
same route of administration or different routes of administration.
The terms “multitarget nucleic acid sequence” or “multitargeted nucleic acid
sequence” have been used interchangeably to mean two or more polynucleotide sequences
wherein some or all polynucleotide sequences comprises either a target sequence, a linker15
sequence, and a self-assembling sequence, or a linker sequence, a target sequence, a linker
sequence, and a self-assembling sequence, or a combination thereof, wherein one
polynucleotide sequence is connected to another polynucleotide sequence by a cleavage
sequence, wherein the multitarget nucleic acid sequence includes a signal sequence upstream
of one or more polynucleotide sequences. In some embodiments, the signal sequence is20
present upstream of the first polynucleotide sequence. In some embodiments, the signal
sequence is present upstream of some polynucleotide sequences. In some embodiments, the
signal sequence is present upstream of each of the polynucleotide sequences. In some
embodiments, the polynucleotide sequence comprises a target sequence, a linker sequence,
and a self-assembling sequence. In some embodiments, the polynucleotide sequence may25
comprise a linker sequence, a target sequence, a linker sequence, and a self-assembling
sequence. Thus, in some embodiments, one linker sequence connects cleavage sequence
with the target sequence and another linker sequence connects the target sequence with the
self-assembling sequence in a polynucleotide sequence. In some embodiments, the linker
sequence connects signal sequence with the polynucleotide sequence. As illustrated in figure30
1 multitarget nucleic acid sequence may comprise multiple repeats of polynucleotide
sequences wherein each polynucleotide sequence may comprise either a target sequence, a
linker sequence, and a self-assembling sequence, or a linker sequence, a target sequence, a
21
linker sequence and a self-assembling sequence, or a combination thereof, such that total
number of polynucleotide sequences in a multitarget nucleic acid sequence are not more
than 100. In some embodiments, the linker sequence connects signal sequence with the first
polynucleotide sequence. In some embodiments, the signal sequence is present upstream of
each of some or all of the polynucleotide sequences. The multitarget nucleic acid sequence5
encodes multitarget peptide.
The terms “multitarget peptide” or “multitargeted peptide” has been used
interchangeably to mean two or more polypeptides wherein some or all polypeptides
comprises either a target peptide, a linker peptide, and a self-assembling peptide, or a linker
peptide, a target peptide, a linker peptide and a self-assembling peptide, or a combination10
thereof, wherein one polypeptide is connected to another polypeptide by a cleavage peptide,
wherein the multitarget peptide includes a signal peptide upstream (amino-terminus) of one
or more polypeptides. In some embodiments, the signal peptide is present on the amino-
terminus of the first polypeptide. In some embodiments, the signal peptide is present on the
amino-terminus of some polypeptides. In some embodiments, the signal peptide is present15
on the amino-terminus of each of the polypeptides. In some embodiments, the polypeptide
may comprise a target peptide, a linker peptide, and a self-assembling peptide. In some
embodiments, the polypeptide may comprise a linker peptide, a target peptide, a linker
peptide, and a self-assembling peptide. Thus, in some embodiments, one linker peptide
connects cleavage sequence with the target sequence and another linker peptide connects the20
target peptide with the self-assembling peptide in a multitarget peptide. In some
embodiments, the multitarget peptide may either comprise a target peptide, a linker peptide,
and a self-assembling peptide, or a linker peptide, a target peptide, a linker peptide, and a
self-assembling peptide, or a combination thereof, such that the total number of polypeptides
in a multitarget peptide are not more than 100. In some embodiments, the linker peptide25
connects signal peptide with the polypeptide. In some embodiments, the signal peptide is
present on the amino-terminus of each of some or all polypeptides. The multitarget peptide
may comprise homologous polypeptides or heterologous polypeptides.
The term “polynucleotide sequence” as used herein means a sequence of nucleotides
that encodes a polypeptide.30
The terms "protein" or "peptide" have been used interchangeably herein and mean a
polymer of amino acids linked through peptide bonds, but does not imply any specific
length. The term also includes fusion proteins, muteins, analogs or modified forms.
22
The term “polypeptide” as used herein means a sequence of amino acids that
comprise either a target peptide, a linker peptide, and a self-assembling peptide or a linker
peptide, a target peptide, a linker peptide, and a self-assembling peptide. In some
embodiments, the polypeptide comprises a target peptide, a linker peptide, and a self-
assembling peptide. In some embodiments, the polypeptide comprises a linker peptide, a5
target peptide, a linker peptide, and a self-assembling peptide. In some embodiments, the
polypeptide may have some residues (amino acids) of cleavage peptide. In some
embodiments, the polypeptide may have signal peptide.
The term “target sequence” as used herein means a sequence of nucleotides that
encodes a target peptide.10
The term “target peptide” as used herein means a sequence of amino acids that has
immunostimulatory or immunomodulatory effect. Target peptide also means a peptide of
interest. In some embodiments, target peptide is an antigen. In some embodiments, the target
peptide in two or more polypeptides may be identical i.e., homologous polypeptides. In some
other embodiments, the target peptides in two or more polypeptides may be different i.e.,15
heterologous polypeptides.
The term “signal sequence” as used herein means a sequence of nucleotides that
encodes a signal peptide.
The term “signal peptide” as used herein means a sequence of amino acids that
transports the multitarget peptide to specific cell organelles. In some embodiments the signal20
peptide transports the multitarget peptide to golgi apparatus or golgi body. The signal peptide
is present on the N-terminus (amino-terminus) of one or more polypeptides. In some
embodiments, the signal peptide is present on the N-terminus of some or all polypeptides.
In some embodiments, the signal peptide is present on the N-terminus of the first
polypeptide. In some embodiments, the signal peptide is present on the N-terminus of some25
polypeptides. In some embodiments, the signal peptide is present on the N-terminus of all
polypeptides. In some embodiments, the signal peptide is encoded by signal sequence. In
some embodiments, the signal peptide is a golgi targeting signal peptide.
The term “cleavage sequence” as used herein means a sequence of nucleotides that
encodes a cleavage peptide.30
The term “cleavage peptide” as used herein means a sequence of amino acids that
facilitates the action of cellular proteases to cleave the multitarget peptide into individual
polypeptides or self cleaves into individual polypeptides. The cleavage peptide is present
23
between any two polypeptides. It connects one polypeptide with another polypeptide, for
example, adjacent polypeptide. The cleavage peptide carries one or more cleavage sites. In
some embodiments, the cleavage peptide is a substrate for proteases. In some embodiments,
cleavage peptide undergoes self-cleavage to result in individual polypeptides. In some
embodiments, the cleavage peptide is a substrate for golgi specific proteases. In some5
embodiments, the cleavage peptide comprises one or more cleavage peptides, for example,
cleavage peptide-1, cleavage peptide-2 and so on. In some embodiments, the cleavage
peptide optionally comprises a linker peptide between two cleavage peptides. In some
embodiments, the cleavage peptide may self-cleave into individual polypeptides or may be
cleaved by the action of cellular proteases.10
The term “linker sequence” as used herein means a sequence of nucleotides that
encodes a linker peptide.
The term “linker peptide” or “peptide linker” have been used interchangeably to
mean a sequence of amino acids that either connects the target peptide with the self-
assembling peptide, connects the signal peptide with the target peptide, connects the15
cleavage peptide with the target peptide, connects signal peptide with the polypeptide, or
connects two cleavage peptides. In some embodiments, the linker peptide connects the signal
peptide with the polypeptide. In some embodiments, the linker peptide connects the target
peptide with the self-assembling peptide in a polypeptide. In some embodiments, one linker
peptide connects cleavage peptide with the target peptide and another linker peptide20
connects target peptide with the self-assembling peptide in a polypeptide. In some
embodiments, one linker peptide connects cleavage peptide with the target peptide and
another linker peptide connects target peptide with the self-assembling peptide. In some
embodiments, one linker peptide connects cleavage peptide with the target peptide and
another linker peptide connects the signal peptide with the target peptide. In some25
embodiments, the linker peptide connects two cleavage peptides. In some embodiments, the
linker peptide is an amino acid linker, a foldon, a scaffold or a combination thereof.
The term “amino acid linker sequence” as used herein means a sequence of
nucleotides that encodes an amino acid linker.
The term “amino acid linker” as used herein means a sequence of amino acids that30
provides structural integrity to polypeptide such that the components of the polypeptide
remain, as far as possible, in their native or stable conformation. In some embodiments,
amino acid linker also helps in orientation of a polypeptide such that the domains or epitopes
24
on the target peptide are exposed or displayed for interaction or communication with cells
or biomolecules or immune system in the absence of foldon or scaffold. In some
embodiments, the amino acid linker connects two cleavage peptides. Some of the non-
limiting examples of amino acid linkers includes, glycine serine linker, glycine proline
linker, glycine threonine linker, alanine serine linker, any combination of two amino acids5
or a combination thereof. In some embodiments, amino acid linker is about 2-49 amino acid
long.
The term “glycine serine linker sequence” as used herein means a sequence of
nucleotides that encodes a glycine serine linker.
The term “glycine serine linker” as used herein means a sequence of amino acid10
comprising one or more glycine (G) and serine (S) in any combinations without any
preference of order or limitation on number of appearances of either glycine or serine. In
some embodiments, the glycine serine linker is few amino acids in length to several amino
acids in length.
The term “foldon sequence” as used herein means a sequence of nucleotides that15
encodes a foldon.
The term “foldon” as used herein means a sequence of amino acids that enables two
or more homologous polypeptides to organise to form an oligomeric complex. In some
embodiments, the foldon also helps in orientation of a polypeptide such that the domains or
epitopes on the target peptide are exposed or displayed for interaction or communication20
with cells or biomolecules or immune system.
The term “scaffold sequence” as used herein means a sequence of nucleotides that
encodes a scaffold.
The term “scaffold” as used herein means a sequence of amino acids that provides
structural and/or functional integrity or support to the target peptide and helps in orientation25
of target peptide such that the domains or epitopes of the target peptide are exposed or
displayed for interaction or communication with cells or biomolecules or immune system.
The term “oligomeric complex” as used herein means a complex formed by two or
more homologous polypeptides. In some embodiments, the oligomeric complex has at least
two homologous polypeptides, at least three homologous polypeptides, at least four30
homologous polypeptides, at least five homologous polypeptides, or at least six homologous
polypeptides and so on.
25
The term “self-assembling sequence” as used herein means a sequence of nucleotides
that encodes a self-assembling peptide.
The term “self-assembling peptide” as used herein means a sequence of amino acids
that enables the polypeptides to self-assemble into polypeptide nanoparticle.
The term "self-assembly” or “self-assemble” or “self-assembling” has been used5
interchangeably to means the ability of polypeptides to undergo multimerization to form a
polypeptide nanoparticle. In some embodiments, the polypeptide nanoparticle may have at
least two polypeptides (dimer or 2-mer), at least three polypeptides (trimer or 3-mer), at least
four polypeptides (tetramer or 4-mer), at least five polypeptides (pentamer or 5-mer), at least
six polypeptides (hexamer or 6-mer), at least seven polypeptides (heptamer or 7-mer), at10
least eight polypeptides (octamer or 8-mer), and so on. In some embodiments, the
polypeptide nanoparticle is up to 100-mers. In some embodiments, hydrogen bonds,
disulfide bonds, hydrophobic interactions, electrostatic interactions, and/or van der Walls
forces combine to maintain self-assembled structure.
The term “multimerization” as used herein means association of two or more units15
of homologous polypeptides or heterologous polypeptides, or oligomeric complex or their
combination.
The term “polypeptide nanoparticle” as used herein means a nanoparticle formed by
self-assembly of polypeptides. In some embodiments, the polypeptide nanoparticle
comprises of two or more homologous polypeptides, or two or more heterologous20
polypeptides, or one or more oligomeric complexes or a combination thereof.
The term “homologous polypeptides” as used herein means polypeptides in a
multitarget peptide that have identical target peptides. For example, if two polypeptides in
the multitarget peptide have identical target peptides they are considered to be homologous
polypeptides.25
The term “heterologous polypeptide” as used herein means polypeptides in a
multitarget peptide that have different target peptides. For example, if two polypeptides in
the multitarget peptide have different or non-identical target peptides, they are considered to
be heterologous polypeptides.
The term “fragment” as used herein, whether in the context of a nucleic acid,30
nucleotide, protein, polypeptide, or peptide, means any length of the nucleic acid, protein,
polypeptide, or peptide sequence except the full length of the respective nucleic acid,
protein, polypeptide, or peptide sequence.
26
The term “variant” as used herein, whether in the context of a nucleic acid,
nucleotide, protein, polypeptide, or peptide sequence, means homologs, orthologs, paralogs,
mutants or analogs of respective nucleic acid, protein, polypeptide, or peptide sequence.
The term “mutant” as used herein, whether in the context of a nucleic acid,
nucleotide, protein, polypeptide, or peptide sequence, means a sequence which is not a wild5
type sequence. A mutant is also understood to mean a nucleic acid, nucleotide, protein,
polypeptide, or peptide sequence that carries a mutation. The term “mutation” as used herein
means, a change or modification in the sequence of nucleic acid or amino acid in comparison
to a reference sequence and includes insertion, deletion, substitution, or a combination
thereof.10
Multitarget nucleic acid sequence and multitarget peptide
In the present disclosure, a multitarget nucleic acid sequence includes two or more
polynucleotide sequences wherein some or all polynucleotide sequences comprises either a
target sequence, a linker sequence, and a self-assembling sequence, or a linker sequence, a15
target sequence, a linker sequence, and a self-assembling sequence, wherein one
polynucleotide sequence is connected to the another polynucleotide sequence by a cleavage
sequence, wherein the multitarget nucleic acid sequence includes a signal sequence upstream
of one or more polynucleotide sequences. In some embodiments, the signal sequence may
be present upstream of all or some polynucleotide sequences. In some embodiments, signal20
sequence is present upstream of the first polynucleotide sequence. In some embodiments,
the signal sequence is present upstream of some polynucleotide sequences. In some
embodiments, signal sequence is present upstream of each of the polynucleotide sequences.
In some embodiments, the polynucleotide sequence comprises a target sequence, a linker
sequence, and a self-assembling sequence. In some embodiments, the polynucleotide25
sequence comprises a linker sequence, a target sequence, a linker sequence, and a self-
assembling sequence. In some embodiments, multitarget nucleic acid sequence may
comprise one polynucleotide sequence comprising a target sequence, a linker sequence and
a self-assembling sequence, and another polynucleotide sequence comprising a linker
sequence, a target sequence, a linker sequence, and a self-assembling sequence. In some30
embodiments, the multitarget nucleic acid sequence may either comprise a target sequence,
a linker sequence and a self-assembling sequence, or a linker sequence, a target sequence, a
linker sequence, and a self-assembling sequence or a combination thereof, such that the total
number of polynucleotide sequences in a multitarget nucleic acid sequences are not more
27
than 100. In some embodiments, the linker sequence connects signal sequence with the
polynucleotide sequence. In some embodiments, one linker sequence connects cleavage
sequence with the target sequence and another linker sequence connects the target sequence
with the self-assembling sequence in a polynucleotide sequence. Some exemplary
illustrations of multitarget nucleic acid sequences are provided in figures 1 and their5
representative encoded multitarget peptides are provided in figures 2 respectively.
The term “nucleic acid” as used herein means a polymer comprising two or more
nucleotides for example, deoxyribonucleotides or ribonucleotides, either in an unmodified
or modified form. The nucleic acid may be either single stranded or double stranded, linear
or circular. The term nucleic acid also encompasses fragments, variants, mutants or codon10
optimized sequences of deoxyribonucleotides or ribonucleotides.
The term "nucleotide” as used herein means a ribonucleotide or
deoxyribonucleotide. If the term nucleotide is used in the context of RNA, it refers to
ribonucleotide, and if it is used in the context of DNA, it refers to deoxyribonucleotide.
In some embodiments, the multitarget nucleic acid sequence is a DNA, an RNA or15
an mRNA. The multitarget nucleic acid sequence may be few nucleotides long to several
thousand nucleotides long.
Deoxyribonucleic acid (DNA)
The term “deoxyribonucleic acid” or “DNA” has been used interchangeably herein20
and means a polymer of deoxyribonucleotides. The DNA may be either single stranded or
double stranded, linear, or circular.
In some embodiments, the multitarget nucleic acid sequence is a DNA. In some
embodiments, the DNA encodes a multitarget peptide described herein.
25
Ribonucleic acid (RNA)
The term “ribonucleic acid” or “RNA” has been used interchangeably herein and
means a polymer of ribonucleotides. The RNA may be either single stranded or double
stranded, linear, or circular. The term RNA also includes messenger RNA (mRNA). In some
embodiments, the multitarget nucleic acid sequence is an mRNA.30
In some embodiments, the mRNA encodes a multitarget peptide as described herein.
In some embodiments, the mRNA may be unmodified or modified or a combination
of both. The modification may be in the nucleobase of the nucleotide, or sugar moiety of the
nucleotide, or the phosphate of the nucleotide.
28
In some embodiments, mRNA is produced using recombinant expression system, or
chemically synthesized or obtained through in vitro transcription. In some embodiments, the
mRNA is obtained through single in vitro transcription (IVT) process.
In some embodiments, the mRNA is circular. In other embodiments, the mRNA is
linear.5
In some embodiments, the mRNA is self-amplifying or self-replicating. Self-
amplifying or self-replicating mRNA as used herein means an mRNA that self-replicate
upon delivery into the cells. Such mRNAs typically contain a replicase sequence, usually
derived from an alphavirus, which enables amplification of the original strand of mRNA
encoding the protein of interest upon delivery into the cells (Beissert, Tim et al. Molecular10
Therapy (2020) 28:119-128).
Target Sequence and target peptide
The multitarget nucleic acid sequence and the multitarget peptide includes target
sequence and target peptide respectively. In some embodiments, the target sequence is a15
DNA or an RNA. In another embodiment, the target sequence is an mRNA. The target
sequence may be modified or unmodified. The target sequence includes codon optimized
sequences, fragments, mutants, variants, or combination thereof. Target sequence may be
obtained from a prokaryote or a eukaryote, a unicellular organism or a multicellular
organism, a virus, a bacterium, a fungus, a protozoan, a worm, a mycoplasma, an animal, a20
human, or a combination thereof.
In some embodiments, the target sequence is a sequence of nucleotides that encodes
a target peptide.
In some embodiments, the target peptide comprises of a sequence of amino acids
that regulates or modulates cellular functions. The term “cellular functions” as used herein25
means various cellular or biological processes such as, but not limited to, biosynthesis, cell
division, cell cycle regulation, cellular metabolism, ion transport, absorption, secretion,
homeostasis, replication, transcription, translation, cell signalling, endocytosis, exocytosis,
phagocytosis, trogocytosis, pyroptosis, apoptosis, DNA replication, DNA repair, protein
synthesis, gene regulation, cell repair, cell growth, cell differentiation, cellular trafficking,30
cell proliferation, metabolic pathways etc.
The terms “regulate” or “modulate” or “regulation” or “modulation” have been used
interchangeably herein and means an act of controlling a cellular or biological process or to
exert a modifying or controlling influence on cellular or biological process.
29
In some aspects, the target peptide is capable of performing one or more functions
such as, but not limited to, immunostimulation, immunomodulation, etc. In some
embodiments, the target peptide means a peptide of interest. In some embodiments, target
peptide is an antigen. In some embodiments, the target peptide is identical in two or more
polypeptides, for example homologous polypeptides. In some embodiments, the target5
peptide is different in two or more polypeptides, for example as in heterologous
polypeptides.
In some embodiments, the target peptide may be few amino acids long to several
hundred amino acids long.
In some embodiments, the antigen may be, but not limited to, those derived from10
Cholera toxoid, tetanus toxoid, diphtheria toxoid, hepatitis B surface antigen, hemagglutinin,
neuraminidase, influenza M protein, PfHRP2, pLDH, aldolase, MSP1, MSP2, AMA1, Der-
p- 1, Der-f-1, Adipophilin, AFP, AIM-2, ART-4, BAGE, alpha-fetoprotein, BCL-2, Bcr-Abl,
BING-4, CEA, CPSF, CT, cyclin DIEp-CAM, EphA2, EphA3, ELF-2, FGF-5, G250,
Gonadotropin Releasing Hormone, HER-2, intestinal carboxyl esterase (iCE), IL13Ralpha2,15
MAGE-1, MAGE-2, MAGE-3, MART-1, MART-2, M-CSF, MDM-2, MMP-2, MUC-1,
NY- EOS-1, MUM-1, MUM-2, MUM-3, p53, PBF, PRAME, PSA, PSMA, RAGE-1,
RNF43, RU1, RU2AS, SART-1, SART-2, SART-3, SAGE-1, SCRN 1, SOX2, SOXIO,
STEAP1, survivin (BIRC5), Telomerase, TGFbetaRl l, TRAG-3, TRP-1, TRP-2, TERT, or
WT1; those derived from a virus, such as Cowpoxvirus, Vaccinia virus, Pseudocowpox20
virus, Human herpesvirus 1, Human herpesvirus 2, Cytomegalovirus, Human adenovirus A-
F, Polyomavirus, Human papillomavirus, Parvovirus, Hepatitis A virus, Hepatitis B virus,
Hepatitis C virus, Human immunodeficiency virus, Orthoreo virus, Rotavirus, Ebolavirus,
parainfluenza virus, influenza virus (e.g., H5N1 influenza virus, influenza A virus, influenza
B virus, influenza C virus), Measles virus, Mumps virus, Rubella virus, Pneumovirus,25
Human respiratory syncytial virus, Rabies virus, California encephalitis virus, Japanese
encephalitis virus, Hantaan virus, Lymphocytic choriomeningitis virus, Epstein-Barr Virus
(EBV), Coronavirus (e.g., MERS-CoV, SARS-CoV-1, SARS-CoV-2, OC43, HKU1, bat
coronavirus, other betacoronavirus), Enterovirus, Rhino virus, Poliovirus, Norovirus,
Flavivirus, Dengue virus, West Nile virus, Yellow fever virus and varicella; those derived30
from a bacterium, such as Anthrax (Bacillus anthracis), Brucella, Bordetella pertussis,
Candida, Chlamydia pneumoniae, Chlamydia psittaci, Cholera, Clostridium botulinum,
Coccidioides immitis, Cryptococcus, Diphtheria, Escherichia coli 0151: H7,
30
Enterohemorrhagic Escherichia coli, Enterotoxigenic Escherichia coli, Haemophilus
influenzae, Helicobacter pylori, Legionella, Leptospira, Listeria, Meningococcus,
Mycoplasma pneumoniae, Mycobacterium, Pertussis, Pneumonia, Salmonella, Shigella,
Staphylococcus, Streptococcus pneumoniae and Yersinia enterocolitica,' or those derived
from a protozoa, e.g., of the genus Plasmodium (Plasmodium falciparum, Plasmodium5
malariae, Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi, or a combination
thereof.
The antigen may be an allergen derived from, without limitation, cells, cell extracts,
proteins, polypeptides, peptides, peptide mimics of polysaccharides and other molecules,
such as small molecules, lipids, glycolipids, and carbohydrates of plants, animals, fungi,10
insects, food, drugs, dust, and mites. Allergens include but are not limited to environmental
aeroallergens; plant pollens (e.g., ragweed/hayfever); weed pollen allergens; grass pollen
allergens; Johnson grass; tree pollen allergens; ryegrass; arachnid allergens (e.g., house dust
mite allergens); storage mite allergens; Japanese cedar pollen/hay fever; mold/fungal spore
allergens; animal allergens (e.g., dog, guinea pig, hamster, gerbil, rat, mouse, etc., allergens);15
food allergens (e.g., crustaceans; nuts; citrus fruits; flour; coffee); insect allergens (e.g.,
fleas, cockroach); venoms: (Hymenoptera, yellow jacket, honey bee, wasp, hornet, fire ant);
bacterial allergens (e.g., streptococcal antigens; parasite allergens such as Ascaris antigen);
viral antigens; drug allergens; hormones (e.g., insulin); enzymes (e.g., streptokinase); and
drugs or chemicals capable of acting as incomplete antigens or haptens (e.g., the acid20
anhydrides and the isocyanates). Where a hapten is used in a composition of the disclosure,
it may be attached to a carrier to form a hapten-carrier adduct. The hapten-carrier adduct is
capable of initiating a humoral immune response, whereas the hapten itself would not elicit
antibody production. Non-limiting examples of haptens are aniline, urushiol (a toxin in
poison ivy), hydralazine, fluorescein, biotin, digoxigenin and dinitrophenol.25
In other embodiments, the antigen is an antigen associated with a disease where it is
desirable to sequester the antigen in circulation, such as for example an amyloid protein
(e.g., Alzheimer's disease).
In some embodiments, the target sequence encoding the target peptide in the
multitarget peptide may be obtained from viruses belonging to the families, for example,30
Coronaviridae, picornaviride, calciviridae, astroviridae, togaviridae, flaviviridae, ,
arteriviridae, rhabndoviridae, filoviridae, paramyxoviridae, bornaviridae,
orthomyxoviridae, bunyaviridae, arenaviridae, reoviridae, retroviridae, polyomaviridae,
31
herpesviridae, poxviridae, papillomaviridae, hepadnaviridae, adenoviridae, parvoviridae,
hepeviridae, circoviridae, or a combination thereof.
In some embodiments, the target sequence encoding the target peptide that may be
incorporated in the multitarget peptide may be obtained from bacteria belonging to genera,
for example, Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia,5
Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus, Helicobacter,
Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas,
Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio, Yersinia, or a
combination thereof.
10
Coronaviridae
Members of the family Coronaviridae are large, enveloped, single stranded RNA
viruses with genomes ranging from 25 to 32 kb and virions of 118 to 140 nm in diameter.
The family Coronaviridae include the following genera viz., alphacoronavirus,
betacoronavirus, deltacoronavirus, gammacoronavirus and torovirus. Most of the15
coronaviruses are responsible for causing mild respiratory infections. However, some of the
coronaviruses have been responsible for deadly outbreaks, for example, severe acute
respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1), Middle Eastern
respiratory syndrome coronavirus (MERS-CoV), and most recently SARS-CoV-2.
In some embodiments, the target sequence encoding the target peptide is obtained or20
derived from coronaviruses comprising SARS-CoV-1, MERS-CoV, SARS-CoV-2, OC43,
HKU1, bat coronavirus, other betacoronavirus, or a combination thereof, including codon
optimized sequences, fragments, mutants, or variants thereof of such target sequences.
All coronaviruses encode four major structural proteins, the spike protein (S), the
membrane protein (M), the envelope protein (E), and the nucleocapsid protein (N). These25
proteins or their fragments can serve as effective target peptides in accordance with the
present disclosure.
In some embodiments, the target sequence encoding the target peptide includes, but
not limited to, spike protein, membrane protein, envelope protein or nucleocapsid protein of
coronaviruses, or a combination thereof, including their codon optimized nucleic acid30
sequences, fragments, mutants, or variants thereof.
In some embodiments, the target sequence encoding the target peptide includes spike
protein or its fragment from a betacoronavirus.
32
Betacoronavirus is a genus within the subfamily Othrocoronavirinae of family
Coronaviridae. The International Committee on Taxonomy of Viruses (ICTV) has classified
the genus Betacoronavirus into 5 subgenera viz., Embecovirus, Sarbecovirus, Merbecovirus,
Nobecovirus and Hibecovirus. The first four of these subgenera were formerly known as
lineages or subgroups A, B, C and D, respectively.5
Betacoronaviruses have been of greater clinical importance as they have been found
to cause outbreaks, for example, the 2002-2003 SARS outbreak caused by SARS-CoV-1 or
SARS-CoV, the 2012 MERS outbreak caused by Middle East Respiratory Virus, and more
recently the 2019-2020 COVID-19 pandemic caused by SARS-CoV-2. The other
betacoronaviruses known to infect human beings are HKU1, and OC43.10
The genome of betacoronaviruses encodes 4 main structural proteins viz spike(S),
membrane(M), envelope(E), and nucleocapsid(N) proteins. The spike protein is the
immunodominant protein among the major structural proteins. It contains two subunits, S1
and S2. The former is further divided into an N-terminal domain (NTD) and a C-terminal
domain (CTD). Both or one of the domains may act as receptor binding domains (RBD)15
interacting with host cell receptors. The RBD contains the receptor-binding motif (RBM).
The S1 subunits are organized to form a trimeric structure. S2 subunit helps the virus to enter
the host cell through membrane fusion. S2 subunit contains fusion peptide (FP), heptad
repeat 1 (HR1), central helix (C-helix), connector domain (CD), stem helix (SH), heptad
repeat 2 (HR2), transmembrane domain and cytoplasmic domain (Dacon, Cherrelle et al.20
Cell Host & Microbe (2023) 31: 1-15; Lan, Jun et al. Nature (2020) 581: 215-220; Wang,
Mei-Yue et al. Frontiers in Cellular and Infection Microbiology (2020) 10:587269).
Receptor Binding Domain (RBD)
S1 subunit of the betacoronaviruses contains the receptor binding domain either on25
the N-terminal region or the C-terminal region. RBD interact with the host cell receptors. In
one of the embodiments, the RBD includes full length S1 subunit of the spike protein or
fragment thereof of a betacoronavirus, including mutant, derivative, or variant thereof that
retains the ability to interact with the host cell receptor. In some embodiments, the receptor
binding domain interacts with angiotensin-converting enzyme 2 (ACE2) receptor or30
dipeptidyl peptidase 4 (DPP4) receptor, 9-O-acetylated sialic acid (9-O-Ac-Sia) receptor, or
combination thereof. In some of the embodiments, the multitarget nucleic acid sequence
encodes multitarget peptide comprising one or more receptor binding domains obtained or
33
derived from one or more betacoronaviruses. In some of the embodiments, the multitarget
nucleic acid sequence encodes multitarget peptide comprising one or more receptor binding
domains obtained or derived from SARS-CoV-1, SARS-CoV-2, MERS-CoV, OC43, HKU1,
bat coronavirus, other betacoronavirus, or a combination thereof.
5
Fusion Peptide
S2 subunit of the spike protein of betacoronavirus contains a region called fusion
peptide which facilitates membrane fusion. In some embodiments, the target sequence of the
present disclosures encodes a fusion peptide of a betacoronavirus. The fusion peptide
includes fragments, mutants, derivatives or variants of fusion peptide. In some of the10
embodiments, the multitarget nucleic acid sequence encodes multitarget peptide comprising
one or more fusion peptides obtained or derived from one or more betacoronaviruses.
Stem Helix
S2 subunit of the spike protein of betacoronavirus contains a region called stem helix15
or S2 stem helix which enables heptad repeats to assume a configuration facilitating fusion
pore formation and viral entry into the cell. In some embodiments, the target sequence of
the present disclosure encodes a stem helix. The stem helix includes fragments, mutants,
derivatives, or variants of fusion peptide. In some of the embodiments, the multitarget
nucleic acid sequence encodes multitarget peptide comprising one or more stem helix20
obtained or derived from one or more betacoronaviruses.
Exemplary target peptides includes, but not limited to, the ones represented by the
following amino acid sequences, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof:
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFST25
FKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGC
VIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNC
YFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
(SEQ ID NO: 1);
RVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFA30
FKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGC
VIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVN
34
CYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
(SEQ ID NO: 2);
RVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFA
FKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGC
VIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNC5
YSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
(SEQ ID NO: 338)
RVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFS
TFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDF
MGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPAL10
NCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNF
(SEQ ID NO: 339)
EAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVND
FTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLIL
ATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDG15
DYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFAN
DTKIASQLGNCVEY (SEQ ID NO: 340)
RADVYRRKPDLPNCNIEAWLNDKSVPSPLNWERKTFSNCNFNMSSLMSFIQADSF
TCNNIDAAKIYGMCFSSITIDKFAIPNGRKVDLQLGNLGYLQSFNYRIDTTATSCQL
YYNLPAANVSVSRFNPSTWNKRFGFIEDSVFKPQPAGVFTDHDVVYAQHCFKAPK20
NFCPCKSNSSLCVGSGPGKNNGIGTCPAGTNYLTCHNLCNPDPITFTGPYKCPQTKS
LVGIGEHCSGLAVKSDHCGGNPCTCQPQAFLGWSADSCVQGDKCNIFANLILHDV
NSGL (SEQ ID NO: 341)
ADVYRRKPDLPNCNIEAWLNDKSVPSPLNWERKTFSNCNFNMSSLMSFIQADSFT
CNNIDAAKIYGMCFSSITIDKFAIPNGRKVDLQLGNLGYLQSFNYRIDTTATSCQLY25
YNLPAANVSVSRFNPSTWNKRFGFIEDSVFKPQPAGVFTDHDVVYAQHCFKAPKN
FCPCKSNSSLCVGSGPGKNNGIGTCPAGTNYLTCHNLCNPDPITFTGPYKCPQTKSL
VGIGEHCSGLAVKSDHCGGNPCTCQPQAFLGWSADSCVQGDKCNIFANLILHDVN
SGL (SEQ ID NO: 342)
RIPDLPDCDIDKWLNNFNVPSPLNWERKIFSNCNFNLSTLLRLVHTDSFSCNNFDES30
KIYGSCFKSIVLDKFAIPNSRRSDLQLGSSGFLQSSNYKIDTTSSSCQLYYSLPAINVT
INNYNPSSWNRRYGFNNFNLSSHSVVYSRYCFSVNNTFCPCAKPSFASSCKSHKPP
SASCPIGTNYRSCESTTVLDHTDWCRCSCLPDPITAYDPRSCSQKKSLVGVGEHCA
35
GFGVDEEKCGVLDGSYNVSCLCSTDAFLGWSYDTCVSNNRCNIFSNFILNGINSGT
TCSND (SEQ ID NO: 343)
RVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFA
FKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGC
VIAWNSNKLDSKPSGNYNYLYRLLRKSKLKPFERDISTEIYQAGNKPCNGVAGPNC5
YSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFN
(SEQ ID NO: 344)
RVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFA
FKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGC
VIAWNSNKLDSKPSGNYNYLYRFLRKSKLKPFERDISTEIYQVGNKPCNGVAGPNC10
YSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
(SEQ ID NO: 345)
RVQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFF
AFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTG
CVIAWNSNKLDSKHSGNYDYWYRSFRKSKLKPFERDISTEIYQAGNKPCKGKGPN15
CYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFN
(SEQ ID NO: 346)
SFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNG (SEQ ID NO: 3);
LQPELDSFKEELDKYFKNHTSPDVDLG (SEQ ID NO: 4);
VNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGI (SEQ ID NO: 5);20
NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSY
GFQPTN (SEQ ID NO: 6);
FSNVTWFHAIHVSGTNGTKRFDN (SEQ ID NO: 7);
KSFTVEKGIYQTSNFRVQP (SEQ ID NO: 8);
SKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYN (SEQ ID NO: 9);25
GTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGD
(SEQ ID NO: 10);
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTAS (SEQ
ID NO: 11);
KTFPPTEPKKDKKKKADETQALPQRQKKQQ (SEQ ID NO: 12);30
VQERIGLFIVNFFIFT (SEQ ID NO: 13);
LVQPALYLYNTGRSVY (SEQ ID NO: 14);
36
MAAPAAPRAVSFADNNDITNTNLSRGRGRNPKPRAAPNNTVSWYTGLTQHGKVPL
TFPPGQGVPLNANSTPAQNAGYWRRQDRKINTGNGIKQLAPRWYFY (SEQ ID NO:
15);
KPNRTSVVSFNEW (SEQ ID NO: 16);
DVANPSTPAYTFTTVKPGAAFSV (SEQ ID NO: 17);5
EVLVTVDGVNFRTVV (SEQ ID NO: 18);
LGYPVDPTFLHRFYSLKAAV (SEQ ID NO: 19);
ALPETSADILVVDEVSMCTNYDLSIINARIKAKHIVY (SEQ ID NO: 20);
TAQADTAGLYTNFRIDVPSAESTGTQSVSVDRESTSTHDGLTEH (SEQ ID NO: 21);
FSDPNMWYL (SEQ ID NO: 22);10
LKPYGGQPVSEYHITLAL (SEQ ID NO: 23);
PAFHRIESTDSIVFTYIPASGYVAALAVNVCLI (SEQ ID NO: 24);
RSIIRTMVLYFLVLYNFLLAIVLVNGVHYPTGSCLIAFLVILIILWFVDRI (SEQ ID NO:
25);
SMKMAPLMLLQLLGR (SEQ ID NO: 26);15
TFFDKTWPRPIDVSKADGIIYPQGRTYSN (SEQ ID NO: 27);
NGFVVRIGAAANSTGTVIISPSTSATI (SEQ ID NO: 28);
VGNFSDGKMGRFFNHTLVLL (SEQ ID NO: 29);
LYGGNMFQFATLPVY (SEQ ID NO: 30);
RSIKSDRKAWAAFYVY (SEQ ID NO: 31);20
CALPDTPSPLTPRSVRSVPGEMRLASIAF (SEQ ID NO: 32);
AIPFAQSIFY (SEQ ID NO: 33);
AQQLVRSESAALSAQ (SEQ ID NO: 34);
PVNGYFIKTNNTRIVDEWSYTGSSFYSPEPITSLNTKYVAPQVTYQNISTNLPPPLLG
NSTIGIDFQDELDEFF (SEQ ID NO: 35);25
SFIEDLLFNKVTLADAGFIKQY (SEQ ID NO: 334);
KQYGDCLGDIAARDLICAQKFN (SEQ ID NO: 335);
KFNGLTVLPPLLTDEMIAQYT (SEQ ID NO: 336);
GWTFGAGAALQIPFAMQMAYRFNGI (SEQ ID NO: 337);
In some embodiments, the target peptide shares at least 70 % identity with the30
sequences described herein above, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
37
Given the above amino acid sequences of the target peptides, a person skilled in the
art would be able to deduce all possible DNA or RNA sequences that encodes the above
target peptides. Such DNA or RNA sequences are deemed to be incorporated in this
disclosure. In some embodiments, the target peptide is encoded by a target sequence which
may be either a DNA, an RNA or an mRNA.5
Herpesviridae
Members of the family Herpesviridae consists of large, enveloped, double stranded
DNA viruses. They are known to infect variety of hosts, including mammals. Nine
herpesviruses have been identified so far that have human as their primary host viz., herpes10
simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human cytomegalovirus
(HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Human herpesviruses 6A
(HHV-6A), 6B (HHV-6B), and 7 (HHV-7), and Kaposi’s sarcoma-associated herpesvirus
(HHV-8) (Pellett, Philip E. and Roizman, Bernard, Chapter 59 – Herpesviridae, Editors(s):
Knipe, David M. and Howley, Peter M., in Fields Virology, 6th edition, 2013 pages 1802-15
1822 ISBN 9781451105636). These viruses cause different clinical manifestations that
range from asymptomatic to severe disease, depending on the host immune status.
In some embodiments, the target sequence encoding the target peptide is obtained or
derived from HSV-1, HSV-2, HCMV, VZV, EBV, HHV-6A, HHV-6B, HHV-7, HHV-8 or a
combination thereof, including codon optimized sequences, fragments, mutants, or variants20
thereof of such target sequences.
In some embodiments, the target peptide includes glycoprotein B, glycoprotein C,
glycoprotein D, glycoprotein E, glycoprotein K, glycoprotein L, or glycoprotein M, of
herpes simplex virus 1 (HSV-1) or herpes simplex virus 2 (HSV-2), or a combination thereof,
including their codon optimized nucleic acid sequences, fragments, mutants or variants25
thereof.
In some embodiments, the target peptide includes glycoprotein B, glycoprotein H,
glycoprotein L, glycoprotein M, or glycoprotein N of human cytomegalovirus (HCMV), or
a combination thereof, including their codon optimized nucleic acid sequences, fragments,
mutants, or variants thereof.30
In some embodiments, the target peptide includes glycoprotein B, glycoprotein C,
glycoprotein H, or glycoprotein L of varicella-zoster virus (VZV), or a combination thereof,
including their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof.
38
In some embodiments, the target peptide includes, but not limited to, glycoprotein
B, glycoprotein H, glycoprotein L, glycoprotein M, glycoprotein N, glycoprotein 42, or
glycoprotein 350 of Epstein-Barr virus (EBV), or a combination thereof, including their
codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Exemplary target peptides includes, but not limited to, the ones represented by the5
following amino acid sequences, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof:
TWVPKPNVEVWPVDPPPPVN (SEQ ID NO: 36);
DEKEGLETTTYITSQEVQNS (SEQ ID NO: 37);
GAQTPEQPAPPATTVQPTAT (SEQ ID NO: 38);10
TTPTSSPPSSPSPPAPSAAR (SEQ ID NO: 39);
RRRDAGNATTPVPPTAPGKS (SEQ ID NO: 40);
YKIVDYDNRGTNPQGERRAF (SEQ ID NO: 41);
LPTWGNWAYPCCHVTQLRAQ (SEQ ID NO: 42);
TPSPGRNRRRSSTSSSSSRS (SEQ ID NO: 43);15
TGVLPAGASSPTNAAAASLT (SEQ ID NO: 44);
TTPTPNATSPTPAVTTPTPN (SEQ ID NO: 45);
TSPTPAVTTPTPNATSPTLG (SEQ ID NO: 46);
TSPTPAGTTSGASPVTPSPS (SEQ ID NO: 47);
TSPTSAVTTPTPNATGPTVG (SEQ ID NO: 48);20
PAPRPGTTSQASGPGNSSTS (SEQ ID NO: 49);
TSPTSAVTTPTPNATSPTLG (SEQ ID NO: 50);
TSPTSAVTTPTPNATSPTLG (SEQ ID NO: 51);
KAPESTTTSPTLNTTGFADP (SEQ ID NO: 52);
TSPPVTTAQATVPVPPTSQP (SEQ ID NO: 53);25
TSAVTTGQHNITSSSTSSMS (SEQ ID NO: 54);
GILTSTSPVATPIPGTGYAY (SEQ ID NO: 55);
THVPTNLTAPASTGPTVSTA (SEQ ID NO: 56);
TNHTLGGTSPTPVVTSQPKN (SEQ ID NO: 57);
NVTKGTPPQNATSPQAPSGQ (SEQ ID NO: 58);30
TAVPTVTSTGGKANSTTGGK (SEQ ID NO: 59);
ETDQMDTIY (SEQ ID NO: 60);
QMDTIYQCY (SEQ ID NO: 61);
39
PTTVMSSIY (SEQ ID NO: 62);
MTAASYARY (SEQ ID NO: 63);
LTSAQSGDY (SEQ ID NO: 64);
ATSVLLSAY (SEQ ID NO: 65);
ALENISDIY (SEQ ID NO: 66);5
LLTTLETLY (SEQ ID NO: 67);
SSSALTGHL (SEQ ID NO: 68);
IADCVAFIY (SEQ ID NO: 69);
FLALGNSFY (SEQ ID NO: 70);
TTDSEEEIF (SEQ ID NO: 71);10
LTEAQDQFY (SEQ ID NO: 72);
IASAIYLMY (SEQ ID NO: 73);
ASAIYLMYV (SEQ ID NO: 74);
CAELYPCTY (SEQ ID NO: 75);
HTFQVPQNY (SEQ ID NO: 76);15
NTREYTFSY (SEQ ID NO: 77);
PTNTTDITY (SEQ ID NO: 78);
FLGNNSILY (SEQ ID NO: 79);
HAEMQNPVY (SEQ ID NO: 80);
In some embodiments, the target peptide shares at least 70 % identity with the20
sequences described herein above, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the
art would be able to deduce all possible DNA or RNA sequences that encodes the above
target peptides. Such DNA or RNA sequences are deemed to be incorporated in this25
disclosure. In some embodiments, the target peptide is encoded by a target sequence which
may be either a DNA, an RNA or an mRNA.
Poxviridae
Members of the family poxviridae consists of large, enveloped, double stranded,30
DNA viruses that replicates generally in the cytoplasm of the host cells. Smallpox (variola)
virus, vaccinia virus, cowpox virus, and monkeypox virus are the most common viruses
within the orthopoxvirus genus of poxviridae family.
40
In some embodiments, the target sequence encoding the target peptide is obtained or
derived from poxviruses such as, but not limited to, smallpox virus, vaccina virus, cowpox
virus or monkeypox virus, or a combination thereof, including codon optimized sequences,
fragments, mutants, or variants thereof of such target sequences.
In some embodiments, the target peptide includes, but not limited to, F9 membrane5
protein of a poxvirus, H3L protein of a poxvirus, A4 protein of a poxvirus, A27 protein of
poxvirus, A33 protein of a poxvirus, A56 protein of a poxvirus, B5 protein of a poxvirus, or
L1 protein of a poxvirus, or a combination thereof, including their codon optimized nucleic
acid sequences, fragments, mutants, or variants thereof.
Exemplary target peptides includes, but not limited to, the ones represented by the10
following amino acid sequences, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof
SNEEFDPVDDGPVSDYVSELY (SEQ ID NO: 81);
LPAVVYSTCTVPTMNNAKLT (SEQ ID NO: 82);15
YISCTANSWNVIPSCQQKCD (SEQ ID NO: 83);
NKINSIVERRSGMSNVVDST (SEQ ID NO: 84);
VAEASTIMVATARSSPEELE (SEQ ID NO: 85);
TKTVPMMNVVTKLQGNTITI (SEQ ID NO: 86);
VHWNKKKYSSYEEAKKHDDG (SEQ ID NO: 87);20
SSSNHEGKPHYITENYRNPY (SEQ ID NO: 88);
VRINFKGGYISGGFLPNEYV (SEQ ID NO: 89);
RSNEEFDPV (SEQ ID NO: 90);
AVVYSTCTV (SEQ ID NO: 91);25
YISCTANSW (SEQ ID NO: 92);
KINSIVERR (SEQ ID NO: 93);
TVAEASTIM (SEQ ID NO: 94);
PMMNVVTKL (SEQ ID NO: 95);
LVHWNKKKY (SEQ ID NO: 96);30
SNHEGKPHY (SEQ ID NO: 97);
GFLPNEYVL (SEQ ID NO: 98);
GKWNPILPTCVRSNE (SEQ ID NO: 99);
41
TLLCVLPAVVYSTCT (SEQ ID NO: 100);
VIGVSYISCTANSWN (SEQ ID NO: 101);
LNFRQDAVNKINSIV (SEQ ID NO: 102);
VAEASTIMVATARSS (SEQ ID NO: 103);
MMNVVTKLQGNTITI (SEQ ID NO: 104);5
GEINLVHWNKKKYSS (SEQ ID NO: 105);
KFRTLLSSSNHEGKP (SEQ ID NO: 106);
GGFLPNEYVLSTIHI (SEQ ID NO: 107);
In some embodiments, the target peptide shares at least 70 % identity with the10
sequences described herein above, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the
art would be able to deduce all possible DNA or RNA sequences that encodes the above
target peptides. Such DNA or RNA sequences are deemed to be incorporated in this15
disclosure. In some embodiments, the target peptide is encoded by a target sequence which
may be either a DNA, an RNA or an mRNA.
Flaviviridae
Members of the family Flaviviridae consists of enveloped, single stranded RNA20
viruses. Dengue virus, Japanese encephalitis virus, zika virus, yellow fever virus, west nile
virus, and hepatitis C virus are the most common viruses within the flavivirus and
hepacivirus genera respectively.
In some embodiments, the target sequence encoding the target peptide is obtained or
derived from dengue virus, Japanese encephalitis virus, zika virus, yellow fever virus, west25
nile virus, or hepatitis C virus, or a combination thereof, including codon optimized
sequences, fragments, mutants, or variants thereof of such target sequences.
In some embodiments, the target peptide includes, but not limited to, capsid protein,
membrane protein, envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B,
NS3, NS4A, NS4B, or NS5) of flaviviruses or hepaciviruses, or a combination thereof,30
including their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof.
In some embodiments, the target peptide includes capsid protein, membrane protein,
envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A,
42
NS4B, or NS5) of Japanese encephalitis virus, or a combination thereof, including their
codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes capsid protein, membrane protein,
envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A,
NS4B, or NS5) of zika virus, or a combination thereof, including their codon optimized5
nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes capsid protein, membrane protein,
envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A,
NS4B, or NS5) of yellow fever virus, or a combination thereof, including their codon
optimized nucleic acid sequences, fragments, mutants, or variants thereof.10
In some embodiments, the target peptide includes capsid protein, membrane protein,
envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A,
NS4B, or NS5) of west nile virus, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes capsid protein, membrane protein,15
envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A,
NS4B, or NS5) of hepatitis C virus, or a combination thereof, including their codon
optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes capsid protein, membrane protein,
envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A,20
NS4B, or NS5) of dengue virus, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
Exemplary target peptides includes, but not limited to, the ones represented by the
following amino acid sequences, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.25
TIPPTAGILKRWGTIKKSKA (SEQ ID NO: 108);
NEPEDIDCWCNSTSTWVTYG (SEQ ID NO: 109);
GLETRTETWMSSEGAWKHVQ (SEQ ID NO: 110);
HGTIVIRVQYEGDGSPCKIP (SEQ ID NO: 111);
GNDTGKHGKEIKITPQSSTT (SEQ ID NO: 112);30
FRCKKNMEGKVVQPENLEYT (SEQ ID NO: 113);
LTGYGTVTMECSPRTGLDFN (SEQ ID NO: 114);
IDGPETAECPNTNRAWNSLE (SEQ ID NO: 115);
43
TDNVHTWTEQYKFQPESPSK (SEQ ID NO: 116);
VMVMVGATMTDDIGMGVTYL (SEQ ID NO: 117);
KNDIPMTGPLVAGGPLTVCY (SEQ ID NO: 118);
WDVPSPPPMGKAELEDGAYR (SEQ ID NO: 119);
DEEREIPERSWNSGHEWVTD (SEQ ID NO: 120);5
QTEKSIEDNPEIEDDIFRKR (SEQ ID NO: 121);
RKTFDSEYAKTRTNDWDFVV (SEQ ID NO: 122);
DNINTPEGIIPSMFEPEREK (SEQ ID NO: 123);
TVIDLDPIPYDPKFEKQLGQ (SEQ ID NO: 124);
AKASREAQKRAAAGIMKNPT (SEQ ID NO: 125);10
KGLTKGGPGHEEPIPMSTYG (SEQ ID NO: 126);
EYTDYMPSMKRFRREEEEAG (SEQ ID NO: 127);
TKPWDVVPMVTQMAMTDTTP (SEQ ID NO: 128);
AYHGSYETKQTGSASSMVNG (SEQ ID NO: 129);
KIKQEHETSWHYDQDHPYKT (SEQ ID NO: 130);15
FTMRYKKATYEPDVDLGSGT (SEQ ID NO: 131);
QGRGPLKLYMALVAFLRFLTI (SEQ ID NO: 132);
NSTSTWVTY (SEQ ID NO: 133);
TTEAELTGY (SEQ ID NO: 134);20
VVQPENLEY (SEQ ID NO: 135);
GTIVIRVQY (SEQ ID NO: 136);
NVHTWTEQY (SEQ ID NO: 137);
MTDDIGMGV (SEQ ID NO: 138);
KVDAIDGEY (SEQ ID NO: 139);25
KAELEDGAY (SEQ ID NO: 140);
VTDFKGKTV (SEQ ID NO: 141);
RTNDWDFVV (SEQ ID NO: 142);
VIDLDPIPY (SEQ ID NO: 143);
YTDYMPSMK (SEQ ID NO: 144);30
LINRFTMRY (SEQ ID NO: 145);
RSLIGNEEY (SEQ ID NO: 146);
KTWAYHGSY (SEQ ID NO: 147);
MTDTTPFGQ (SEQ ID NO: 148);
44
ASSMVNGVF (SEQ ID NO: 149);
MSTYGWNLV (SEQ ID NO: 150);
QEHETSWHY (SEQ ID NO: 151);
WGTIKKSKAINVLRGFR (SEQ ID NO: 152);5
MSSEGAWKH (SEQ ID NO: 153);
WTEQYKFQPESPSKLASAIQKAHEEG (SEQ ID NO: 154);
WNSLEVEDY (SEQ ID NO: 155);
RDLGRVMVMVGATMTD (SEQ ID NO: 156);
VTYLALLAAFKVRPT (SEQ ID NO: 157);10
IPMTGPLVAGGPLTVCYV (SEQ ID NO: 158);
IFRKRRLTIMDLHPGAG (SEQ ID NO: 159);
GVLWDVPSPPPMGKA (SEQ ID NO: 160);
FLLVAHYAIIGPALQAKASREAQKRAAA (SEQ ID NO: 161);
15
In some embodiments, the target peptide shares at least 70 % identity with the
sequences described herein above, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the
art would be able to deduce all possible DNA or RNA sequences that encodes the above20
target peptides. Such DNA or RNA sequences are deemed to be incorporated in this
disclosure. In some embodiments, the target peptide is encoded by a target sequence which
may be either a DNA, an RNA or an mRNA.
Togaviridae25
Members of the family Togaviridae consists of enveloped, single stranded RNA
viruses. The family togaviridae consists of two genera viz., the alphaviruses and the
rubiviruses. Alphaviruses cause severe human illnesses including persistent arthritis and
fatal encephalitis and are responsible for emerging human diseases. Some alphaviruses (e.g.,
Chikungunya (CHIKV), Ross River (RRV), Mayaro (MAYV), Semliki Forest (SFV),30
Sindbis (SINV), and O'nyong-nyong (ONNV)) cause acute inflammatory musculoskeletal
and joint-associated syndromes, which can become chronic, whereas others (Eastern
(EEEV), Western (WEEV), and Venezuelan (VEEV) equine encephalitis viruses) cause
45
infection in the brain and neurological disease (Holmes, Autumn C. et al. 2020 PLoS
Pathogens 16(10): e1008876).
In some embodiments, the target sequence encoding the target peptide is obtained or
derived from Chikungunya (CHIKV), Ross River (RRV), Mayaro (MAYV), Semliki Forest
(SFV), Sindbis (SINV), and O'nyong-nyong (ONNV) viruses, Eastern (EEEV), Western5
(WEEV), or Venezuelan (VEEV) equine encephalitis viruses, or a combination thereof,
including codon optimized sequences, fragments, mutants, or variants thereof of such target
sequences.
In some embodiments, the target peptide includes, but not limited to, capsid protein,
or envelope protein such as E1, E2 and E3 of alphaviruses, or a combination thereof,10
including their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof.
In some embodiments, the target peptide includes, but not limited to, domain A,
domain B, or domain C of E2 protein of alphaviruses, or a combination thereof, including
their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.15
In some embodiments, the target peptide includes, but not limited to capsid protein,
or envelope protein such as E1, E2, and E3 of chikungunya virus, or a combination thereof,
including their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof.
In some embodiments, the target peptide includes, but not limited to, domain A,20
domain B, or domain C of E2 protein of chikungunya virus, or a combination thereof,
including their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof.
Exemplary target peptides includes, but not limited to, the ones represented by the
following amino acid sequences, or a combination thereof, including their codon optimized25
nucleic acid sequences, fragments, mutants, or variants thereof.
KGRVVAIVL (SEQ ID NO: 162);
IDNADLAKL (SEQ ID NO: 163);
FKRSSKYDL (SEQ ID NO: 164);
LLANTTFPC (SEQ ID NO: 165);30
YEKEPEETL (SEQ ID NO: 166);
LLANTTFPC (SEQ ID NO: 167);
GTLKIQVSL (SEQ ID NO: 168);
46
RNEATDGTL (SEQ ID NO: 169);
TMTVVVVSV (SEQ ID NO: 170);
LKIQVSLQI (SEQ ID NO: 171);
AALILIVVL (SEQ ID NO: 172);
LQISFSTAL (SEQ ID NO: 173);5
LILIVVLCV (SEQ ID NO: 174);
VLFSVGSTL (SEQ ID NO: 175);
YAVHAPTSL (SEQ ID NO: 176);
RSAEDPERL (SEQ ID NO: 177);
YVVKRITMS (SEQ ID NO: 178);10
FACHSGTLL (SEQ ID NO: 179);
KSISRRCTL (SEQ ID NO: 180);
CHSGTLLAL (SEQ ID NO: 181);
GLEISARTV (SEQ ID NO: 182);
MQMKVNYNH (SEQ ID NO: 183);15
VNSVAIPLL (SEQ ID NO: 184);
VTRLGVNSV (SEQ ID NO: 185);
VLLPNVHTL (SEQ ID NO: 186);
RLFKLGKPL (SEQ ID NO: 187);
VKIIDAVVS (SEQ ID NO: 188);20
STKDNFNVYKATRPYLAHC (SEQ ID NO: 189);
TDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERAGL (SEQ ID NO: 190);
HHDPPVIGREKFHSRPQHGKELPCST (SEQ ID NO: 191);
ATTEEIEVHMPPDTPDRT (SEQ ID NO: 192);
GNVKITVNGQTVRYKCNC (SEQ ID NO: 193);25
LTTTDKVINNCKVDQCHAAVTNHKKW (SEQ ID NO: 194);
HAAVTNHKKWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYOK
NQV (SEQ ID NO: 195);
PTVTYGKNQVIMLLYPDHPTLLSYRN (SEQ ID NO: 196);
RNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNWEPYKYWPQLSTHGT30
(SEQ ID NO: 197);
LLSMVGMAAGMCHCARRRCITPYELTPGATVPFL (SEQ ID NO: 198);
LAHCPDCGEGHSCHS (SEQ ID NO: 199);
47
ADAERAGLFV (SEQ ID NO: 200);
THPFHHDPPV (SEQ ID NO: 201);
EIEVHMPPDT (SEQ ID NO: 202);
GEEPNYQEEW (SEQ ID NO: 203);
VPTEGLEVTW (SEQ ID NO: 204);5
GNNEPYKYWP (SEQ ID NO: 205);
HCPDCGEGHSCHSPV (SEQ ID NO: 206);
RIRNEATDGTLKI (SEQ ID NO: 207);
IKTDDSHDWTKLRY (SEQ ID NO: 208);
KLRYMDNHIPADAGRA (SEQ ID NO: 209);10
GLFVRTSAPCTITGTM (SEQ ID NO: 210);
GFTDSKISHSCTHPFHHD (SEQ ID NO: 211);
PVIGREKFHSRPQHGKELPC (SEQ ID NO: 212);
TYVQSTAATTEEIEVHMPP (SEQ ID NO: 213);
DTPDRTLMSQQSGNVKIT (SEQ ID NO: 214);15
VNGRTVRYKCNCGGSNEG (SEQ ID NO: 215);
DKVINNCKVDQCHAAVTNHK (SEQ ID NO: 216);
NSPLVPRNAELGDRKGK (SEQ ID NO: 217);
RVPKARNPTVTYGKNQ (SEQ ID NO: 218);
NMGEEPNYQEEWVMHK (SEQ ID NO: 219);20
VPTEGLEVTWGNNEPY (SEQ ID NO: 220);
LSTNGTAHGHPHE (SEQ ID NO: 221);
HEIILYYYELYP (SEQ ID NO: 222);
In some embodiments, the target peptide shares at least 70 % identity with the
sequences described herein above, or a combination thereof, including their codon optimized25
nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the
art would be able to deduce all possible DNA or RNA sequences that encodes the above
target peptides. Such DNA or RNA sequences are deemed to be incorporated in this
disclosure. In some embodiments, the target peptide is encoded by a target sequence which30
may be either a DNA, an RNA or an mRNA.
Retroviridae
48
Members of the family retroviridae consists of enveloped single stranded RNA
viruses. It constitutes a large family of viruses that predominantly infect both human and
animal vertebrate hosts and causes wide spectrum of diseases ranging from malignancies to
immune deficiencies and neurologic disorders. Retroviriade family is organized into several
genera viz., alpharetrovirus, betraretrovirus, gammaretrovirus, deltaretrovirus,5
epsilonretrovirus, lentivirus and spumavirus. Of these genera, lentiviruses have been the
subject of considerable interest the most prominent among them being the human
immunodeficiency virus (HIV).
In some embodiments, the target sequence encoding the target peptide is obtained or
derived from alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus,10
epsilonretrovirus, lentivirus, spumavirus, or a combination thereof, including codon
optimized sequences, fragments, mutants, or variants thereof of such target sequences.
In some embodiments, the target peptide includes, but not limited to, gag, pol or env
proteins of retroviruses, or a combination thereof, including their codon optimized nucleic
acid sequences, fragments, mutants, or variants thereof.15
In some embodiments, the target peptide includes, p17Gag, p24Gag, p7Gag, p6Gag,
gp12Env, gp41Env, or pol proteins from lentiviruses, or a combination thereof, including their
codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes, p17Gag, p24Gag, p7Gag, p6Gag,
gp12Env, gp41Env, or pol proteins from human immunodeficiency virus (HIV), or a20
combination thereof, including their codon optimized nucleic acid sequences, fragments,
mutants, or variants thereof.
Exemplary target peptides includes, but not limited to, the ones represented by the
following amino acid sequences, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.25
PEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPAQ
AGPIAPGQIREPRGSDIAGTTSSLQEQITWMTXNPPIPVGEIYKRWIILGLNKIVRMY
SPVGILDIRQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKT
ILKALGPAATLEEMMTACQGVG (SEQ ID NO: 223);30
MGARASVLSGGKLDTWERIRLRPGGKKKYALKHLIWASRELERFKLNPGLLETSE
GCKQIIGQLQPSIQTGSEEIRSLYNTVATLYCVHERIEVKDTKEAVEKMEEEQNKSK
KKAQQAADSSQVSQNY (SEQ ID NO: 224);
49
GSLAEEEVVIRSENFTDNAKTIIVQLNESVEINCTRPNNNTSKSIPIGPGRAFYATDRI
IGDIRQAHCNLSRTKWDSTLEKIVIKLREQVGNKTIVFNQS (SEQ ID NO: 225);
MGAASLTLTVQARQLLSGIVQQQNDLLRAIEAQQHLLQLTVWGIKQLQARVLAVE5
RYLKDQQLLGIWGCSGKLICTTAVPWNSSWSNKSQDQIWHNMTWMEWEREIENY
TDLIYTLIEKSQNQQEKNEQELLELDKWASLWNW (SEQ ID NO: 226);
KIRLRPGGK (SEQ ID NO: 227);
KYKLKHIVW (SEQ ID NO: 228);
GHQAAMQMLKETI (SEQ ID NO: 229);10
EIYKRWIIL (SEQ ID NO: 230);
ILGLNKIV (SEQ ID NO: 231);
RMYSPTSIL (SEQ ID NO: 232);
KLWVTVYYGVPVWR (SEQ ID NO: 233);
IISLWDQSLK (SEQ ID NO: 234);15
QMAVFIHHFKRK (SEQ ID NO: 235);
NTSVITQACPKVSFEPEPIHYCAPA (SEQ ID NO: 236);
GFAILKCNNKT (SEQ ID NO: 237);
SLYNTVATL (SEQ ID NO: 238);
In some embodiments, the target peptide shares at least 70 % identity with the20
sequences described herein above, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the
art would be able to deduce all possible DNA or RNA sequences that encodes the above
target peptides. Such DNA or RNA sequences are deemed to be incorporated in this25
disclosure. In some embodiments, the target peptide is encoded by a target sequence which
may be either a DNA, an RNA or an mRNA.
Paramyxoviridae
Members of the paramyxoviridae family consists of enveloped, single stranded RNA30
viruses. Diseases caused by these viruses continue to produce high mortality and morbidity
across the world. The family Paramyxoviridae is classified into two subfamilies, the
paramyxovirinae and the penumovirinae. The former contains seven genera: respirovirus,
50
rubulavirus, Moribilivirus, Henipavirus, Aqiaparamyxovirus, Avulavirus, and Ferlavirus,
while the latter contains two genera: Penumovirus and Metapenumovirus.
Some of the important members of paramyxoviridae that are known to cause disease
outbreaks are: Mumps virus (MuV), Parainfluenza virus type 5 (PIV5), Human
parainfluenza virus type 2, types 4a and 4b (HPIV2/4a/4b), Newcastle disease virus (NDV),5
Human parainfluenza virus type 1 and type 3 (HPIV1/3), Nipah virus (NiV), Measles virus
(MeV), Human respiratory syncytial virus A2, B1, S2, (HRSV), and Human
metapneumovirus (HMPV).
In some embodiments, the target sequence encoding the target peptide is obtained or
derived from Mumps virus (MuV), Parainfluenza virus type 5 (PIV5), Human parainfluenza10
virus type 2, types 4a and 4b (HPIV2/4a/4b), Newcastle disease virus (NDV), Human
parainfluenza virus type 1 and type 3 (HPIV1/3), Nipah virus (NiV), Measles virus (MeV),
Human respiratory syncytial virus A2, B1, S2, (HRSV), or Human metapneumovirus
(HMPV), or a combination thereof, including codon optimized sequences, fragments,
mutants, or variants thereof of such target sequences.15
In some embodiments, the target peptide includes, but not limited to, nucleocapsid
protein, P protein, V protein, W protein, D protein, I protein, C protein, L protein, M protein,
H (hemagglutinin) protein, HN (hemagglutinin-neuraminidase) protein, G protein, F protein,
or a combination thereof, of Mumps virus (MuV), Parainfluenza virus type 5 (PIV5), Human
parainfluenza virus type 2, types 4a and 4b (HPIV2/4a/4b), Newcastle disease virus (NDV),20
Human parainfluenza virus type 1 and type 3 (HPIV1/3), Nipah virus (NiV), Measles virus
(MeV), Human respiratory syncytial virus A2, B1, S2, (HRSV), Human metapneumovirus
(HMPV), or a combination thereof, including their codon optimized nucleic acid sequences,
fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes, but not limited to, nucleocapsid25
protein, P protein, V protein, W protein, D protein, I protein, C protein, L protein, M protein,
H (hemagglutinin) protein, HN (hemagglutinin-neuraminidase) protein, G protein, F protein
of human respiratory syncytial virus A2, B1, S2 (HRSV), or a combination thereof,
including their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof.30
Exemplary target peptides includes, but not limited to, the ones represented
by the following amino acid sequences, or a combination thereof, including their codon
optimized nucleic acid sequences, fragments, mutants, or variants thereof.
51
KSIAQITLSILA (SEQ ID NO: 239);
YLTQNPQLGISF (SEQ ID NO: 240);
TTKQRQNKPPNK (SEQ ID NO: 241);
CSICSNNPTCWA (SEQ ID NO: 242);
CSNNPTCWAICK (SEQ ID NO: 243);5
SSEGNISPSQVY (SEQ ID NO: 244);
ETVIEFQQKNNR (SEQ ID NO: 245);
DTPCWKLHTSPL (SEQ ID NO: 246);
SVSFFPLAETCK (SEQ ID NO: 247);
FFPLAETCKVQS (SEQ ID NO: 248);10
SLYVKGEPIINF (SEQ ID NO: 249);
CKARSTPVTLSK (SEQ ID NO: 250);
RSTPVTLSKDQL (SEQ ID NO: 251);
AIIFIASANNKVTLT (SEQ ID NO: 252);
IIFIASANNKVTLTT (SEQ ID NO: 253);15
In some embodiments, the target peptide shares at least 70 % identity with the
sequences described herein above, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the
art would be able to deduce all possible DNA or RNA sequences that encodes the above20
target peptides. Such DNA or RNA sequences are deemed to be incorporated in this
disclosure. In some embodiments, the target peptide is encoded by a target sequence which
may be either a DNA, an RNA or an mRNA.
Papillomaviridae25
Members of the family Papillomaviridae are small non-enveloped, double stranded
DNA viruses. The most common genus in the family papillomaviridae is papillomavirus.
Human papillomaviruses are the most prominent papillomaviruses which are known to
cause papillomas or warts, and are responsible for causing cervical cancer, vaginal cancer,
vulvar cancer, penile cancer, anal cancer, and oropharyngeal cancer.30
In some embodiments, the target sequence encoding the target peptide is obtained or
derived from papillomavirus, preferably human papillomavirus, including codon optimized
sequences, fragments, mutants, or variants thereof of such target sequences.
52
In some embodiments, the target sequence encoding the target peptide is obtained or
derived from human papillomaviruses selected from the group comprising HPV1, HPV2,
HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV10, HPV11, HPV 12, HPV13,
HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23,
HPV24, HPV 25, HPV26, HPV27, HPV28, HPV29, HPV29, HPV30, HPV31, HPV32,5
HPV33, HPV34, HPV35, HPV36, HPV37, HPV38, HPV39, HPV40, HPV41, HPV42,
HPV43, HPV44, HPV45, HPV46, HPV47, HPV48, HPV49, HPV50, HPV51, HPV52,
HPV53, HPV54, HPV55, HPV56, HPV57, HPV58, HPV59, HPV60, HPV61, HPV62,
HPV63, HPV64, HPV65, HPV66, HPV67, HPV68, HPV69, HPV70, HPV71, HPV72,
HPV73, HPV74, HPV75, HPV76, HPV77, HPV78, HPV79, HPV80, HPV81, HPV82,10
HPV83, HPV84, HPV85, HPV86, HPV87, HPV88, HPV89, HPV90, HPV91, HPV92,
HPV93, HPV94, HPV95, HPV96, HPV97, HPV98, HPV99, HPV100, HPV101, HPV102,
HPV103, HPV104, HPV105, HPV106, HPV107, HPV108, HPV109, HPV110, HPV111,
HPV112, HPV113, HPV114, HPV115, HPV116, HPV117, HPV118, HPV119, HPV120, or
a combination thereof.15
In some embodiments, the target peptide includes, but not limited to, early (E)
protein or late (L) protein or a combination thereof, of papillomavirus, preferably human
papillomavirus, including codon optimized sequences, fragments, mutants, or variants
thereof.
In some embodiments, the target peptide includes, but not limited to, E1, E2, E4, E5,20
E6, or E7 protein of papillomavirus, preferably human papillomavirus, or a combination
thereof, including codon optimized sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes, but not limited to, L1 (major
capsid) protein, L2 (minor capsid) protein, of papillomavirus, preferably human
papillomavirus, or a combination thereof, including codon optimized sequences, fragments,25
mutants, or variants thereof.
In some embodiments, the target peptide includes, L1 (major capsid) protein of
papillomavirus, preferably human papillomavirus, that has lost the ability to self-assemble
into a virus like particle. In some embodiments, the L1 protein is modified or mutated such
that it loses its ability to self-assemble into a virus like particle.30
In some embodiments, the target peptide includes, L2 (minor capsid) protein of
papillomavirus, preferably human papillomavirus, including codon optimized sequences,
fragments, mutants, or variants thereof.
53
Exemplary target peptides includes, but not limited to, the ones represented by the
following amino acid sequences, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
QLYKTCKQAGTCPPDIIPKV (SEQ ID NO: 353)
KTCKQAGTCPPDIIPKVEG (SEQ ID NO: 354)5
GGLGIGTGSGTGGRTGYIPL (SEQ ID NO: 355)
RTGYIPLGTRPPT (SEQ ID NO: 356)
LVEETSFIDAGAP (SEQ ID NO: 357)
MAHSRARRRKRASATQLYQTCKLTGTCPPDVIPKVEHNTIADQILKWGSLGVFFG
GLGIGTGSGTGGRTGYVPLGTSAKPSITSGPMARPPVVVEPVAPSDPSIVSLIEESAII10
NAGAPEIVPPAHGGFTITSSETTTPAILDVSVTSHTTTSIFRNPVFTEPSVTQPQPPVE
ANGHILISAPTITSHPIEEIPLDTFVISSSDSGPTSSTPVPGTAPRPRVGLYSRALHQVQ
VTDPAFLSTPQRLITYDNPVYEGEDVSVQFSHDSIHNAPDEAFMDIIRLHRPAIASRR
GLVRYSRIGQRGSMHTRSGKHIGARIHYFYDISPIAQAAEEIEMHPLVAAQEDTFDI
YAESFEPDINPTQHPVTNISDTYLTSTPNTVTQPWGNTTVPLSIPNDLFLQSGPDITFP15
TAPMGTPFSPVTPALPTGPVFITGSGFYLHPAWYFARKRRKRIPLFFSDVAA (SEQ ID
NO: 358)
MKPRARRRKRASATQLYQTCKATGTCPPDVIPKVEHTTIADQILKWGSLGVFFGGL
GIGTGAGSGGRAGYIPLGSSPKPAITGGPAARPPVLVEPVAPSDPSIVSLIEESAIINA20
GAPEVVPPTQGGFTITSSESTTPAILDVSVTNHTTTSVFQNPLFTEPSVIQPQPPVEA
NGHILISAPTITSQHVEDIPLDTFVVSSSDSGPTSSTPLPRAFPRPRVGLYSRALQQVQ
VRDPAFLSTPQRLVTYDNPVYEGEDVSLQFTHESIHNAPDEAFMDIIRLHRPAITSR
RGLVRFSRIGQRGSMYTRSGQHIGARIHYFQDISPVTQAAEEIELHPLVAAENDTFDI
YAEPFDPIPDPVQHSVTQSYLTSTPNTLSQSWGNTTVPLSIPSDWFVQSGPDITFPTA25
SMGTPFSPVTPALPTGPVFITGSDFYLHPTWYFARRRRKRIPLFFTDVAA (SEQ ID
NO: 359)
MRHKRSAKRTKRASATQLYKTCKQAGTCPPDIIPKVEGKTIADQILQYGSMGVFFG
GLGIGTGSGTGGRTGYIPLGTRPPTATDTLAPVRPPLTVDPVGPSDPSIVSLVEETSFI30
DAGAPTPVPSIPPDVSGFSITTSTDTTPAILDINNTVTTVTTHNNPTFTDPSVLQPPTP
AETGGHFTLSSSTISTHNYEEIPMDTFIVSTNPNTVTSSTPIPGSRPVARLGLYSRTTQ
QVKVVDPAFVTAPTKLITYDNPAYEGIDVDNTFYFPSNDNSINIAPDPDFLDIVALHR
54
PALTSRRTGIRYSRIGNKQTLRTRSGKSIGAKVHYYYDLSTINPAEEIELQTITPSTYT
TTSHAASPTSINNGLYDIYADDFITDTVTTPVPAIPSTSLSGYIPANTTIPFGGAYNIPL
VSGPDIPINTTDQTPSLIPIVPGSPQYTIIADGGDFYLHPSYYMLRKRRKRLPYFFSD
VSLAA (SEQ ID NO: 360)
5
MVSHRAARRKRASVTDLYKTCKQSGTCPSDVVNKVEGTTLADKILQWSSLGIFLG
GLGIGTGSGTGGRTGYIPLGGRSNTVVDVGPTRPPVVIEPVGPTDPSIVTLIEDSSVV
TSGAPRPTFTGTSGFDITSAGTTTPAVLDITPSSTSVSISTTNFTNPAFSDPSIIEVPQTG
EVSGNVFVGTPTSGTHGYEEIPLQTFASSGTGEEPISSTPLPTVRRVTGPRLYSRAYQ
QVSVANPEFLTRPSSLITYDNPAFEPMDTTLTFEPRSNVPDSDFMDIIRLHRPASTSRR10
GTVRFSRLGQRATMFTRSGTQIGARVHFYHDISPIAPSPEYIELQPLVSATEDNGLFD
IYADDIDPALPVPSRPTTSSAVSTYSPTISSASSYSNVTVPLTSSWDVPVYTGPDITLP
STTSVWPIVSPTDPASTQYIGIHGTHYYLWPLYYFIPKKRKRVPYFFADGFVAA (SEQ
ID NO: 361)
15
MRSKRSTKRTKRASATQLYQTCKAAGTCPSDVIPKIEHTTIADQILRYGSMGVFFG
GLGIGSGSGTGGRTGYVPLSTRPSTVSEASIPIRPPVSIDPVGPLDPSIVSLVEESGIVD
VGAPAPIPHPPTTSGFDIATTADTTPAILDVTSVSTHENPTFTDPSVLQPPTPAETSGH
LLLSSSSISTHNYEEIPMDTFIVSTNNENITSSTPIPGVRRPARLGLYSKATQQVKVID
PTFLSAPKQLITYENPAYETVNAEESLYFSNTSHNIAPDPDFLDIIALHRPALTSRRNT20
VRYSRLGNKQTLRTRSGATIGARVHYYYDISSINPAGESIEMQPLGASATTTSTLND
GLYDIYADTDFTVDTPATHNVSPSTAVQSTSAVSAYVPTNTTVPLSTGFDIPIFSGPD
VPIEHAPTQVFPFPLAPTTPQVSIFVDGGDFYLHPSYYMLKRRRKRVSYFFTDVSVA
A (SEQ ID NO: 362)
25
MRHKRSTRRKRASATQLYQTCKATGTCPPDVIPKVEGSTIADQILKYGSLGVFFGG
LGIGTGSGSGGRTGYVPIGTDPPTAAIPLQPIRPPVTVDTVGPLDSSIVSLIEETSFIEA
GAPAPSIPTPSGFDVTTSADTTPAIINVSSVGESSIQTISTHLNPTFTEPSVLHPPAPAE
ASGHFIFSSPTVSTQSYENIPMDTFVVSTDSSNVTSSTPIPGSRPVARLGLYSRNTQQ
VKVVDPAFLTSPHKLITYDNPAFESFDPEDTLQFQHSDISPAPDPDFLDIIALHRPAIT30
SRRHTVRFSRVGQKATLKTRSGKQIGARIHYYQDLSPIVPLDHTVPNEQYELQPLH
DTSTSSYSINDGLYDVYADDVDNVHTPMQHSYSTFATTRTSNVSIPLNTGFDTPVM
55
SGPDIPSPLFPTSSPFVPISPFFPFDTIVVDGADFVLHPSYFILRRRRKRFPYFFTDVRV
AA (SEQ ID NO: 363)
MVSHRAARRKRASATDLYKTCKQSGTCPPDVINKVEGTTLADRILQWSSLGIFLGG
LGIGTGSGSGGRTGYVPLGGRSNTVVDVGPTRPPVVIDPVGPTDPSIVTLVEESSVV5
SSGAPVPTFTGTSGFEITSSGTTTPAVLDITPTVDSVSISSTSFTNPAFSDPSIIEVPQTG
EVSGNIFVGTPTSGSHGYEEIPLQTFASSGSGTEPISSTPLPTVRRVAGPRLYSRANQ
QVRVSTSQFLTRPSSLVTFDNPAYEPLDTTLSFEPTSNVPDSDFMDIIRLHRPALSSRR
GTVRFSRLGQRATMFTRSGKQIGGRVHFYHDISPIAATEEIELQPLLSATDDSDLFD
VYADFPPPASTTPSTINKSFTYPKYSLTMPSTAASSYSNVTVPLTSAWDVPIYTGPDII10
LPSHTPMWPSTSPTNAATSTYIGIHGTQYYLWPWYYYFPKKRKRIPYFFADGFVAA
(SEQ ID NO: 364)
MRYRRSTRHKRASATQLYQTCKASGTCPPDVIPKVEGTTIADQLLKYGSLGVFFGG
LGIGTGAGSGGRAGYVPLSTRPPTSSITTSTIRPPVTVEPIGPLEPSIVSMIEETTFIES15
GAPAPSIPSATGFDVTTSANNTPAIINVTSIGESSVQSVSTHLNPTFTEPSIIQPPAPAE
ASGHVLFSSPTISTHTYEEIPMDTFVTSTDSSSVTSSTPIPGSRPTTRLGLYSRATQQV
KVVDPAFMSSPQKLVTYNNPVFEGVDTDETIIFDRSQLLPAPDPDFLDIIALHRPALT
SRRGTVRFSRLGNKATLRTRSGKQIGARVHYYHDISPIQPAEVQEDIELQPLLPQSV
SPYTINDGLYDVYADSLQQPTFHLPSTLSTHNNTFTVPINSGIDFVYQPTMSIESGPD20
IPLPSLPTHTPFVPIAPTAPSTSIIVDGTDFILHPSYFLLRRRRKRFPYFFTDVRVAA
(SEQ ID NO: 365)
MRHKRSTRRKRASATQLYQTCKASGTCPPDVIPKVEGTTIADQILRYGSLGVFFGG
LGIGTGSGTGGRTGYVPLGSTPPSEAIPLQPIRPPVTVDTVGPLDSSIVSLIEESSFIDA25
GAPAPSIPTPSGFDITTSADTTPAILNVSSIGESSIQTVSTHLNPSFTEPSVLRPPAPAEA
SGHLIFSSPTVSTHSYENIPMDTFVISTDSGNVTSSTPIPGSRPVARLGLYSRNTQQV
KVVDPAFLTSPHRLVTYDNPAFEGFNPEDTLQFQHSDISPAPDPDFLDIVALHRPALT
SRRGTVRYSRVGQKATLRTRSGKQIGAKVHYYQDLSPIQPVQEQVQQQQQFELQS
LNTSVSPYSINDGLYDIYADDADTIHDFQSPLHSHTSFATTRTSNVSIPLNTGFDTPLV30
SLEPGPDIASSVTSMSSPFIPISPLTPFNTIIVDGADFMLHPSYFILRRRRKRFPYFFAD
VRVAA (SEQ ID NO: 366)
56
KRAAPKDIYPSCKISNTCPPDIQNKIEHTTIADKILQYGSLGVFLGGLGIGTARGSGG
RIGYTPLGEGGGVRVATRPT (SEQ ID NO: 367)
KRASATQLYQTCKLTGTCPPDVIPKVEHNTIADQILKWGSLGVFFGGLGIGTGSGT
GGRTGYVPLGTSAKPSITSGPM (SEQ ID NO: 368)5
KRDSVTHIYQTCKQAGTCPPDVINKVEQTTVADNILKYGSAGVFFGGLGISTGRGT
GGATGYVPLGEGPGVRVGGTPT (SEQ ID NO: 369)
SATQLYQTCKAAGTCPSDVIPKIEHTTIADQILRYGSMGVFFGGLGIGSGSGTGGRT10
GYVPLSTRPSTVSEASIPRA (SEQ ID NO: 370)
MAHSRARRRKRASATQLYQTCKLTGTCPPDVIPKVEHNTIADQILKWGSLGVFFG
GLGIGTGSGTGGRTGYVPLGTSAKPSITSGPMARPPVVVEPVAPS (SEQ ID NO: 371)
15
MKPRARRRKRASATQLYQTCKATGTCPPDVIPKVEHTTIADQILKWGSLGVFFGG
LGIGTGAGSGGRAGYIPLGSSPKPAITGGPAARPPVLVEPVAPSD (SEQ ID NO: 372)
MRHKRSAKRTKRASATQLYKTCKQAGTCPPDIIPKVEGKTIADQILQYGSMGVFF
GGLGIGTGSGTGGRTGYIPLGTRPPTATDTLAPVRPPLTVDPVGP (SEQ ID NO: 373)20
MVSHRAARRKRASVTDLYKTCKQSGTCPSDVVNKVEGTTLADKILQWSSLGIFLG
GLGIGTGSGTGGRTGYIPLGGRSNTVVDVGPTRPPVVIEPVGPTD (SEQ ID NO: 374)
MRSKRSTKRTKRASATQLYQTCKAAGTCPSDVIPKIEHTTIADQILRYGSMGVFFG25
GLGIGSGSGTGGRTGYVPLSTRPSTVSEASIPIRPPVSIDPVGP (SEQ ID NO: 375)
MRHKRSTRRKRASATQLYQTCKATGTCPPDVIPKVEGSTIADQILKYGSLGVFFGG
LGIGTGSGSGGRTGYVPIGTDPPTAAIPLQPIRPPVTVDTVGPL (SEQ ID NO: 376)
30
MVSHRAARRKRASATDLYKTCKQSGTCPPDVINKVEGTTLADRILQWSSLGIFLG
GLGIGTGSGSGGRTGYVPLGGRSNTVVDVGPTRPPVVIDPVGPTD (SEQ ID NO:
377)
MRYRRSTRHKRASATQLYQTCKASGTCPPDVIPKVEGTTIADQLLKYGSLGVFFG35
GLGIGTGAGSGGRAGYVPLSTRPPTSSITTSTIRPPVTVEPIGPL (SEQ ID NO: 378)
MRHKRSTRRKRASATQLYQTCKASGTCPPDVIPKVEGTTIADQILRYGSLGVFFGG
LGIGTGSGTGGRTGYVPLGSTPPSEAIPLQPIRPPVTVDTVGPL (SEQ ID NO: 379)
40
MAHSRARRRKRASATQLYQTCKLTGTCPPDVIPKVEHNTIADQILKWGSLGVFFG
GLGIGTGSGTGGRTGYVPLGTSAKPSITSGPMARPPVVVEPVAPSDPSIVSLIEESAII
NAGAPEIVPPAHGGFTITSSETTTPAILDVSVTSHTTTSIFRNPVFTEPSVTQPQPPVE
ANGHILISAPTITSHPIEEIPLDTFVI (SEQ ID NO: 380)
45
MKPRARRRKRASATQLYQTCKATGTCPPDVIPKVEHTTIADQILKWGSLGVFFGG
LGIGTGAGSGGRAGYIPLGSSPKPAITGGPAARPPVLVEPVAPSDPSIVSLIEESAIIN
AGAPEVVPPTQGGFTITSSESTTPAILDVSVTNHTTTSVFQNPLFTEPSVIQPQPPVE
ANGHILISAPTITSQHVEDIPLDTFVVS (SEQ ID NO: 381)
50
57
MRHKRSAKRTKRASATQLYKTCKQAGTCPPDIIPKVEGKTIADQILQYGSMGVFF
GGLGIGTGSGTGGRTGYIPLGTRPPTATDTLAPVRPPLTVDPVGPSDPSIVSLVEETS
FIDAGAPTPVPSIPPDVSGFSITTSTDTTPAILDINNTVTTVTTHNNPTFTDPSVLQPP
TPAETGGHFTLSSSTISTHNYEEIPMDT (SEQ ID NO: 382)
5
MVSHRAARRKRASVTDLYKTCKQSGTCPSDVVNKVEGTTLADKILQWSSLGIFLG
GLGIGTGSGTGGRTGYIPLGGRSNTVVDVGPTRPPVVIEPVGPTDPSIVTLIEDSSVV
TSGAPRPTFTGTSGFDITSAGTTTPAVLDITPSSTSVSISTTNFTNPAFSDPSIIEVPQT
GEVSGNVFVGTPTSGTHGYEEIPLQTF (SEQ ID NO: 383)
10
MRSKRSTKRTKRASATQLYQTCKAAGTCPSDVIPKIEHTTIADQILRYGSMGVFFG
GLGIGSGSGTGGRTGYVPLSTRPSTVSEASIPIRPPVSIDPVGPLDPSIVSLVEESGIV
DVGAPAPIPHPPTTSGFDIATTADTTPAILDVTSVSTHENPTFTDPSVLQPPTPAETS
GHLLLSSSSISTHNYEEIPMDTFIVST (SEQ ID NO: 384)
15
MRHKRSTRRKRASATQLYQTCKATGTCPPDVIPKVEGSTIADQILKYGSLGVFFGG
LGIGTGSGSGGRTGYVPIGTDPPTAAIPLQPIRPPVTVDTVGPLDSSIVSLIEETSFIEA
GAPAPSIPTPSGFDVTTSADTTPAIINVSSVGESSIQTISTHLNPTFTEPSVLHPPAPAE
ASGHFIFSSPTVSTQSYENIPMDT (SEQ ID NO: 385)
20
MVSHRAARRKRASATDLYKTCKQSGTCPPDVINKVEGTTLADRILQWSSLGIFLG
GLGIGTGSGSGGRTGYVPLGGRSNTVVDVGPTRPPVVIDPVGPTDPSIVTLVEESSV
VSSGAPVPTFTGTSGFEITSSGTTTPAVLDITPTVDSVSISSTSFTNPAFSDPSIIEVPQ
TGEVSGNIFVGTPTSGSHGYEEIPLQTF (SEQ ID NO: 386)
25
MRYRRSTRHKRASATQLYQTCKASGTCPPDVIPKVEGTTIADQLLKYGSLGVFFG
GLGIGTGAGSGGRAGYVPLSTRPPTSSITTSTIRPPVTVEPIGPLEPSIVSMIEETTFIE
SGAPAPSIPSATGFDVTTSANNTPAIINVTSIGESSVQSVSTHLNPTFTEPSIIQPPAPA
EASGHVLFSSPTISTHTYEEIPMDT (SEQ ID NO: 387)
30
MRHKRSTRRKRASATQLYQTCKASGTCPPDVIPKVEGTTIADQILRYGSLGVFFGG
LGIGTGSGTGGRTGYVPLGSTPPSEAIPLQPIRPPVTVDTVGPLDSSIVSLIEESSFIDA
GAPAPSIPTPSGFDITTSADTTPAILNVSSIGESSIQTVSTHLNPSFTEPSVLRPPAPAE
ASGHLIFSSPTVSTHSYENIPMDT (SEQ ID NO: 388)
35
In some embodiments, the target peptide shares at least 70 % identity with the
sequences described herein above, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the
art would be able to deduce all possible DNA or RNA sequences that encodes the above40
target peptides. Such DNA or RNA sequences are deemed to be incorporated in this
disclosure. In some embodiments, the target peptide is encoded by a target sequence which
may be either a DNA, an RNA or an mRNA.
Self-assembling sequence and self-assembling peptide45
58
The multitarget nucleic acid sequence and multitarget peptide includes self-
assembling sequence and self-assembling peptide respectively. The self-assembling
sequence comprises of a sequence of nucleotides, either deoxyribonucleotides or
ribonucleotides, that encodes a self-assembling peptide. The self-assembling sequence
includes codon optimized sequences, fragments, mutants, variants or a combination thereof.5
In some embodiments, the self-assembling sequence is a DNA or an RNA or an mRNA.
Any self-assembling peptide that is capable of self-assembling into a polypeptide
nanoparticle can be employed in accordance with the present disclosure.
In some embodiments self-assembling peptide may be a full-length protein or its
fragment, mutants, or variant thereof.10
In some embodiments, the self-assembling peptide includes, but not limited to,
lumazine synthase from Aquifex species, hepatitis B surface antigen (HBsAg) from
Hepatitis B Virus, hepatitis B core antigen (HBcAg) from Hepatitis B virus, human
papillomavirus L1 (HPV L1) protein, matrix protein M1 from influenza A virus, ferritin,
riboflavin synthase, or a combination thereof, including their codon optimized nucleic acid15
sequences, fragments, mutants, or variants thereof.
In some embodiments, the self-assembling peptide is a ferritin peptide.
Ferritin is one of the ubiquitous proteins found in nature. It is produced by all living
organisms including archaea, bacteria, algae, higher plants, and animals. Each ferritin
protein is generally composed of 24 subunits or peptides which self-assembles into a ferritin20
nanoparticle.
In some aspects, the multitarget nucleic acid sequence and multitarget peptide
includes ferritin sequence and ferritin peptide respectively. The ferritin sequence comprises
of a sequence of nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes a
ferritin peptide. In some embodiments, the ferritin sequence is a DNA or an RNA or an25
mRNA.
Any ferritin peptide that is capable of self-assembling into a nanoparticle can be
employed in accordance with the present disclosure. In some embodiments, the ferritin
peptide is obtained or derived from Helicobacter pylori ferritin, including their codon
optimized nucleic acid sequences, fragments, mutants, or variants thereof.30
Exemplary self-assembling peptides includes, but not limited to, the ones
represented by the following amino acid sequences, or a combination thereof, including
codon optimized sequences, fragments, mutants, or variants thereof:
59
LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAK
KLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDH
ATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGI (SEQ ID NO:
331);
LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAK5
KLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDH
ATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIRRKR (SEQ
ID NO: 333);
LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAK
KLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDH10
ATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS
(SEQ ID NO: 254);
MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSW
EIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVI15
TADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR (SEQ ID NO: 255);
MTKKVGIVDTTFARVDMASIAIKKLKELSPNIKIIRKTVPGIKDLPVACKKLLEEEG
CDIVMALGMPGKAEKDKVCAHEASLGLMLAQLMTNKHIIEVFVHEDEAKDDKEL
DWLAKRRAEEHAENVYYLLFKPEYLTRMAGKGLRQGFEDAGPARE (SEQ ID NO:20
256);
MTEKEKMLAEKWYDANFDQYLINERARAKDICFELNHTRPSATNKRKELIDQLFQ
TTTDNVSISIPFDTDYGWNVKLGKNVYVNTNCYFMDGGQITIGDNVFIGPNCGFY
TATHPLNFHHRNEGFEKAGPIHIGSNTWFGGHVAVLPGVTIGEGSVIGAGSVVTKDI25
PPHSLAVGNPCKVVRKIDNDLPSETLNDETIK (SEQ ID NO: 257);
MENTTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFQGGAPTCPGQNSQSPT
SNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLLPGTSTT
GTGPCRTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSWL30
SLLVPFVQWFAGLSPTVWLSVIWMMWYRGPSLYNTLSPFLPLLPISFCLWVYI (SEQ
ID NO: 258);
MIFVLGGCRHKLVCSPAPCNFFHLCLIISCSCPTVHASKLCLGWLWGMHIDPYKEF
GASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELM35
60
NLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSF
GVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRE
SQC (SEQ ID NO: 259);
MSLWLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFPIKKPN5
NNKILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQP
LGVGISGHPLLNKLDDTENASAYAANAGVDNRECISMDYKQTQLCLIGCKPPIGEH
WGKGSPCTNVAVNPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQANKSEVPLD
ICTSICKYPDYIKMVSEPYGDSLFFYLRREQMFVRHLFNRAGAVGENVPDDLYIKG
SGSTANLASSNYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTVV10
DTTRSTNMSLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTADVMTYI
HSMNSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPKEDPLKKYTFW
EVNLKEKFSADLDQFPLGRKFLLQAGLKAKPKFTLGKRKATPTTSSTSTTAKRKKR
KL (SEQ ID NO: 260);
15
MSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEALMEWLKTRPILSPLTK
GILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDRAVKLYRKLKREITFHGA
KEIALSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQMVT
TTNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMEVASQARQMVQAMRAIG
THPRSSAGLKDDLLENLQAYQKRMGVQMQRFK (SEQ ID NO: 261);20
In some embodiments, the self-assembling peptide shares at least 70 % identity with
the sequences described herein above, or a combination thereof, including their codon
optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the self-assembling peptides, a person
skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes25
the above self-assembling peptides. Such DNA or RNA sequences are deemed to be
incorporated in this disclosure. In some embodiments, the self-assembling peptide is
encoded by the self-assembling sequence which may be either a DNA, an RNA or an mRNA.
Linker sequence and linker peptide30
The multitarget nucleic acid sequence and multitarget peptide includes linker
sequence and linker peptide respectively. The linker sequence comprises of a sequence of
nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes a linker peptide.
The linker sequence includes codon optimized sequences, fragments, mutants, variants, or
61
combination thereof. In some embodiments, the linker sequence is a DNA or an RNA or an
mRNA.
In some embodiments, the linker peptide connects the target peptide with the self-
assembling peptide in a polypeptide. In some embodiments, the linker peptide connects the
signal peptide with a polypeptide. In some other embodiments, the linker peptide connects5
the signal sequence with the first polypeptide. In some embodiments, one linker peptide
connects the cleavage peptide with the target peptide and another linker peptide connects
the target peptide with the self-assembling peptide in a polypeptide. In some embodiments,
the linker peptide connects two cleavage peptides. Any suitable linker peptides can be
employed in accordance with the present disclosure.10
In some embodiments, linker peptide is an amino acid linker, a foldon, a scaffold or
a combination thereof. In some embodiments, linker peptide consists of a combination of an
amino acid linker and a foldon. In some embodiments, linker peptide consists of a
combination of an amino acid linker and a scaffold. In some embodiments, linker peptide
consists of a combination of a foldon and scaffold. In some embodiments, linker peptide15
consists of a combination of an amino acid linker, a foldon, and a scaffold.
In some embodiments, the amino acid linker comprises of about 2-49 amino acids,
2-40 amino acids, 2-40 amino acids, 2-20 amino acids, 2-15 amino acids, or 2-10 amino
acids. In some embodiments, the amino acid linker may comprise a glycine serine linker, a
glycine proline linker, a glycine threonine linker, an alanine serine linker etc.20
The glycine proline linker comprises of glycine (G) and proline (P) amino acids
consecutively without any preference of order of appearance of either glycine or proline. In
some embodiments, the glycine proline linker is 2-49 amino acids in length.
The glycine threonine linker comprises of glycine (G) and threonine (T) amino acids25
consecutively without any preference of order of appearance of either glycine or threonine.
In some embodiments, the glycine threonine linker is 2-49 amino acids in length.
The alanine serine linker comprises of alanine (A) and serine (S) amino acids
consecutively without any preference of order of appearance of either alanine or serine. In
some embodiments, the alanine serine linker is 2-49 amino acids in length.30
The glycine serine linker comprises of glycine (G) and serine (Serine) amino acids
consecutively without any preference of order of appearance of either glycine or serine. In
some embodiments, the glycine serine linker is 2-49 amino acids in length.
62
Exemplary glycine serine linkers includes, but not limited to, the ones represented
by the following amino acid sequences, or their combinations, including their codon
optimized nucleic acid sequences, fragments, mutants, or variants thereof:
GSG (SEQ ID NO: 350)
GSGG (SEQ ID NO: 330);5
GGSGG (SEQ ID NO: 262);
GGSGGGGSGG (SEQ ID NO: 263);
GGSGGGGSGGGGSGG (SEQ ID NO: 264);
SGGSGG (SEQ ID NO: 265);
GGGGSGGGGS (SEQ ID NO: 266);10
GGGGSGGGGSGGGGS (SEQ ID NO: 267);
In some embodiments, the glycine serine linkers share at least 70 % identity with the
sequences described herein above, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the glycine serine linkers, a person skilled15
in the art would be able to deduce all possible DNA or RNA sequences that encodes the
above glycine serine linkers. Such DNA or RNA sequences are deemed to be incorporated
in this disclosure. In some embodiments, the glycine serine linker is encoded by a glycine
serine linker sequence which may be either a DNA, an RNA or an mRNA.
20
In some embodiments, the linker peptide is a foldon. A foldon comprises of a
sequence of amino acids encoded by a foldon sequence. The foldon sequence includes codon
optimized sequences, fragments, mutants, variants or a combination thereof. A foldon
enables two or more homologous polypeptides to organise to form an oligomeric complex.
In some embodiments, foldon also helps in orientation of a polypeptide such that the25
domains or epitopes on the target peptide are exposed or displayed for interaction or
communication with cells or biomolecules or immune system.
Exemplary foldons includes, but not limited to, the ones represented by the following
amino acid sequences, or a combination thereof, including their codon optimized nucleic
acid sequences, fragments, mutants, or variants thereof:30
YIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 268);
HENEISHHAKEIERLQKEIERHKQSIKKLKQSE (SEQ ID NO: 269);
63
In some embodiments, the foldon shares at least 70 % identity with the sequences
described herein above, or a combination thereof, including their codon optimized nucleic
acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the foldons, a person skilled in the art
would be able to deduce all possible DNA or RNA sequences that encodes the above foldons.5
Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some
embodiments, the foldon is encoded by a foldon sequence which may be either a DNA, an
RNA or an mRNA.
In some embodiments, the linker peptide is a scaffold. A scaffold comprises of a10
sequence of amino acids encoded by a scaffold sequence. The scaffold sequence includes
codon optimized sequences, fragments, mutants, variants, or combination thereof. A scaffold
provides structural and/or functional integrity or support to the target peptide and may also
helps in orientation of target peptide such that the domains or epitopes of the target peptide
are exposed or displayed for interaction or communication with cells or biomolecules or15
immune system.
Exemplary scaffolds includes, but not limited to, the ones represented by the
following amino acid sequences, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof:
VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDA20
QAPK (SEQ ID NO: 270);
QDSTSDLIPAPPLSKVPLQQNFQDNQFHGKWYVVGKAGNHDLREDKDPRKMQAT
IYELKEDKSYNVTNVRFVHKKCNYRIWTFVPGSQPGEFTLGNIKSWPGLTSWLVR
VVSTNYNQHAMVFFKRVYQNRELFEITLYGRTKELTNELKENFIRFSKSLGLPENHI
VFPVPIDQCIDGSAWSHPQFEK (SEQ ID NO: 271);25
VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTA
TISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 272);
GCPRILMRCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 273);
MRGSHHHHHHGSDLGKKLLEAARAGQDDEVRILMANGADVNATDNDGYTPLHL
AASNGHLEIVEVLLKNGADVNASDLTGITPLHLAAATGHLEIVEVLLKHGADVNA30
YDNDGHTPLHLAAKYGHLEIVEVLLKHGADVNAQDKFGKTAFDISIDNGNEDLAE
ILQ (SEQ ID NO: 274);
MRGSHHHHHHGSVKVKFFWNGEEKEVDTSKIVWVKRAGKSVLFIYDDNGKNGY
GDVTEKDAPKELLDMLARAEREKKL (SEQ ID NO: 275);
64
MLPAPKNLVVSEVTEDSARLSWDDPAAFYESFLIQYQESEKVGEAIVLTVPGSERS
YDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGHHHHHH (SEQ ID
NO: 276);
ETDICKLPKDEGTCRDFILKWYYDPNTKSCARFWYGGCGGNENKFGSQKECEKV
CAPV (SEQ ID NO: 277);5
MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVVAGTNYYIK
VRAGDNKYMHLKVFKSLPGQNEDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 278);
PCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCA (SEQ ID NO: 279);
MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSD
YNIQKESTLHLVLRLRGG (SEQ ID NO: 280);10
MGSIIFLEDRAFQGRIYGCTTDCPNLQPYFSRCNSIVVQSGCWMIYERPNYQGHQY
FLRRGEYPDYQQWMGLSDSIRSCCLIPPHSGAYRMKIYDRDELRGQMSELTDDCL
SVQDRFHLTEIHSLNVLEGSWILYEMPNYRGRQYLLRPGEYRRFLDWGAPNAKVG
SLRRVMDLYLEHHHHHH (SEQ ID NO: 281);
15
AGHRIAWLLMMGHPRQQLAIIFGIGVSTLYRYFPA (SEQ ID NO: 282);
AFSKSEEARHSSLERECIEEICDHAEAWDIMM (SEQ ID NO: 283);
RECDYCGTDIEPGTGGMAVHGDGATTHFCSHRCAWDAMMGAEARNLEWTDTAR
(SEQ ID NO: 284);
CSQNEYFDSLLHACIPCQLRCSGAPHRCAWDCMM (SEQ ID NO: 285);20
EHIPGTLAARLSHRAAWDLMMHSLDASQGTATGPRGIFTAEDALKLVQLKQTGK;
TFPTYKCGHRFAWDCMMGSGLNGAACFAVKIADLPVYSCECAIGFMGQRCEYKE
(SEQ ID NO: 286);
ACYGHRCAWDCMMLGFSSGKCINSKCKCYK (SEQ ID NO: 287);
GEYVVEKVLDKRVVKGKVEYLLKWKGFSDEDNTWEPDENLDGHRLAWDFMM;25
ADVYEVEAILADRVNKNGINEYYIKWAGYDWYDNTWEPEQNLFGAGHRLAWW
MMR (SEQ ID NO: 288);
AGTIKITQTRSAIGRLPAHKATLLGLGLRRIGHTVEREDGHRIAWDIMMVSFMVKV
EG (SEQ ID NO: 289);
30
GIPCGESCGSPCISSAIGCSCKLINTNGSWHIVCYRN (SEQ ID NO: 290);
GKCPETFDAWYCLNDAHCFAVLINTNGSWHIVYSCECAIGFMGQRCEYKE (SEQ
ID NO: 291);
65
QEEADRTVFVGNLEARVREEILYELFLQAGPLTKVTICKDREGKPKSFGFVCFKHPE
SVSYAIALAGLINLNGSWIIVSGPSSG (SEQ ID NO: 292);
NEEDAGKMFVGGLSWDTSKKDLKDYFTKFGEVVDCTIKMDPNTGRSRGFGFILF
KDAASVEKVLDAGLHNLNGSWIIPKKA (SEQ ID NO: 293);
SGNIFIKNLDKSIDNKALYDTFSAFGNILSCKVVCDEQGSKGYGFVHFETQEAAER5
AIAKMGLMNLNGSWVIVGRFKSRKE (SEQ ID NO: 294);
PSRVVYLGSIPYDQTEEQILDLCSNVGPVINLKMMFDPQTGRSKGYAFIEFRDLESS
ASAVGALGLYNLNGSWLICGYSSNSDISGVSLEHHHH (SEQ ID NO: 295);
LAILVFGYPETMANQVIAYFQEFGTILEDFEVLRKPQAMTVGLQDRQFVPIFSGNS
WTKITYDNPASAVDALAEGLANFNGSWLLVIPYTKDAVERLQ (SEQ ID NO: 296);10
RLVNCNGSWLIGLDRPPYPGAKGEDIYNNVSRKAWDEWQKHQTMLINERRLNM
MNAEDRKFLQQEMDKFLSGEDY (SEQ ID NO: 297);
FAVESIEKLRNRNGSWEILVKWRGWSPKYNTWEPEENIG (SEQ ID NO: 298);
MRDFFVITNSLYNFNGSWYIKGAVLHVSPTQKRAFWVIADQENFIKQVNKNIEYVE
KQASPAFLQRIVEIYQVKFEGKNVG (SEQ ID NO: 299);15
In some embodiments, the scaffold peptide shares at least 70 % identity with the
sequences described herein above, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the scaffold peptides, a person skilled in
the art would be able to deduce all possible DNA or RNA sequences that encodes the above20
scaffold peptides. Such DNA or RNA sequences are deemed to be incorporated in this
disclosure. In some embodiments, the target peptide is encoded by the target sequence which
may be either a DNA, an RNA, or an mRNA.
In some embodiments, the linker peptide consists of a glycine serine linker followed25
by a foldon. In another embodiment, the linker peptide consists of a foldon followed by a
glycine serine linker. In some embodiments, the linker peptide consists of a glycine serine
linker followed by a foldon and another glycine serine linker. In some embodiments, the
linker peptide consists of a foldon followed by a glycine serine linker and another foldon.
30
Cleavage sequence and cleavage peptide
The multitarget nucleic acid sequence and multitarget peptide includes cleavage
sequence and cleavage peptide respectively. The cleavage sequence comprises of a sequence
of nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes a cleavage
66
peptide. The cleavage sequence includes codon optimized sequences, fragments, mutants,
variants or a combination thereof. In some embodiments, the cleavage sequence is a DNA
or an RNA or an mRNA.
The cleavage peptide connects one polypeptide with another polypeptide, for
example, the adjacent polypeptide. The cleavage peptide carries one or more cleavage sites.5
In some embodiments, the cleavage peptide comprises one or more cleavage peptides, for
example cleavage peptide-1, cleavage peptide-2 and so on. In some embodiments, the
cleavage peptide optionally comprises a linker peptide between two cleavage peptides.
In some embodiments, the cleavage peptide facilitates the action of cellular proteases
to cleave the multitarget polypeptide into individual polypeptides or self cleaves into10
individual polypeptides. In some embodiments, the resulting polypeptides may comprise
either a target peptide, a linker peptide and a self-assembling peptide or a linker peptide,
target peptide, linker peptide and a self-assembling peptide or a combination thereof. In
some embodiments, the polypeptide, in addition to these peptides, may also have some
residues (amino acids) of cleavage peptide. Any cleavage peptide that is susceptible to the15
action of cellular proteases or a cleavage peptide that has the ability to undergo self-cleavage
can be employed in accordance with the present disclosure.
In some embodiments, the cleavage peptide is a golgi specific cleavage peptide i.e.,
susceptible to action of golgi specific proteases.
Exemplary cleavage peptide includes, but not limited to, the one represented by the20
following amino acid sequence, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof:
RRKRSVS (SEQ ID NO: 300);
GIRRKRSVSH (SEQ ID NO: 301);
VQREKRAVGI (SEQ ID NO: 302);25
SIRHKREPSV (SEQ ID NO: 303);
KRRQRRRPPQ (SEQ ID NO: 304);
KIRRRRDVVD (SEQ ID NO: 305);
HNRTKRSTDG (SEQ ID NO: 306);
RKRRKRELET (SEQ ID NO: 307);30
THRTRRSTSD (SEQ ID NO: 308);
SRRKRRSAST (SEQ ID NO: 309);
67
NLRRRRDLVD (SEQ ID NO: 310);
LRRRRRDAGN (SEQ ID NO: 311);
ATNFSLLKQAGDVEENPGP (SEQ ID NO: 347)
EGRGSLLTCGDVEENPGP (SEQ ID NO: 348)
QCTNYALLKLAGDVESNPGP (SEQ ID NO: 349)5
In some embodiments, the cleavage peptide shares at least 70 % identity with the
sequence described herein above, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the cleavage peptide, a person skilled in10
the art would be able to deduce all possible DNA or RNA sequences that encodes the above
cleavage peptide. Such DNA or RNA sequences are deemed to be incorporated in this
disclosure. In some embodiments, the cleavage peptide is encoded by a cleavage sequence
which may be either a DNA, an RNA or an mRNA.
15
Signal sequence and signal peptide
The multitarget nucleic acid sequence and multitarget peptide includes signal
sequence and signal peptide respectively. The signal sequence comprises of a sequence of
nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes a signal peptide.
The signal sequence includes codon optimized sequences, fragments, mutants, variants, or20
a combination thereof. In some embodiments, the signal sequence is a DNA or an RNA or
an mRNA.
The signal peptide is present upstream (N-terminus or amino-terminus) of one or
more polypeptides. In some embodiments, the signal peptide may be present on the N-
terminus of all or some polypeptides. In some embodiments, the signal peptide is present on25
the N-terminus of the first polypeptide. In some embodiments, the signal peptide is present
upstream (N-terminus) of some polypeptides. In some embodiments, the signal peptide is
present upstream (N-terminus) of each of the polypeptides.
In some embodiments, the signal peptide transports the multitarget peptide to cell
organelles. In some embodiments, the signal peptide transports the multitarget peptide to30
golgi body or golgi apparatus. Any signal peptide that transports the multitarget peptide to
golgi bodies can be employed in accordance with the present disclosure. In some
embodiments, the signal peptide is a golgi targeting signal peptide i.e., directs the multitarget
peptide to golgi complex.
68
Exemplary signal peptide includes, but not limited to, the one represented by the
following amino acid sequence, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof:
MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAA (SEQ ID NO: 312);
MDMRAPAGIFGFLLVLFPGYRS (SEQ ID NO: 313);5
MKWVTFISLLFLFSSAYS (SEQ ID NO: 314);
MDWTWRVFCLLAVTPGAHP (SEQ ID NO: 315);
MAWSPLFLTLITHCAGSWA (SEQ ID NO: 316);
MTRLTVLALLAGLLASSRA (SEQ ID NO: 317);
MARPLCTLLLLMATLAGALA (SEQ ID NO: 318);10
MRSLVFVLLIGAAFA (SEQ ID NO: 319);
MSRLFVFILIALFLSAIIDVMS (SEQ ID NO: 320);
MGMRMMFIMFMLVVLATTVVS (SEQ ID NO: 321);
MRAFLFLTACISLPGVFG (SEQ ID NO: 322);
MKFQSTLLLAAAAGSALA (SEQ ID NO: 323);15
MASSLYSFLLALSIVYIFVAPTHS (SEQ ID NO: 324);
MKTHYSSAILPILTLFVFLSINPSHG (SEQ ID NO: 325);
MESVSSLFNIFSTIMVNYKSLVLALLSVSNLKYARG (SEQ ID NO: 326);
MKAAQILTASIVSLLPIYTSA (SEQ ID NO: 327);
MIKLKFGVFFTVLLSSAYA (SEQ ID NO: 328);20
MGVKVLFALICIAVAEA (SEQ ID NO: 329);
In some embodiments, the signal peptide shares at least 70 % identity with the
sequence described herein above, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.25
Given the above amino acid sequences of the signal peptide, a person skilled in the
art would be able to deduce all possible DNA or RNA sequences that encodes the above
signal peptide. Such DNA or RNA sequences are deemed to be incorporated in this
disclosure. In some embodiments, the signal peptide is encoded by a signal sequence which
may be either a DNA, an RNA or an mRNA.30
Synthesis of multitarget nucleic acid sequences
Multitarget nucleic acid sequence according to the present disclosure can be either a
DNA or an RNA or an mRNA. The multitarget nucleic acid sequence as described herein
69
can be synthesized by molecular biology or genetic engineering techniques well known in
the art, for example, using recombinant expression system, chemical synthesis, or in vitro
transcription (IVT).
In some embodiments, the multitarget nucleic acid sequence is obtained through a
single IVT process. In some embodiments, the multitarget nucleic acid sequence obtained5
through single IVT process is an mRNA.
In some embodiments, the multitarget nucleic acid sequence is a messenger RNA
(mRNA). The mRNA encodes a multitarget peptide as described herein.
Typically, an mRNA includes at least a coding region (which encodes the multitarget
peptide), a 5’ UTR, a 3’ UTR, a 5’ cap and a 3’ poly(A) tail. UTR (untranslated regions)10
flanks the coding region or open reading frame (ORF). The 5’ UTR and the 3’ UTR are
sections of the mRNA before the start codon and after the stop codon respectively. The 5’
UTR has a cap (5’ cap) consisting of altered nucleotides. mRNA also contains a
polyadenylated region at its 3’ end having adenine nucleotides called poly(A) tail.
In some embodiments, the mRNA may be unmodified or modified or a combination15
of both. The modification may be in the nucleobase of the nucleotide, or sugar moiety of the
nucleotide, or the phosphate of the nucleotide. In some embodiments, unmodified mRNA
may comprise naturally occurring nucleosides, for example, adenosine, guanosine, cytidine,
and uridine. mRNA may comprise one or more modified nucleosides, for example,
adenosine analog, guanosine analog, cytidine analog, or uridine analog.20
In some embodiments, the one or more modified nucleosides is a nucleoside analog
selected from 2-aminoadenosine, 3-methyl adenosine, 7-deazaadenosine, 7-deazaguanosine,
8-oxoadenosine, or 8-oxoguanosine or a combination thereof.
In some embodiments, the one or more modified nucleosides is a uridine analog
selected from propynyl-uridine, pseudouridine, C5-bromouridine, C5-fluorouridine, C5-25
iodouridine, C5-propynyl-uridine, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-
thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 3-methyl-uridine, 5-
carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 1-methyl-3-(3-amino-3-
carboxypropyl)pseudouridine, 2-thio-2’-O-methyl-uridine, 5-methoxycarbonylmethyl-2’-
O-methyl-uridine, 5-carboxymethylaminomethyl-2’-O-methyl-uridine, 3,2’-O-dimethyl-30
uridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine, 1-
taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurino-4-thio-
pseudouridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-
70
pseudouridine, 1-methyl-1deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine,
dihydro-uridine, dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-dihydro-
pseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, or
4-methoxy-2-thio-pseudouridine or a combination thereof.
In some embodiments, the one or more modified nucleosides is a cytidine analog5
selected from 5-methylcytidine, C5-propynyl-cytidine, C5-methylcytidine,
pseudoisocytidine, 1-methyl-pseudoisocytidine, pyrrolo-pseudoisocytidine, 4-thio-
pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-
pseudoisocytidine, 1-methyl-1-1deaza-pseudoisocytidine, 4-methoxy-1-methyl-
pseudoisocytidine or a combination thereof.10
Methods for making modified nucleosides are well known in the art (WO
2020168466, US8278036; US8691966; US8748089; US8835108; US9750824;
US10232055; WO2007024708; WO2012135805; WO2013052523; WO2011012316)
In some embodiments, the modified nucleoside is pseudouridine, for example, 1-
methyl-pseudouridine, 1-propynyl-pseudouridine, 1-carboxymethyl-pseudouridine, 1-15
methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 4-methoxy-pseudouridine, or 4-
methoxy-2-thio-pseudouridine 4-thio-pseudouridine, 2-thio-pseudouridine, 4-thio-1-
methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, dihydro-pseudouridine or a
combination thereof.
In some embodiments, mRNA is produced using recombinant expression system,20
chemically synthesized, or obtained through in vitro transcription.
In some embodiments, the multitarget nucleic acid sequence is obtained through a
single IVT process.
mRNAs according to the present disclosure may be synthesized via in vitro
transcription (IVT). Briefly, IVT is typically performed with a DNA template containing a25
promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and
magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA
polymerase), DNase I, pyrophosphatase, and/or RNase inhibitor. The exact conditions may
vary according to the specific application. Methods of making mRNA through IVT reaction
is well known in the art (see for example, Beckert, Bertrand and Masquida, Benoit Methods30
in Molecular Biology (2011) 703, 29-41; Brunelle, Julie L. and Green Rachel Methods in
Enzymology (2013) 530, 101-114; Kamakaka, Rohinton T. and Kraus W. Lee Current
Protocols in Cell Biology (1999) 11.6.1-11.6.17; Kanwal, Fariha et al. Cellular Physiology
71
and Biochemistry (2018) 48:1915-1927; WO2018157153; WO2020185811;
WO2022082001).
In some embodiments, the in vitro transcription occurs in a single batch. In some
embodiments, IVT reaction includes capping and tailing reactions either co-transcriptionally
or separately. A cap analog is added to the in vitro transcription reaction and will be5
incorporated at the 5’ end of the mRNA during the reaction. Alternative method of capping
involves adding the cap post-transcriptionally through an enzymatic reaction. The poly (A)
tail can be incorporated into the DNA template sequence, and thus the poly (A) tail will be
incorporated into the mRNA by T7 RNA polymerase during the in vitro transcription.
Alternative method of tailing involves adding the poly (A) tail post-transcriptionally through10
an enzymatic reaction. In some embodiments, capping and tailing reactions are performed
co-transcriptionally i.e., during the IVT reaction. In some embodiments, capping and tailing
reactions are performed separately from IVT reaction i.e., post transcriptionally.
mRNA produced as a result of IVT reaction may be purified using techniques well
known in the art, such as, centrifugation, filtration and/or chromatographic techniques. The15
purification of mRNA may be accomplished before capping and tailing steps are performed
or after capping and tailing. The synthesized mRNA may be purified by ethanol precipitation
or filtration or chromatography methods. In some embodiments, tangential flow filtration is
used to purify mRNA. In some embodiments, mRNA is purified by chromatographic step.
In other embodiments, mRNA is purified by a combination of filtration and chromatography20
steps.
In some embodiments, a suitable mRNA sequence is an mRNA sequence encoding
a protein, peptide, polypeptide. In some embodiments, a suitable mRNA sequence is codon
optimized for efficient expression in a host cell or organism. Codon optimization typically
includes modifying a naturally-occurring or wild-type nucleic acid sequence encoding a25
peptide, polypeptide or protein to achieve the highest possible expression of peptide,
polypeptide, protein or an antibody without altering the amino acid sequence.
In some embodiments, the mRNA is circular. In other embodiments, the mRNA is
linear.
In some embodiments, the mRNA is self-amplifying or self-replicating.30
The multitarget nucleic acid sequence as described herein, express multitarget
peptide.
Polypeptide nanoparticle
72
The multitarget peptide comprises multiple repeats of polypeptide comprising either
a target peptide, a linker peptide, and a self-assembling peptide or a linker peptide, target
peptide, a linker peptide, and a self-assembling peptide, or a combination thereof,
interspersed with cleavage peptide (see illustration in figures). In some embodiments, the
total number of polypeptides present in a multitarget peptide may be up to 100 polypeptides.5
In some embodiments, one or more polypeptides in the multitarget peptide may have
identical target peptides (homologous polypeptides). In some embodiments, one or more
polypeptides in the multitarget peptide may have different target peptides (heterologous
polypeptides).
A multitarget peptide as described herein is encoded by a multitarget nucleic acid10
sequence as described herein. Each multitarget peptide comprises two or more polypeptides,
wherein some or all polypeptides comprises either a target peptide, a linker peptide, and a
self-assembling peptide or a linker peptide, a target peptide, a linker peptide, and a self-
assembling peptide or a combination thereof. The polypeptides are connected with each
other through a cleavage peptide. The multitarget peptide includes a signal peptide upstream15
(N-terminus) of one or more polypeptides. In some embodiments, the multitarget peptide
includes a signal peptide upstream (N-terminus) of each of all or some polypeptides. In some
embodiments, the multitarget peptide optionally includes a signal peptide upstream (N-
terminus) of each polypeptide. In some embodiments, the multitarget peptide includes a
signal peptide upstream (N-terminus) of some polypeptides. In some embodiments, the20
multitarget peptide includes a signal peptide upstream (N-terminus) of all polypeptides.
The signal peptide transports the multitarget peptide to golgi body or golgi apparatus.
The cellular proteases act on the cleavage sites present in the cleavage peptides or the
cleavage peptide undergoes self-cleavage and cleaves the multitarget peptide into individual
polypeptides comprising either a target peptide, a linker peptide, and a self-assembling25
peptide, or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide,
or a signal peptide, a target peptide, a linker peptide, and a self-assembling peptide, or a
signal peptide, a linker peptide, a target peptide, a linker peptide, and a self-assembling
peptide, or a combination thereof. The polypeptides may additionally also have some
residues (amino acids) of cleavage peptide.30
In some embodiments, the linker peptide may be an amino acid linker, a foldon, a
scaffold, or a combination thereof.
In some embodiments, the polypeptides may be homologous polypeptides.
73
In some embodiments, two or more homologous polypeptides may organise to form
an oligomeric complex. In some embodiments, the oligomeric complex may comprise at
least two homologous polypeptides, at least three homologous polypeptides, at least four
homologous polypeptides, at least five homologous polypeptides, or at least six homologous
polypeptides and so on.5
In some embodiments, the polypeptides may be heterologous polypeptides.
A polypeptide nanoparticle is formed by self-assembly of two or more homologous
polypeptides, or two or more heterologous polypeptides, or one or more oligomeric
complexes, or their combination.
In some embodiments, a polypeptide nanoparticle comprises homologous10
polypeptides, heterologous polypeptides, oligomeric complex, or a combination thereof.
In some embodiments, the polypeptide nanoparticles may be symmetrical, non-
symmetrical, asymmetrical, or a combination thereof.
In some embodiments, the polypeptide nanoparticles may be icosahedral, helical,
spherical, rod-like or a combination thereof.15
In some embodiments, the polypeptide nanoparticles may be enveloped or non-
enveloped or a combination thereof.
In some embodiments, the polypeptide nanoparticles may be single layered or multi-
layered or a combination thereof.
In some of the embodiments, the polypeptide nanoparticle may comprise at least 220
or up to 100 polypeptides.
In some embodiments, the polypeptide nanoparticle may comprise polypeptides
between 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-99.
In one of the embodiments, the polypeptide nanoparticle may comprise at least 2 or
up to 100 homologous polypeptides.25
In some embodiments, the polypeptide nanoparticle may comprise at least 2 or up to
100 heterologous polypeptides.
In some embodiments, the polypeptide nanoparticle may comprise two or more
oligomeric complexes such that total number of polypeptides in the polypeptide nanoparticle
are not more than 100.30
In some embodiments, the polypeptide nanoparticle may comprise some
homologous polypeptides and some heterologous polypeptides such that the total number of
polypeptides in the polypeptide nanoparticle are not more than 100.
74
In some embodiments, the polypeptide nanoparticle may comprise some
homologous polypeptides and some oligomeric complexes such that the total number of
polypeptides in the polypeptide nanoparticle are not more than 100.
In some embodiments, the polypeptide nanoparticle may comprise some
heterologous polypeptides and some oligomeric complexes such that the total number of5
polypeptides in the polypeptide nanoparticle are not more than 100. In some embodiments,
the polypeptide nanoparticle may comprise some homologous polypeptides, some
heterologous polypeptides, some oligomeric complexes, or their combination such that the
total number of polypeptides in the polypeptide nanoparticle are not more than 100.
10
Lipid nanoparticles (LNP) composition
The multitarget nucleic acid sequences as described herein may be encapsulated in a
lipid nanoparticle composition.
In some embodiments, the lipid nanoparticle composition comprises lipid
components, ionizable polymer, or a combination thereof and a multitarget nucleic acid15
sequence as described herein.
In some embodiments, the lipid nanoparticle composition comprises lipid
components such as a cationic lipid, a phospholipid, a sterol, a PEG-lipid and a multitarget
nucleic acid sequence as described herein.
In another embodiment, the lipid nanoparticle composition comprises an ionizable20
polymer, a cationic lipid, a phospholipid, a sterol, a PEG-lipid and a multitarget nucleic acid
sequence as described herein.
Lipid Components
Lipid components of the lipid nanoparticle compositions may include one or more25
lipids, such as a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.
Cationic lipid
Cationic lipid refers to a lipid that has a net positive charge at a selected pH. Cationic
lipids generally consist of a hydrophilic head group that carries the charge and a hydrophobic30
tail.
Exemplary cationic lipid for use in the lipid nanoparticle compositions include, but
are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-
dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-
75
dimethylammonium bromide(DDAB); N-(2,3dioleoyloxy)propyl)-N,N,N-
trimethylammonium chloride (DOTAP); 3-(N—(N',N'-dimethylaminoethane)-
carbamoyl)cholesterol (DC-Chol), N-(l-(2,3-dioleoyloxy)propyl)N-2-
(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (DOSPA),
dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dioleoyl-5
3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine
(DODMA), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide (DMRIE), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 3-
dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc-
tadecadienoxy)propane (Clin-DMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-10
dimethyl-l-(cis,cis-9',12'-octadecadienoxy)propane (CpLin-DMA), 2,3-Dilinoleoyloxy-
N,N-dimethylpropylamine (DLin-DAP), 1,2-N,N'-Dilinoleylcarbamyl-3-
dimethylaminopropane (DLincarb-DAP), l,2-Dilinoleoylcarbamyl-3-
dimethylaminopropane (DLin-CDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-
dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-15
(dimethylamino)butanoate (DLin-MC3-DMA), heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-
6-undecoxyhexyl)amino]octanoate (SM-102), 6-[6-(2-hexyldecanoyloxy)hexyl-(4-
hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), nonyl 8-[(8-heptadecan-9-
yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate (SLP-0001) or a combination thereof.
Cationic lipids with amine head group are preferred cationic lipids. The amine group20
can be at primary, secondary or tertiary position. The cationic lipid may comprise one
(monoamine) or more (polyamine) such amine groups.
In some embodiments, the cationic lipids are positively charged at acidic pH i.e., pH
1.0 to pH 6.9. In certain embodiments, the cationic lipids are neutral at certain pH i.e., around
physiological pH (pH 7.0 to pH 7.5). A cationic lipid that can exist in a positively charged25
or neutral form depending on the pH is referred to as ionizable lipid. Preferred cationic lipids
are ionizable such that they can exist in a positively charged or neutral form depending on
pH. For example, an ionizable lipid may be neutral around physiological pH (pH 7.0 to pH
7.5), and cationic around acidic pH (pH 1.0 to pH 6.9).
In some embodiments, cationic lipid present in the lipid nanoparticle compositions30
is an ionizable lipid.
Methods of making cationic lipid and/or ionizable lipid or imparting the cationic
lipid the ability to behave as an ionizable lipid are well known in the art (WO2005121348;
76
WO2009127060; WO2009086558; WO2010042877; WO2010144740; WO2011075656;
WO2017049245; WO2017075531; WO2018118102; WO2015199952; Reynier P. et al.
Journal of Drug Targeting (2004) 12: 25-38; Sabnis, Staci et al. Molecular Therapy (2018)
26: 1509-1519).
The proportion of cationic lipid present in the lipid nanoparticle compositions is from5
about 25 mol % to about 70 mol % or any range therein.
In some embodiments, the proportion of cationic lipid present in the lipid
nanoparticle compositions is from about 25 mol % to about 70 mol %, from about 25 mol
% to about 65 mol %, from about 25 mol % to about 60 mol %, from about 25 mol % to
about 55 mol %, from about 25 mol % to about 50 mol %, from about 25 mol % to about 4810
mol %, from about 25 mol % to about 46 mol %, from about 25 mol % to about 45 mol %,
from about 25 mol % to about 44 mol %, from about 25 mol % to about 43 mol %, from
about 25 mol % to about 42 mol %, from about 25 mol % to about 41 mol %, from about 25
mol % to about 40 mol %, or any range therein.
In some embodiments, the proportion of cationic lipid present in the lipid15
nanoparticle compositions is about 25 mol %, about 26 mol %, about 27 mol %, about 28
mol %, about 29 mol %, about 30 mol %, about 31 mol %, about 32 mol %, about 33 mol
%, about 34 mol %, about 35 mol %, about 36 mol %, about 37 mol %, about 40 mol %,
about 41 mol %, about 42 mol %, about 43 mol %, about 44 mol %, about 45 mol %, about
46 mol %, about 47 mol %, about 48 mol %, about 49 mol %, about 50 mol % about 51 mol20
%, about 52 mol %, about 53 mol %, about 54 mol %, about 55 mol %, about 56 mol %,
about 57 mol %, about 58 mol %, about 59 mol %, about 60 mol %, about 61 mol %, about
62 mol %, about 63 mol %, about 64 mol %, about 65 mol %, about 66 mol %, about 67 mol
%, about 68 mol %, about 69 mol %, about 70 mol % or any portion or fraction thereof.
25
Phospholipids
Phospholipid includes a lipid containing a hydrophilic head with a phosphate group
and a hydrophobic tail composed of fatty acid chains attached to a glycerol or sphingosine
backbone.
Exemplary phospholipids for use in the lipid nanoparticle compositions include, but30
are not limited to, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-
sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-
phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-
77
palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-
glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-
glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16
Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-
phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-5
glycero-3-phosphoethanolamine (DOPE), 1,2-diphytanoyl-sn-glycero-3-
phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine,
1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-
phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-
didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-10
phospho-rac-(1-glycerol) sodium salt (DOPG), 1-myristoyl-2-stearoyl-sn-glycero-3-
phosphocholine (MSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC),
1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-stearoyl-2-myristoyl-sn-
glycero-3-Phosphocholine (SMPC), 1-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine
(SPPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1-stearoyl-2-15
docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC), sphingomyelin, or a combination
thereof.
The proportion of phospholipid present in the lipid nanoparticle compositions is from
about 2 mol % to about 30 mol % or any range therein.
In some embodiments, the proportion of phospholipid present in the lipid20
nanoparticle compositions is from about 2 mol % to about 30 mol %, from about 2 mol %
to about 28 mol %, from about 2 mol % to about 26 mol %, from about 2 mol % to about 24
mol %, from about 2 mol % to about 22 mol %, from about 2 mol % to about 20 mol %,
from about 3 mol % to about 19 mol %, from about 3 mol % to about 18 mol %, from about
3 mol % to about 17 mol %, from about 3 mol % to about 16 mol %, from about 3 mol % to25
about 15 mol %, from about 3 mol % to about 14 mol %, from about 3 mol % to about 13
mol %, from about 3 mol % to about 12 mol %, or any range therein.
In some embodiments, the proportion of phospholipid present in the lipid
nanoparticle compositions is about 2 mol %, about 3 mol %, about 4 mol %, about 5 mol %,
about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about 10 mol %, about 1130
mol %, about 12 mol %, about 13 mol %, about 14 mol %, about 15 mol %, about 16 mol
%, about 17 mol %, about 18 mol %, about 19 mol %, about 20 mol %, about 21 mol %,
about 21 mol %, about 22 mol %, about 23 mol %, about 24 mol %, about 25 mol %, about
78
26 mol %, about 27 mol %, about 28 mol %, about 29 mol %, about 30 mol %, or any portion
or fraction thereof.
Sterol
Lipid nanoparticle composition disclosed herein may include sterol and/or sterol5
derivatives. The term “sterol” as used herein include, but not limited to, cholesterol,
sitosterol, fecosterol, ergosterol, campesterol, stigmasterol or their derivatives. In some
embodiments, lipid nanoparticle composition comprises cholesterol and/or cholesterol
derivatives. Non-limiting examples of cholesterol and cholesterol derivatives include 5α-
cholestanol, 5β-coprostanol, cholesteryl-(2’-hydroxy)-ethyl ether, cholesteryl-(4’-hydroxy)-10
butyl ether, 6-ketocholestanol, 5α-cholestane, cholestenone, 5α-cholestanone, 5β-
cholestanone, cholesteryl decanoate, or mixtures thereof. Methods of making cholesterol
and cholesterol derivatives are well known in the art (WO2009127060; WO2019152557).
The proportion of sterol present in the lipid nanoparticle compositions may be from
about 30 mol % to about 65 mol % or any range therein.15
In some embodiments, the proportion of sterol present in the lipid nanoparticle
compositions is from about 30 mol % to about 65 mol %, from about 31 mol % to about 60
mol %, from about 32 mol % to about 60 mol %, from about 33 mol % to about 60 mol %,
from about 34 mol % to about 60 mol %, from about 35 mol % to about 60 mol %, or any
range therein.20
In some embodiments, the proportion of sterol present in the lipid nanoparticle
compositions is about 30 mol %, about 31 mol %, about 32 mol %, about 33 mol %, about
34 mol %, about 35 mol %, about 36 mol %, about 37 mol %, about 38 mol %, about 39 mol
%, about 40 mol %, about 41 mol %, about 42 mol %, about 43 mol %, about 44 mol %,
about 45 mol %, about 46 mol %, about 47 mol %, about 48 mol %, about 49 mol %, about25
50 mol %, about 51 mol %, about 52 mol %, about 53 mol %, about 54 mol %, about 55 mol
%, about 56 mol %, about 57 mol %, about 58 mol %, about 59 mol %, about 60 mol %,
about 61 mol %, about 62 mol %, about 63 mol %, about 64 mol %, about 65 mol % or any
portion or fraction thereof.
30
PEG-lipid
The term PEG-lipid, pegylated lipid, PEG linked lipid, PEG conjugated lipid, PEG-
lipid conjugate, PEG modified lipid have been used interchangeably to mean polyethylene
glycol linked to a lipid moiety. The lipid moiety may be linked directly to the PEG molecule
79
or through a linker. In some embodiments, a PEG-lipid comprises a PEG-modified
phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides,
PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified
dialkylglycerols, and/or PEG-modified cholesterol, and/or mixtures thereof. The methods of
making PEG-lipid are well known to persons skilled in the art, see for example,5
US20030077829; US2005008689; US5885613; US7404969; WO2005026372;
WO2009086558).
In some embodiments, PEG-lipid is selected from mPEG-Dimyristoyl glycerol
(mPEG-DMG), mPEG-N,N-Ditetradecylacetamide (mPEG-DTA or ALC0159), mPEG-
Cholesterol (mPEG-CLS), mPEG-DSPE, mPEG-DMPE, mPEG-DPPE, mPEG-DLPE,10
mPEG-DOPE, mPEG-DPPC, mPEG-DSPC, 1,2-Distearoyl-sn-Glycero-3-
Phosphoethanolamine with conjugated methoxyl poly(ethylene glycol) (mPEG-DSPE), 1,2-
dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) or
mixtures thereof.
The PEG moiety of the PEG-lipid may comprise an average molecular weight15
ranging from 0.5 kDa to 10 kDa. In some embodiments, the PEG-lipid has an average
molecular weight of about 0.5 kDa to 5 kDa, about 0.5 kDa to 4 kDa, 0.5 kDa to 3 kDa, 0.5
kDa to 2 kDa. In preferred embodiments, the PEG-lipid has an average molecular weight of
about 0.5 kDa to about 2 kDa.
The proportion of PEG-lipid present in the lipid nanoparticle compositions may be20
from about 0.2 mol % to about 2.0 mol % or any range therein.
In some embodiments, the proportion of PEG-lipid present in the lipid nanoparticle
compositions is from about 0.2 mol % to about 2.0 mol %, from about 0.2 mol % to about
1.8 mol %, from about 0.2 mol % to about 1.5 mol %, or any range therein.
In some embodiments, the proportion of PEG-lipid present in the lipid nanoparticle25
compositions is about 0.2 mol %, about 0.3 mol %, about 0. 4 mol %, about 0.5 mol %,
about 0.6 mol %, about 0.7 mol %, about 0.8 mol %, about 0.9 mol %, about 1.0 mol %,
about 1.1 mol %, about 1.2 mol %, about 1.3 mol %, about 1.4 mol %, about 1.5 mol %,
about 1.6 mol %, about 1.7 mol %, about 1.8 mol %, about 1.9 mol %, or about 2.0 mol %
or any portion or fraction thereof.30
In some embodiments, the lipid nanoparticle composition additionally may contain
an ionizable polymer.
80
Ionizable polymer
As used herein the term “polymer” means a compound formed from a plurality of
repeating units called monomers. Polymers are produced through a process called
polymerization wherein two or more monomers are linked through chemical bonds to form
the polymer. In some embodiments, the polymer is branched or unbranched. In some5
embodiments, the polymer may be homopolymer, i.e., consisting of same type of repeat units
or monomers, or heteropolymer, i.e., consisting of more than one type of repeat units or
monomers. The terms heteropolymer and copolymer have been used interchangeably herein.
The term “ionizable polymer” as used herein means, a polymer that can exist in a
positively charged or neutral form depending on the pH of the solution or environment, for10
example, ionizable polymer will be cationic (positively charged) around acidic pH (pH 1.0
to pH 6.9) and neutral (no charge) around physiological pH (pH 7.0 to pH 7.5).
In some embodiments, the ionizable polymer is a biocompatible polymer or
biodegradable polymer. The term “biocompatible polymer” and “biodegradable polymer”
have been used interchangeably to mean a polymer that is substantially free from any15
deleterious effects when introduced into a living or biological system. Such polymers are
capable of undergoing degradation when introduced into the living or biological systems
and are not expected to produce significant toxicity or immunological response.
In some embodiments, the lipid nanoparticle compositions comprise an ionizable
polymer. The ionizable polymer may be selected from chitosan, chitosan derivatives,20
cellulose derivatives, poly-L-lysine (PLL), poly-L-glutamic acid, protamine,
polyethyleneimine, their derivatives, or a combinations thereof.
In some embodiments, the ionizable polymer is positively charged at acidic pH i.e.,
pH 1.0 to 6.9 and is neutral around physiological pH (pH 7.0 to 7.5).
The proportion of ionizable polymer present in the lipid nanoparticle compositions25
may be from about 1 mol % to about 25 mol %.
In some embodiments, the proportion of ionizable polymer present in the lipid
nanoparticle compositions is from about 1 mol % to about 25 mol %, 1 mol % to about 25
mol %, from about 1 mol % to about 24 mol %, from about 1 mol % to about 23 mol %,
from about 1 mol % to about 22 mol %, from about 1 mol % to about 21 mol %, from about30
1 mol % to about 20 mol %, from about 1 mol % to about 19 mol %, or from about 1 mol %
to about 18 mol %, from about 1 mol % to about 17 mol %, from about 1 mol % to about 16
mol %, from about 1 mol % to about 15 mol % or any range therein.
81
In some embodiment, the proportion of ionizable polymer present in the lipid
nanoparticle compositions is from about 1 mol % to about 25 mol %, from about 1 mol %
to about 24 mol %, from about 1 mol % to about 23 mol %, from about 1 mol % to about 22
mol %, from about 1 mol % to about 21 mol %, from about 1 mol % to about 20 mol %,
from about 1 mol % to about 19 mol %, from about 1 mol % to about 18 mol %, from about5
1 mol % to about 17 mol %, from about 1 mol % to about 16 mol %, from about 1 mol % to
about 15 mol %, or any range therein.
In some embodiments, the proportion of ionizable polymer present in the lipid
nanoparticle compositions is about 1 mol %, is about 2 mol %, about 3 mol %, about 4 mol
%, 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about 10 mol10
%, about 11 mol %, about 12 mol %, about 13 mol %, about 14 mol %, about 15 mol %,
about 16 mol %, about 17 mol %, about 18 mol %, about 19 mol %, about 20 mol %, about
21 mol %, about 22 mol %, about 23 mol %, about 24 mol %, about 25 mol %, or any portion
or fraction thereof.
In some embodiments, the preferred ionizable polymer comprises chitosan, chitosan15
derivatives, cellulose derivatives, or a combination thereof.
Chitosan
Chitosan is a natural polymer composed of glucosamine units. Chitosan is
chemically poly-β-(1-4)-2-amino-2-deoxy-D-glucose. Chitosan is prepared by partial20
deacetylation of chitin, which is commonly carried out by alkaline hydrolysis. Thus,
chitosan may contain acetylated units (N-acetyl-D-glucosamine) as well as deacetylated
units (β-(1→4)-linked D-glucosamine). Typically, chitosan molecule has greater than 60%
degree of deacetylation when compared to chitin. The molecular weight of chitosan typically
varies between 10 kDa to 1000 kDa. Chitosan nanoparticles have been used for drug25
delivery, including delivery of nucleic acids. However, chitosan nanoparticles alone are
insufficient in effective delivery of nucleic acids (Ragelle, Héloïse et al. Journal of
Controlled Release (2013) 172: 207-218).
In some embodiments, the lipid nanoparticle composition comprises an ionizable
polymer such as chitosan along with lipid components and nucleic acid.30
In some embodiments, the ionizable polymer is chitosan or its derivatives. In some
embodiments, the ionizable polymer includes chitosan derivatives or dialdehyde chitosan
derivatives or a combination thereof.
82
The chitosan or chitosan derivatives employed in the present disclosure may have a
molecular weight from about 25 kDa to about 400 kDa.
In some embodiments, the molecular weight of chitosan or its derivatives is from
about 25 kDa to 375 Kda, from about 30 kDa to about 350 kDa, from about from about 35
kDa to about 325 kDa, from about 40 kDa to about 300 kDa, from about 40 kDa to about5
250 kDa, from about 40 kDa to about 225 kDa, from about 40 kDa to about 220 kDa, from
about 40 kDa to about 210 kDa, or from about 40 kDa to about 200 kDa or any range therein.
The proportion of chitosan or its derivatives or a combination thereof present in the
lipid nanoparticle compositions may be from about 1 mol % to about 25 mol %.
In some embodiments, the proportion of chitosan or its derivatives or a combination10
thereof present in the lipid nanoparticle compositions is from about 1 mol % to about 25 mol
%, , from about 1 mol % to about 24 mol %, from about 1 mol % to about 23 mol %, from
about 1 mol % to about 22 mol %, from about 1 mol % to about 21 mol %, from about 1
mol % to about 20 mol %, from about 1 mol % to about 19 mol %, or from about 1 mol %
to about 18 mol %, from about 1 mol % to about 17 mol %, from about 1 mol % to about 1615
mol %, from about 1 mol % to about 15 mol % or any range therein.
In some embodiments, the proportion of chitosan or its derivatives or a combination
thereof present in the lipid nanoparticle compositions is from about 1 mol % to about 25 mol
%, preferably about 1 mol % to 20 mol %, most preferably about 1 mol % to about 15 mol20
%.
In some embodiments, the proportion of chitosan or its derivatives or a combination
thereof present in the lipid nanoparticle compositions is about 1 mol %, about 2 mol %,
about 3 mol %, about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol
%, about 9 mol %, about 10 mol %, about 11 mol %, about 12 mol %, about 13 mol %, about25
14 mol %, about 15 mol %, about 16 mol %, about 17 mol %, about 18 mol %, about 19 mol
%, about 20 mol %, about 21 mol %, about 22 mol %, about 23 mol %, about 24 mol %,
about 25 mol %, or any portion or fraction thereof.
Cellulose30
Cellulose is a polymer composed of a linear chain of d-glucose units linked via β-
1,4 glycosidic bonds. It typically contains repeating glucose units ranging from few
hundreds to several thousands. Native cellulose is not ideal for mRNA delivery and
therefore, requires modification in accordance with the present disclosure. In some
83
embodiments, cellulose is modified (Jelkmann et al. Biomacromolecules (2018) 19: 4059-
4067; Lee, Hye Ji et al. International Journal of Biosciences Biochemistry and
Bioinformatics (2019) 9: 134-140). Cellulose and cellulose derived materials have been used
for drug delivery of small molecules (Amalin Kavitha, K. Thomas Paul, Parambath
Anilkumar, Chapter 18 - Cellulose-derived materials for drug delivery applications,5
Editor(s): Faruq Mohammad, Hamad A. Al-Lohedan, Mohammad Jawaid, In Micro and
Nano Technologies, Sustainable Nanocellulose and Nanohydrogels from Natural Sources,
Elsevier, 2020, Pages 367-390, ISBN 9780128167892).
In some embodiments, the lipid nanoparticle composition comprises cellulose
derivatives. Cellulose derivatives may be dialdehyde cellulose derivatives.10
The proportion of cellulose derivatives or dialdehyde cellulose derivatives or a
combination thereof present in the lipid nanoparticle compositions may be from about 1 mol
% to about 25 mol %.
In some embodiments, the proportion of cellulose derivatives or dialdehyde cellulose
derivatives or combination thereof present in the lipid nanoparticle compositions is from15
about 1 mol % to about 25 mol %, from about 1 mol % to about 24 mol %, from about 1
mol % to about 23 mol %, from about 1 mol % to about 22 mol %, from about 1 mol % to
about 21 mol %, from about 1 mol % to about 20 mol %, from about 1 mol % to about 19
mol %, or from about 1 mol % to about 18 mol %, from about 1 mol % to about 17 mol %,
from about 1 mol % to about 16 mol %, from about 1 mol % to about 15 mol % or any range20
therein.
In some embodiments, the proportion of cellulose derivatives or dialdehyde cellulose
derivatives or combination thereof present in the lipid nanoparticle compositions is from
about 1 mol % to about 25 mol %, preferably about 1 mol % to about 20 mol %, most25
preferably about 1 mol % to about 15 mol % or any range therein.
In some embodiments, the proportion of cellulose or its derivatives or combination
thereof present in the lipid nanoparticle compositions is about 1 mol %, about 2 mol %,
about 3 mol %, about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol
%, about 9 mol %, about 10 mol %, about 11 mol %, about 12 mol %, about 13 mol %, about30
14 mol %, about 15 mol %, about 16 mol %, about 17 mol %, about 18 mol %, about 19 mol
%, about 20 mol %, about 21 mol %, about 22 mol %, about 23 mol %, about 24 mol %,
about 25 mol %, or any portion or fraction thereof.
84
Method of treatment
In some aspects, provided herein is a method of treating or preventing a disease,
comprising administering to a subject in need thereof the multitarget nucleic acid sequence
as described herein.
In some aspects, provided herein is a method of treating or preventing a disease,5
comprising administering to a subject in need thereof the lipid nanoparticle composition
comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid and the multitarget nucleic
acid sequence as described herein.
In some aspects, provided herein is a method of treating or preventing a disease,
comprising administering to a subject in need thereof the lipid nanoparticle composition10
comprising an ionizable polymer, a cationic lipid, a phospholipid, a sterol, a PEG-lipid and
the multitarget nucleic acid sequence as described herein.
In some aspects, the disclosure relates to use of a lipid nanoparticle composition
comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and the multitarget nucleic
acid sequence as described herein in the manufacture of a medicament for the treatment or15
prevention of a disease in a subject.
In some embodiments, the multitarget nucleic acid sequence is present in
biologically effective amount or therapeutically effective amount. In some embodiments,
the biologically effective amount of multitarget nucleic acid sequence is between 0.1 μg to
2000 μg, 0.1 μg to 1800 μg, 0.1 μg to 1600 μg, 0.1 μg to 1600 μg, 0.1 μg to 1400 μg, 0.120
μg to 1200 μg, 0.1 μg to 1000 μg, 0.1 μg to 950 μg, 0.1 μg to 900 μg, 0.1 μg to 850 μg, 0.1
μg to 800 μg, 0.1 μg to 750 μg, 0.1 μg to 700 μg, 0.1 μg to 650, 0.1 μg to 600, 0.1 μg to
550, 0.1 μg to 500 μg, 0.1 to 450 μg, 0.1 μg to 400 μg, 0.1 μg to 350 μg, 0.1 to 300 μg, 0.1
to 200 μg or any range therein. In some embodiments, the biologically effective amount of
multitarget nucleic acid sequence is from about 0.1 μg to 1000 μg, 0.1 μg to 950 μg, 0.1 μg25
to 900 μg, 0.1 μg to 850 μg, 0.1 μg to 800 μg, 0.1 μg to 750 μg, 0.1 μg to 700 μg, 0.1 μg to
650, 0.1 μg to 600, 0.1 μg to 550, 0.1 μg to 500 μg or any range therein.
In some embodiments, the biologically effective amount of multitarget nucleic acid
sequence is 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7, μg, 0.8 μg, 0.9 μg, 1 μg, 2
μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 4030
μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 100 μg, 110 μg,
120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 220 μg, 240 μg,
250 μg, 260 μg, 280 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 600 μg, 700 μg, 800 μg,
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900 μg, 1000 μg, 1100 μg, 1200 μg, 1300 μg, 1400 μg, 1500 μg, 1600 μg, 1700 μg, 1800
μg, 1900 μg, 2000 μg or any portion or fraction thereof.
In one aspect, provided herein is a nucleic acid comprising a plurality of
polynucleotide sequences, wherein some or all polynucleotide sequence of the plurality5
comprises either a target sequence, a linker sequence, and a self-assembling sequence or a
linker sequence, a target sequence, a linker sequence and a self-assembling sequence or a
combination thereof, wherein each polynucleotide sequence of the plurality is connected to
an adjacent polynucleotide sequence of the plurality by a cleavage sequence, and wherein
the nucleic acid further comprises a signal sequence upstream of one or more of the10
polynucleotide sequences of the plurality. In another aspect, provided herein is a nucleic acid
comprising a plurality of polynucleotide sequences, wherein each polynucleotide sequence
of the plurality comprises a target sequence, a linker sequence, and a self-assembling
sequence or a second linker sequence, a second target sequence, a third linker sequence, and
a second self-assembling sequence, wherein each polynucleotide sequence of the plurality15
is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence,
and wherein the nucleic acid further comprises a signal sequence upstream of one or more
of the polynucleotide sequences of the plurality. In another aspect, provided herein a nucleic
acid comprising a plurality of polynucleotide sequences, wherein each polynucleotide
sequence of the plurality comprises a target sequence, a linker sequence, and a self-20
assembling sequence, wherein each polynucleotide sequence of the plurality is connected to
an adjacent polynucleotide sequence of the plurality by a cleavage sequence, and wherein
the nucleic acid further comprises a signal sequence upstream of one or more of the
polynucleotide sequences of the plurality. In yet another aspect, provided herein is a nucleic
acid comprising a plurality of polynucleotide sequences, wherein each polynucleotide25
sequence of the plurality comprises a linker sequence, a target sequence, a linker sequence,
and a self-assembling sequence, wherein each polynucleotide sequence of the plurality is
connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence,
and wherein the nucleic acid further comprises a signal sequence upstream of one or more
of the polynucleotide sequences of the plurality. In one aspect, provided herein is a nucleic30
acid comprising a first plurality of polynucleotide sequences and a second plurality of
polynucleotide sequences, each polynucleotide sequence of the first plurality comprises a
first target sequence, a first linker sequence, and a first self-assembling sequence, wherein
each polynucleotide sequence of the second plurality comprises a second linker sequence, a
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second target sequence, a third linker sequence, and a second self-assembling sequence,
wherein each polynucleotide sequence of the first plurality and each polynucleotide
sequence of the second plurality is connected to an adjacent polynucleotide sequence of the
first plurality or an adjacent polynucleotide sequence of the second plurality by a cleavage
sequence, and wherein the first polynucleotide sequence in the nucleic acid is a5
polynucleotide sequence of the first plurality or a polynucleotide sequence of the second
plurality, and wherein the nucleic acid further comprises a signal sequence upstream of the
first polynucleotide sequence of the first plurality or the second polynucleotide sequence of
the second plurality, or a combination thereof. In another aspect, provided herein is a nucleic
acid encoding a plurality of polypeptides, wherein some or all polypeptides of the plurality10
comprises either a target peptide, a linker peptide, and a self-assembling peptide or a linker
peptide, a target peptide, a linker peptide and a self-assembling peptide or a combination
thereof, wherein each polypeptide of the plurality is connected to an adjacent polypeptide of
the plurality by a cleavage peptide, and wherein the nucleic acid further comprises a signal
peptide on the amino-terminus of one or more of the polypeptides of the plurality. In another15
aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein some
or all polypeptides of the plurality comprises either a target peptide, a linker peptide, and a
self-assembling peptide or a second linker peptide, a second target peptide, a third linker
peptide and a second self-assembling peptide or combination thereof, wherein each
polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a20
cleavage peptide, and wherein the nucleic acid further comprises a signal peptide on the
amino-terminus of the first polypeptide of the plurality or the second polypeptide of the
plurality or a combination thereof. In one aspect, provided herein is a nucleic acid encoding
a plurality of polypeptides, wherein each polypeptide of the plurality comprises a target
peptide, a linker peptide, and a self-assembling peptide, wherein each polypeptide of the25
plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide, and
wherein the nucleic acid further encodes a signal peptide on the amino-terminus of one or
more of the polypeptides of the plurality. In another aspect, provided herein is a nucleic acid
encoding a plurality of polypeptides, wherein each polypeptide of the plurality comprises a
linker peptide, a target peptide, a linker peptide, and a self-assembling peptide, wherein each30
polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a
cleavage peptide, and wherein the nucleic acid further encodes a signal peptide on the
amino-terminus of one or more of the polypeptides of the plurality. In yet another aspect,
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provided herein is nucleic acid encoding a first plurality of polypeptides and a second
plurality of polypeptides, wherein each polypeptide of the first plurality comprises a target
peptide, a linker peptide, and a self-assembling peptide, wherein each polypeptide of the
second plurality comprises a second linker peptide, a second target peptide, a third linker
peptide, and a second self-assembling peptide, wherein each polypeptide of the first plurality5
and each polypeptide of the second plurality is connected to an adjacent polypeptide of the
first plurality or an adjacent polypeptide of the second plurality by a cleavage peptide, and
wherein the first polypeptide encoded by the nucleic acid is a polypeptide of the first
plurality or a polypeptide of the second plurality, and wherein the nucleic acid further
encodes a signal peptide on the amino-terminus of the first polypeptide of the first plurality10
or the second polypeptide of the second plurality or a combination thereof.
Embodiments
Some of the embodiments of the present disclosure are set out in the following
numbered paragraphs:15
1. A nucleic acid comprising a plurality of polynucleotide sequences, wherein some or
all polynucleotide sequence of the plurality comprises either a target sequence, a linker
sequence, and a self-assembling sequence or a linker sequence, a target sequence, a linker
sequence and a self-assembling sequence or a combination thereof, wherein each20
polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence
of the plurality by a cleavage sequence, and wherein the nucleic acid further comprises a
signal sequence upstream of one or more of the polynucleotide sequence of the plurality.
2. A nucleic acid comprising a plurality of polynucleotide sequences, wherein each25
polynucleotide sequence of the plurality comprises a target sequence, a linker sequence, and
a self-assembling sequence, wherein each polynucleotide sequence of the plurality is
connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence,
and wherein the nucleic acid further comprises a signal sequence upstream of one or more
of the polynucleotide sequence of the plurality.30
3. A nucleic acid comprising a plurality of polynucleotide sequences, wherein each
polynucleotide sequence of the plurality comprises a linker sequence, a target sequence, a
linker sequence, and a self-assembling sequence, wherein each polynucleotide sequence of
the plurality is connected to an adjacent polynucleotide sequence of the plurality by a
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cleavage sequence, and wherein the nucleic acid further comprises a signal sequence
upstream of one or more of the polynucleotide sequence of the plurality.
4. A nucleic acid encoding a plurality of polypeptides, wherein some or all polypeptides
of the plurality comprises either a target peptide, a linker peptide, and a self-assembling
peptide or a linker peptide, a target peptide, a linker peptide and a self-assembling peptide5
or a combination thereof, wherein each polypeptide of the plurality is connected to an
adjacent polypeptide of the plurality by a cleavage peptide, and wherein the nucleic acid
further comprises a signal peptide on the amino-terminus of one or more of the polypeptide
of the plurality.
5. A nucleic acid encoding a plurality of polypeptides, wherein each polypeptide of the10
plurality comprises a target peptide, a linker peptide, and a self-assembling peptide, wherein
each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by
a cleavage peptide, and wherein the nucleic acid sequence further encodes a signal peptide
on the amino-terminus of one or more of the polypeptide of the plurality.
6. A nucleic acid encoding a plurality of polypeptides, wherein each polypeptide of the15
plurality comprises a linker peptide, a target peptide, a linker peptide, and a self-assembling
peptide, wherein each polypeptide of the plurality is connected to an adjacent polypeptide
of the plurality by a cleavage peptide, and wherein the nucleic acid sequence further encodes
a signal peptide on the amino-terminus of one or more of the polypeptide of the plurality.
7. The nucleic acid according to any of the preceding paragraphs, wherein total number20
of the polynucleotide sequences is not more than 100.
8. The nucleic acid according to any of the preceding paragraphs, wherein total number
of the polynucleotide sequences is between 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70,
70-80, 80-90, or 90-99.
9. The nucleic acid according to any of the preceding paragraphs, wherein the nucleic25
acid is a DNA or an RNA.
10. The nucleic acid according to paragraph 9, wherein the RNA is an mRNA.
11. The nucleic acid according to any of the preceding paragraph wherein the linker
sequence encodes a linker peptide.
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12. The nucleic acid according to paragraph 11, wherein the linker peptide is an amino
acid linker, a foldon, a scaffold or a combination thereof.
13. The nucleic acid according to paragraph 12, wherein the amino acid linker comprises
2 to 49 amino acids.
14. The nucleic acid according to paragraph 13, wherein the amino acid linker is a5
glycine serine linker, a glycine proline linker, a glycine threonine linker, an alanine serine
linker, any combination of two amino acids, or a combination thereof.
15. The nucleic acid according to any of the preceding paragraphs, wherein the linker
peptide has an amino acid sequence of any one of SEQ ID NOs: 262-299, 330, and 350.
16. The nucleic acid according to any of the preceding paragraphs, wherein the self-10
assembling sequence encodes a self-assembling peptide.
17. The nucleic acid according to paragraph 16, wherein the self-assembling peptide is
a lumazine synthase from Aquifex species, a hepatitis B surface antigen (HBsAg) from
Hepatitis B Virus, a hepatitis B core antigen (HBcAg) from Hepatitis B virus, a human
papillomavirus L1 (HPV L1) protein, a matrix protein M1 from influenza A virus, a ferritin,15
a riboflavin synthase, or a combination thereof, including their codon optimized nucleic acid
sequences, fragments, mutants, or variants thereof.
18. The nucleic acid according to paragraph 17, wherein the ferritin comprises of a
ferritin subunit or a ferritin peptide.
19. The nucleic acid according to paragraph 18, wherein the ferritin peptide is derived20
from Helicobacter pylori ferritin.
20. The nucleic acid according to any of the preceding paragraphs, wherein the self-
assembling peptide has an amino acid sequence of any one of SEQ ID NOs: 254-261, 331
and 333.
21. The nucleic acid according to any of the preceding paragraphs, wherein the cleavage25
sequence encodes one or more cleavage peptide.
22. The nucleic acid according to paragraph 21, wherein the one or more cleavage
peptides are optionally connected to each other by a linker peptide.
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23. The nucleic acid according paragraph 22, wherein the cleavage peptide is a golgi
specific cleavage peptide or a self-cleaving peptide.
24. The nucleic acid according to any of the preceding paragraphs, wherein the cleavage
peptide has an amino acid sequence of any one of SEQ ID NOs: 300-311 and 347-349.
25. The nucleic acid according to any of the preceding paragraphs, wherein the signal5
sequence encodes a signal peptide.
26. The nucleic acid according to paragraph 25, wherein the signal peptide is present on
the amino-terminus of one or more of the polypeptides of the plurality.
27. The nucleic acid according paragraph 26, wherein the nucleic acid further encodes a
second signal peptide on the amino-terminus of all or some polypeptides of the plurality.10
28. The nucleic acid according to any of the preceding paragraphs, wherein the signal
peptide has an amino acid sequence of any one of SEQ ID NOs: 312-329.
29. The nucleic acid according to any of the preceding paragraphs, wherein the target
sequence encodes a target peptide.
30. The nucleic acid sequence according to paragraph 29, wherein the target peptide is15
encoded by a codon optimized nucleic acid sequence, or fragments, mutants, or variants
thereof.
31. The nucleic acid according to paragraph 30, wherein the target peptide is obtained
from a prokaryote, a eukaryote, a unicellular organism, a multicellular organism, a virus, a
bacterium, a fungus, a protozoan, a worm, a mycoplasma, an animal, a human or a20
combination thereof.
32. The nucleic acid according to paragraph 31, wherein the virus is selected from the
family comprising picornaviride, calciviridae, astroviridae, togaviridae, flaviviridae,
coronaviridae, arteriviridae, rhabndoviridae, filoviridae, paramyxoviridae, bornaviridae,
orthomyxoviridae, bunyaviridae, arenaviridae, reoviridae, retroviridae, polyomaviridae,25
herpesviridae, poxviridae, papillomaviridae, hepadnaviridae, adenoviridae, parvoviridae,
hepeviridae, circoviridae or a combination thereof.
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33. The nucleic acid according to paragraph 31, wherein the bacterium is selected from
the genus comprising Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia,
Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus, Helicobacter,
Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas,
Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio, Yersinia, or a5
combination thereof.
34. The nucleic acid according to paragraph 32, wherein the virus is selected from the
family comprising coronaviridae, herpesviridae, poxviridae, flaviviridae, togaviridae,
retroviridae, paramyxoviridae, or a combination thereof.
35. The nucleic acid according to paragraph 31, wherein the virus is alphacoronavirus,10
betacoronavirus, deltacoronavirus, gammacoronavirus, torovirus or a combination thereof.
36. The nucleic acid according to paragraph 35, wherein the betacoronavirus is a SARS-
CoV-1, a SARS-CoV-2, a MERS-CoV, an OC43, a HKU1, a bat coronavirus, other
betacoronavirus, or a combination thereof.
37. The nucleic acid according to paragraph 30, wherein the target peptide is a spike15
protein, a membrane protein, an envelope protein or a nucleocapsid protein of a coronavirus
or a combination thereof.
38. The nucleic acid according to paragraph 37, wherein the target peptide is a spike
protein, or its fragment thereof.
39. The nucleic acid according to paragraph 38, wherein the target peptide is a receptor20
binding domain, a fusion peptide, a stem helix of the spike protein, or a combination thereof,
including their codon optimized nucleic acid sequences, fragments, mutants or variants
thereof.
40. The nucleic acid according to paragraph 39, wherein the target peptide is a receptor
binding domain obtained or derived from a betacoronavirus comprising SARS-CoV-1,25
SARS-CoV-2, MERS-CoV, OC43, HKU1, bat coronavirus, other betacoronavirus or a
combination thereof.
41. The nucleic acid according to paragraph 30, wherein the target peptide is a
glycoprotein B, a glycoprotein C, a glycoprotein D, a glycoprotein E, a glycoprotein K, a
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glycoprotein L, or a glycoprotein M, of a herpes simplex virus 1 (HSV-1) or a herpes simplex
virus 2 (HSV-2), or a combination thereof, including their codon optimized nucleic acid
sequences, fragments, mutants or variants thereof.
42. The nucleic acid according to paragraph 30, wherein the target peptide is a
glycoprotein B, a glycoprotein H, a glycoprotein L, a glycoprotein M, or a glycoprotein N5
of a human cytomegalovirus (HCMV), or a combination thereof, including their codon
optimized nucleic acid sequences, fragments, mutants, or variants thereof.
43. The nucleic acid according to paragraph 30, wherein the target peptide is a a
glycoprotein B, a glycoprotein C, a glycoprotein H, or a glycoprotein L of a varicella-zoster
virus (VZV), or a combination thereof, including their codon optimized nucleic acid10
sequences, fragments, mutants, or variants thereof.
44. The nucleic acid according to paragraph 30, wherein the target peptide is a
glycoprotein B, a glycoprotein H, a glycoprotein L, a glycoprotein M, a glycoprotein N, a
glycoprotein 42, or a glycoprotein 350 of an Epstein-Barr virus (EBV), or a combination
thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or15
variants thereof.
45. The nucleic acid according to paragraph 30, wherein the target peptide is a F9
membrane protein of a poxvirus, a H3L protein of a poxvirus, an A4 protein of a poxvirus,
an A27 protein of poxvirus, an A33 protein of a poxvirus, an A56 protein of a poxvirus, a
B5 protein of a poxvirus, or a L1 protein of a poxvirus, or a combination thereof, including20
their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
46. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid
protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1,
NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of flaviviruses, hepaciviruses, or a combination
thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or25
variants thereof.
47. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid
protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1,
NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a Japanese encephalitis virus, or a
93
combination thereof, including their codon optimized nucleic acid sequences, fragments,
mutants, or variants thereof.
48. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid
protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1,
NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a zika virus, or a combination thereof,5
including their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof.
49. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid
protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1,
NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a yellow fever virus, or a combination thereof,10
including their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof.
50. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid
protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1,
NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a awest nile virus, or a combination thereof,15
including their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof.
51. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid
protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1,
NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a hepatitis C virus, or a combination thereof,20
including their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof.
52. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid
protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1,
NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a dengue virus, or a combination thereof,25
including their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof.
53. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid
protein, or an envelope protein such as E1, E2 and E3 protein of an alphaviruses, or a
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combination thereof, including their codon optimized nucleic acid sequences, fragments,
mutants, or variants thereof.
54. The nucleic acid according to paragraph 30, wherein the target peptide is a domain
A, a domain B, or a domain C of the E2 protein of an alphaviruses, or a combination thereof,
including their codon optimized nucleic acid sequences, fragments, mutants, or variants5
thereof.
55. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid
protein, or an envelope protein such as E1, E2 and E3 protein of a chikungunya virus, or a
combination thereof, including their codon optimized nucleic acid sequences, fragments,
mutants, or variants thereof.10
56. The nucleic acid according to paragraph 30, wherein the target peptide is a gag, a pol
or an env proteins of retroviruses, or a combination thereof, including their codon optimized
nucleic acid sequences, fragments, mutants, or variants thereof.
57. The nucleic acid according to paragraph 30, wherein the target peptide is a p17Gag, a
p24Gag, a p7Gag, a p6Gag, a gp12Env, a gp41Env, or a pol proteins from lentiviruses, or a15
combination thereof, including their codon optimized nucleic acid sequences, fragments,
mutants, or variants thereof.
58. The nucleic acid according to paragraph 30, wherein the target peptide is a p17Gag, a
p24Gag, a p7Gag, a p6Gag, a gp12Env, a gp41Env, or a pol proteins from a human
immunodeficiency virus (HIV), or a combination thereof, including their codon optimized20
nucleic acid sequences, fragments, mutants, or variants thereof.
59. The nucleic acid according to paragraph 30, wherein the target peptide is a
nucleocapsid protein, a P protein, a V protein, a W protein, a D protein, an I protein, a C
protein, a L protein, a M protein, a H (hemagglutinin) protein, a HN (hemagglutinin-
neuraminidase) protein, a G protein or a F protein, or a combination thereof, of a Mumps25
virus (MuV), a Parainfluenza virus type 5 (PIV5), a Human parainfluenza virus type 2, types
4a and 4b (HPIV2/4a/4b), a Newcastle disease virus (NDV), a Human parainfluenza virus
type 1 and type 3 (HPIV1/3), a Nipah virus (NiV), a Measles virus (MeV), a Human
respiratory syncytial virus A2, B1, S2, (HRSV), or a Human metapneumovirus (HMPV), or
95
a combination thereof, including their codon optimized nucleic acid sequences, fragments,
mutants, or variants thereof.
60. The nucleic acid according to paragraph 30, wherein the target peptide is a
nucleocapsid protein, a P protein, a V protein, a W protein, a D protein, an I protein, a C
protein, a L protein, a M protein, a H (hemagglutinin) protein, a HN (hemagglutinin-5
neuraminidase) protein, a G protein or a F protein of human respiratory syncytial virus A2,
B1, S2 (HRSV), or a combination thereof, including their codon optimized nucleic acid
sequences, fragments, mutants, or variants thereof.
61. The nucleic acid according to paragraph 30, wherein the target peptide is an E1, an E2,
an E4, an E5, an E6, an E7, a L1 or a L2 protein of papillomavirus, preferably a human10
papillomavirus, or a combination thereof, including their codon optimized nucleic acid
sequences, fragments, mutants, or variants thereof.
62. The nucleic acid according to any of the preceding paragraphs, wherein the target
peptide has an amino acid sequence of any one of SEQ ID NOs: 1-253, 334-337, 338-346,
and 353-388.15
63. A lipid nanoparticle composition comprising a cationic lipid, a phospholipid, a sterol,
a PEG-lipid, and the nucleic acid according to any of the preceding paragraphs.
64. The lipid nanoparticle composition according to paragraph 63, wherein the cationic
lipid comprises an ionizable lipid.
65. The lipid nanoparticle composition according to paragraph 64, wherein the ionizable20
lipid is present in an amount from 25 mol percent to 70 mol percent.
66. The lipid nanoparticle composition according to paragraph 63, wherein the
phospholipid is present in an amount from 2 mol percent to about 30 mol percent.
67. The lipid nanoparticle composition according to paragraph 63, wherein the sterol is
present in an amount from 30 mol percent to about 65 mol percent.25
68. The lipid nanoparticle composition according to paragraph 63, wherein the PEG-
lipid is present in an amount from 0.2 mol percent to about 2.0 mol percent.
96
69. The lipid nanoparticle composition according to paragraph 63, additionally
comprising an ionizable polymer.
70. The lipid nanoparticle composition according to paragraph 69, wherein the ionizable
polymer is present in an amount from 1 mol percent to 25 mol percent.
71. The lipid nanoparticle composition according to paragraph 70, wherein the ionizable5
polymer is selected from the group comprising a chitosan, a cellulose derivative, a poly-L-
lysine, a poly-L-glutamic acid, and/or their derivatives or a combination thereof.
72. A method of treating or preventing a disease, comprising administrating to a subject
in need thereof the nucleic acid according to any one of claims 1-62.
73. A method of treating or preventing a disease, comprising administrating to a subject10
in need thereof the lipid nanoparticle composition according to any one of paragraphs 63-
71.
74. Use of the nucleic acid sequence according to any one of paragraphs 1-62, in the
manufacture of a medicament for the treatment or prevention of a disease in a subject.
75. Use of a lipid nanoparticle composition according to any one of paragraphs 63-71 in15
the manufacture of a medicament for the treatment or prevention of a disease in a subject.
76. A multitarget peptide encoded by the nucleic acid of any one of paragraphs 1-62.
77. A multitarget peptide comprising two or more polypeptides, wherein some or all
polypeptides comprises either a target peptide, a linker peptide, and a self-assembling
peptide, or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide20
or a combination thereof, wherein one polypeptide is connected to another polypeptide by a
cleavage peptide, wherein the multitarget peptide includes a signal peptide on the amino-
terminus of one or more of the polypeptides.
78. The multitarget peptide according to paragraph 77, wherein the linker peptide is an
amino acid linker, a foldon, a scaffold or a combination thereof.25
79. The multitarget peptide according to paragraph 78, wherein the amino acid linker
comprises 2-49 amino acids.
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80. The multitarget peptide according to paragraph 78, wherein the amino acid linker is
selected form the group comprising a glycine serine linker, a glycine proline linker, a glycine
threonine linker, an alanine serine linker, any combination of two amino acids, or a
combination thereof.
81. The multitarget peptide according to paragraph 77, wherein the self-assembling5
peptide is selected from the group comprising a lumazine synthase from Aquifex species, a
hepatitis B surface antigen (HBsAg) from Hepatitis B Virus, a hepatitis B core antigen
(HBcAg) from Hepatitis B virus, a human papillomavirus L1 (HPV L1) protein, a matrix
protein M1 from influenza A virus, a ferritin, a riboflavin synthase, or a combination thereof,
including their codon optimized nucleic acid sequences, fragments, mutants, or variants10
thereof.
82. The multitarget peptide according to paragraph 81, wherein the ferritin comprises a
ferritin subunit or a ferritin peptide.
83. The multitarget peptide according to paragraph 82, wherein the ferritin peptide is
derived from Helicobacter pylori ferritin.15
84. The multitarget peptide according to paragraph 77, wherein the cleavage peptide is
a golgi specific cleavage peptide or a self-cleaving cleavage peptide or a combination
thereof.
85. The multitarget peptide according to paragraph 77, wherein the signal peptide may
be present on the amino-terminus of all or some polypeptides.20
86. The multitarget peptide according to paragraph 77, wherein the target peptide is
obtained or derived from a prokaryote, a eukaryote, a unicellular organism, a multicellular
organism, a virus, a bacterium, a fungus, a protozoan, a worm, a mycoplasma, an animal, a
human or a combination thereof.
87. The multitarget peptide according to paragraph 86, wherein the virus is selected from25
the family comprising picornaviride, calciviridae, astroviridae, togaviridae, flaviviridae,
coronaviridae, arteriviridae, rhabndoviridae, filoviridae, paramyxoviridae, bornaviridae,
orthomyxoviridae, bunyaviridae, arenaviridae, reoviridae, retroviridae, polyomaviridae,
98
herpesviridae, poxviridae, papillomaviridae, hepadnaviridae, adenoviridae, parvoviridae,
hepeviridae, circoviridae or a combination thereof.
88. The multitarget peptide according to paragraph 86, wherein the bacterium is selected
from the genus comprising Bacillus, Bordetella, Borrelia, Brucella, Campylobacter,
Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus,5
Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria,
Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio,
Yersinia, or a combination thereof.
89. The multitarget peptide according to paragraph 87, wherein the virus is selected from
the family comprising coronaviridae, herpesviridae, poxviridae, flaviviridae, togaviridae,10
retroviridae, paramyxoviridae, or a combination thereof.
90. The multitarget peptide according to paragraph 89, wherein the virus is
alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus, torovirus or a
combination thereof.
91. The multitarget peptide according to paragraph 90, wherein the betacoronavirus is15
selected from the group comprising a SARS-CoV-1, a SARS-CoV-2, a MERS-CoV, an
OC43, a HKU1, a bat coronavirus, other betacoronavirus, or a combination thereof.
92. The multitarget peptide according to paragraph 77, wherein the target peptide is a
spike protein, a membrane protein, an envelope protein or a nucleocapsid protein of
coronaviruses, or a combination thereof, including their codon optimized nucleic acid20
sequences fragments, mutants or variants thereof.
93. The multitarget peptide according to paragraph 92, wherein the target peptide is a
spike protein or its fragment thereof.
94. The multitarget peptide according to paragraph 93, wherein the target peptide is a
receptor binding domain, a fusion peptide, a stem helix of the spike protein, or a combination25
thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or
variants thereof.
95. The multitarget peptide according to paragraph 77, wherein the target peptide is a
glycoprotein B, a glycoprotein C, a glycoprotein D, a glycoprotein E, a glycoprotein K, a
99
glycoprotein L, or a glycoprotein M of a herpes simplex virus 1 (HSV-1) or a herpes simplex
virus 2 (HSV-2), or a combination thereof, including their codon optimized nucleic acid
sequences, fragments, mutants or variants thereof.
96. The multitarget peptide according to paragraph 77, wherein the target peptide is a
glycoprotein B, a glycoprotein H, a glycoprotein L, a glycoprotein M, or a glycoprotein N5
of a human cytomegalovirus (HCMV), or a combination thereof, including their codon
optimized nucleic acid sequences, fragments, mutants, or variants thereof.
97. The multitarget peptide according to paragraph 77, wherein the target peptide is a
glycoprotein B, a glycoprotein C, a glycoprotein H, or a glycoprotein L of a varicella-zoster
virus (VZV), or a combination thereof, including their codon optimized nucleic acid10
sequences, fragments, mutants, or variants thereof.
98. The multitarget peptide according to paragraph 77, wherein the target peptide is a
glycoprotein B, a glycoprotein H, a glycoprotein L, a glycoprotein M, a glycoprotein N, a
glycoprotein 42, a glycoprotein 350 of an Epstein-Barr virus (EBV), or a combination
thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or15
variants thereof.
99. The multitarget peptide according to paragraph 77, wherein the target peptide is a F9
membrane protein of a poxvirus, a H3L protein of a poxvirus, an A4 protein of a poxvirus,
an A27 protein of poxvirus, an A33 protein of a poxvirus, an A56 protein of a poxvirus, a
B5 protein of a poxvirus, or a L1 protein of a poxvirus, or a combination thereof, including20
their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
100. The multitarget peptide according to paragraph 77, wherein the target peptide is a
capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such
as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of flaviviruses, hepaciviruses, or a
combination thereof, including their codon optimized nucleic acid sequences, fragments,25
mutants, or variants thereof.
101. The multitarget peptide according to paragraph 77, wherein the target peptide is a
capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such
as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a Japanese encephalitis virus, or a
100
combination thereof, including their codon optimized nucleic acid sequences, fragments,
mutants, or variants thereof.
102. The multitarget peptide according to paragraph 77, wherein the target peptide is a
capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such
as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a zika virus, or a combination thereof,5
including their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof.
103. The multitarget peptide according to paragraph 77, wherein the target peptide is a
capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such
as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a yellow fever virus, or a combination10
thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or
variants thereof.
104. The multitarget peptide according to paragraph 77, wherein the target peptide is a
capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such
as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a west nile virus, or a combination15
thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or
variants thereof.
105. The multitarget peptide according to paragraph 77, wherein the target peptide is a
capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such
as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a hepatitis C virus, or a combination20
thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or
variants thereof.
106. The multitarget peptide according to paragraph 77, wherein the target peptide is a
capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such
as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a dengue virus, or a combination25
thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or
variants thereof.
107. The multitarget peptide according to paragraph 77, wherein the target peptide is a
capsid protein, or an envelope protein such as E1, E2 and E3 protein of an alphaviruses, or
101
a combination thereof, including their codon optimized nucleic acid sequences, fragments,
mutants, or variants thereof.
108. The multitarget peptide according to paragraph 77, wherein the target peptide is a
domain A, a domain B, a domain C of the E2 protein of an alphaviruses, or a combination
thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or5
variants thereof.
109. The multitarget peptide according to paragraph 77, wherein the target peptide is a
capsid protein, or an envelope protein such as E1, E2 and E3 protein of a chikungunya virus,
or a combination thereof, including their codon optimized nucleic acid sequences,
fragments, mutants, or variants thereof.10
110. The multitarget peptide according to paragraph 77, wherein the target peptide is a
gag, a pol and an env proteins of retroviruses, or a combination thereof, including their codon
optimized nucleic acid sequences, fragments, mutants, or variants thereof.
111. The multitarget peptide according to paragraph 77, wherein the target peptide is a
p17Gag, a p24Gag, a p7Gag, a p6Gag, a gp12Env, a agp41Env, or a pol proteins from lentiviruses,15
or a combination thereof, including their codon optimized nucleic acid sequences,
fragments, mutants, or variants thereof.
112. The multitarget peptide according to paragraph 77, wherein the target peptide is a
p17Gag, a p24Gag, a p7Gag, a p6Gag, a gp12Env, a gp41Env, or a pol proteins from a human
immunodeficiency virus (HIV), or a combination thereof, including their codon optimized20
nucleic acid sequences, fragments, mutants, or variants thereof.
113. The multitarget peptide according to paragraph 77, wherein the target peptide is a
nucleocapsid protein, a P protein, a V protein, a W protein, a D protein, an I protein, a C
protein, a L protein, a M protein, a H (hemagglutinin) protein, a HN (hemagglutinin-
neuraminidase) protein, a G protein, a F protein, or a combination thereof, of a Mumps virus25
(MuV), a Parainfluenza virus type 5 (PIV5), a Human parainfluenza virus type 2, types 4a
and 4b (HPIV2/4a/4b), a Newcastle disease virus (NDV), a Human parainfluenza virus type
1 and type 3 (HPIV1/3), a Nipah virus (NiV), a Measles virus (MeV), a Human respiratory
syncytial virus A2, B1, S2, (HRSV), or a Human metapneumovirus (HMPV), or a
102
combination thereof, including their codon optimized nucleic acid sequences, fragments,
mutants, or variants thereof.
114. The multitarget peptide according to paragraph 77, wherein the target peptide is a
nucleocapsid protein, a P protein, a V protein, a W protein, a D protein, an I protein, a C
protein, a L protein, a M protein, a H (hemagglutinin) protein, a HN (hemagglutinin-5
neuraminidase) protein, a G protein or a F protein of a human respiratory syncytial virus A2,
B1, S2 (HRSV), or a combination thereof, including their codon optimized nucleic acid
sequences, fragments, mutants, or variants thereof.
115. The multitarget peptide according to paragraph 77, wherein the target peptide is an
E1, an E2, an E4, an E5, an E6, an E7, a L1, or a L2 protein of a papillomavirus, preferably10
a human papillomavirus, or a combination thereof, including their codon optimized nucleic
acid sequences, fragments, mutants, or variants thereof.
116. The multitarget peptide according to any one of paragraphs 77-115, wherein total
number of the polypeptides are not more than 100.
117. The multitarget peptide according to paragraph 116, wherein total number of the15
polypeptides is between 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or
90-99.
118. A Polypeptide nanoparticle comprising at least 2 or up to 100 polypeptides according
to any one of paragraphs 1 to 62 or 77-115.
119. The polypeptide nanoparticle according to paragraph 118, wherein the polypeptides20
are homologous polypeptides, heterologous polypeptides, oligomeric complex or
combination thereof.
120. The polypeptide nanoparticle according to paragraph 118, wherein the polypeptide
nanoparticle is icosahedral, helical, spherical, rod-like or a combination thereof.
25
Examples
Example 1: Synthesis of multitarget nucleic acid sequence (mRNA)
Plasmid DNA construction
103
The multitarget nucleic acid sequence comprising of signal sequence, three repeats
of polynucleotide sequences, each separated by cleavage sequence, was codon optimized for
human expression. The multitarget nucleic acid sequence was synthesised by Twist
BioSciences, USA. Each polynucleotide sequence consisted of a different target sequence.
The first polynucleotide sequence consisted of a target sequence (encoding receptor binding5
domain (RBD) of SARS-CoV-2), linker sequence (glycine serine linker sequence encoding
glycine serine linker, foldon sequence encoding foldon, & glycine serine linker sequence
encoding glycine serine linker) and a self-assembling sequence (ferritin sequence encoding
ferritin). The second polynucleotide sequence consisted of a linker sequence (scaffold
sequence encoding scaffold, and glycine serine linker sequence encoding glycine serine10
linker), a target sequence (encoding fusion peptide of SARS-CoV-2), a linker sequence
(glycine serine linker sequence encoding glycine serine linker, scaffold sequence encoding
scaffold, and glycine serine linker sequence encoding glycine serine linker), and a self-
assembling sequence (ferritin sequence encoding ferritin). The third polynucleotide
sequence consisted of a linker sequence (scaffold sequence encoding scaffold, and glycine15
serine linker sequence encoding glycine serine linker), a target sequence (encoding stem
helix of SARS-CoV-2), a linker sequence (glycine serine linker sequence encoding glycine
serine linker, scaffold sequence encoding scaffold, and glycine serine linker sequence
encoding glycine serine linker) and a self-assembling sequence (ferritin sequence encoding
ferritin). A signal sequence (golgi target signal sequence) was included upstream of the first20
polynucleotide sequence. 5’ UTR, cap, and 3’ UTR sequences were also included in the
multitarget nucleic acid sequence. The multitarget nucleic acid sequence construct was
inserted between HindIII and BamHI restriction sites of pTwist Kan High Copy (Twist
BioScience, USA). Sequence of the multitarget peptide encoded by the above multitarget
nucleic acid sequence, is provided Table 1.25
Table 1 shows Sequence of the multitarget peptide encoded by the above multitarget
nucleic acid sequence.
Signal Peptide MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAA (SEQ ID NO: 312)
First
Polypeptide
Target Peptide (RBD of
SARS-CoV-2)
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSV
LYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG
KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPF
ERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVV
LSFELLHAPATVCGPKKSTNLVKNKCVNF (SEQ ID NO: 1)
Linker
Peptide
Glycine
serine linker GSGG (SEQ ID NO: 330)
104
Foldon YIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 268)
Glycine
serine linker GGGGSGGGGS (SEQ ID NO: 266)
Self-Assembling
peptide (ferritin
peptide)
LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAA
EEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISE
SINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNEN
HGLYLADQYVKGI (SEQ ID NO: 331)
Cleavage Peptide RRKRSVS (SEQ ID NO: 300)
Second
Polypeptide
Linker
Peptide
Scaffold HENEISHHAKEIERLQKEIERHKQSIKKLKQSE (SEQ ID NO: 269)
Glycine
serine linker GGGGSGGGGSGGGGS (SEQ ID NO: 267)
Target Peptide (Fusion
Peptide of SARS-CoV-2)
SFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNG (SEQ ID
NO: 3)
Linker
Peptide
Glycine
serine linker GGGGSGGGGSGGGGS (SEQ ID NO: 267)
Scaffold HENEISHHAKEIERLQKEIERHKQSIKKLKQSE (SEQ ID NO: 269)
Glycine
serine linker GGGGSGGGGS (SEQ ID NO: 266)
Self-Assembling
peptide (ferritin
peptide)
LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAA
EEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISE
SINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNEN
HGLYLADQYVKGI (SEQ ID NO: 331)
Cleavage Peptide RRKRSVS (SEQ ID NO: 300)
Third
Polypeptide
Linker
Peptide
Scaffold HENEISHHAKEIERLQKEIERHKQSIKKLKQSE (SEQ ID NO: 269)
Glycine
serine linker GGGGSGGGGSGGGGS (SEQ ID NO: 267)
Target Peptide (Stem
Helix of SARS-CoV-2) LQPELDSFKEELDKYFKNHTSPDVDLG (SEQ ID NO: 4)
Linker
Peptide
Glycine
serine linker GGGGSGGGGSGGGGS (SEQ ID NO: 267)
Scaffold HENEISHHAKEIERLQKEIERHKQSIKKLKQSE (SEQ ID NO: 269)
Glycine
serine linker GGGGSGGGGS (SEQ ID NO: 266)
Self-Assembling
peptide (ferritin
peptide)
LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAA
EEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISE
SINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNEN
HGLYLADQYVKGIRRKR (SEQ ID NO: 333)
Amino acid sequence of different peptides present in the multitarget peptide encoded
by the multitarget nucleic acid sequence of example 1 is separately identified in:
Complete sequence of the multitarget peptide encoded by the multitarget nucleic acid5
of example 1
MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAARVQPTESIVRFPNITNLCPFGEV
FNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYA
DSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYL10
YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPY
RVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFGSGGYIPEAPRDGQAYVRKDG
EWVLLSTFLGGGGSGGGGSLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGA
105
GLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISES
INNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVK
GIRRKRSVSHENEISHHAKEIERLQKEIERHKQSIKKLKQSEGGGGSGGGGSGGGG
SSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGGGGGSGGGGS
GGGGSHENEISHHAKEIERLQKEIERHKQSIKKLKQSEGGGGSGGGGSLSKDIIKLLN5
EQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENN
VPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWY
VAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIRRKRSVSHENEISHHAKEI
ERLQKEIERHKQSIKKLKQSEGGGGSGGGGSGGGGSLQPELDSFKEELDKYFKN
HTSPDVDLGGGGGSGGGGSGGGGSHENEISHHAKEIERLQKEIERHKQSIKKLKQ10
SEGGGGSGGGGSLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLF
LFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQH
ISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHG
LYLADQYVKGIRRKR (SEQ ID NO: 332)
*The different bolded, underlined, and italicized sequences correspond to the different15
sequences in Table 1.
Plasmid DNA isolation and linearization
Plasmid DNA was obtained by standard techniques. Briefly, the plasmid DNA was
introduced into E. coli DH5 alpha cells (Stellar™ Competent Cells Catalogue no. 636763,20
Takara Bio) by giving heat shock for 60-90 seconds at 42 oC, followed by cold shock for 5
min on ice. The cells were transferred to LB (Lauria-Bertani) medium in a tube and
incubated for 1 h at 37 oC on a thermal shaker at 600-900 rpm. The cells were recovered and
centrifuged at 4000 g for 2 min, pellet was collected and resuspended in the same medium.
Resuspended cells were transferred to agar plate containing LB medium supplemented with25
kanamycin and cells were spread evenly using sterile glass beads and incubated for 14-16 h
at 37 oC. Well grown colonies were picked and resuspended in LB medium supplemented
with kanamycin and colony PCR was performed to check the presence of pDNA. The
colonies were further cultured overnight and NucleoSpin® Plasmid Miniprep kit (catalogue
no. 740588, Takara Bio Inc.) was used to isolate the pDNA. The pDNA was linearized by30
digestion with BbsI-HF restriction enzyme (catalogue no. R3539L, New England Biolabs
Inc,). pDNA was quantified using NanoDrop™ One/OneC Microvolume UV-Vis
Spectrophotometer (Catalogue no. ND-ONE-W, Thermo Scientific).
Invitro transcription (IVT)35
IVT was performed following standard procedure described in HiScribe™ T7 High
Yield RNA Synthesis Kit (Catalogue no. E2040S, New England BioLabs Inc.). Briefly, IVT
mix containing four ribonucleotide triphosphates (ATP, UTP, GTP, CTP), NEB T7 buffer,
106
NEB T7 enzyme mix, CleanCap® Reagent AG (3' OMe) (Catalogue no, N-7413, TriLink
BioTechnologies), and linearized plasmid DNA was taken in a PCR tube containing nuclease
free sterile water. The mixture was incubated for 37 oC for about 2 hours. This was followed
by DNase I (Catalogue no. M0303S, New England BioLabs Inc.) treatment to degrade any
plasmid DNA. The mRNA was purified as per instructions given in Monarch® RNA5
Cleanup Kit (Catalogue no. T2050L, New England BioLabs Inc.). Poly A tail was added to
purified mRNA following standard post tailing procedure using E. coli Poly(A) Polymerase
(Catalogue no. M0276L, New England BioLabs). The mRNA with poly A tail was purified
using Monarch® RNA Cleanup Kit (Catalogue no. T2050L, New England BioLabs Inc.).
The RNA was quantified by Qubit™ RNA BR, Assay Kits (Catalogue no. Q10210, Thermo10
Scientific) using Qubit 4 Fluorometer (Thermo Scientific).
Transfection
HEK293T cells (Catalogue no. CRL3216, ATCC) were seeded at 0.25 – 1 x 106 cells
in a 6 well plate containing Gibco DMEM medium, high glucose, pyruvate (Catalogue no.15
11995065, ThermoFisher Scientific) supplemented with 10% fetal bovine serum and 100
units/mL penicillin-streptomycin, and cultured to achieve 70-80% confluency. Transfection
mix containing multitarget nucleic acid (mRNA, 3 ug of example 1) and Lipofectamine™
2000 (9 uL) in 100 uL serum free medium was incubated for 15 min at room temperature
and added to the cells and left for incubation for 24 and 48 hours. Spent media was collected20
by aspiration, and cells were collected with ice-cold 1x PBS (1 mL) with hard pipetting. The
cells were centrifuged at 4000 g for 5 min at 4 °C. Excess PBS was aspirated. The cells were
suspended gently in 300 μL of 1x NETN lysis buffer with inhibitors and kept on ice for 20
min. The lysate was centrifuged at 16,000 g for 20 min at 4 °C. The supernatant was
aspirated into microtubes.25
Western Blot
6x Laemmli buffer was added to each sample (supernatant or cell lysate) to obtain a
final concentration of 1x. The samples were heated at 95 °C for 5 min and centrifuged at
10,000 g for 5 min. About 21 μL of cell lysate and 42 μL of supernatant along with molecular30
weight standard were added to the gel (NuPAGE 4-12% Bis-Tris Gel) and the gel was run
using a mops-SDS running buffer. The proteins were transferred onto the PVDF membrane
using iBlot 2 blotting system. The blots were blocked using 5% skimmed milk in 1x TBST
107
for 1 h and incubated with primary antibody (SARS-CoV-2 (2019-nCoV) Spike Antibody,
Rabbit PAb Catalogue no. 40589-T62, Sino Biological Inc., or SARS-CoV-2 spike RBD
polyclonal antibody (Catalogue no. E-AB-V-1006 Elab Sciences) for 1 h at room
temperature. The membrane was washed thrice, and incubated with secondary antibody (anti
rabbit IgG HRP Catalogue no. 7074S, manufacturer) for 1 h. The membrane was washed5
thrice and 50 μL luminol and 50 μL peroxide solution was used for signal development. The
image was captured using ChemiDoc™ Imaging System (Bio-Rad). The results are shown
in figures 3a and 3b.
ELISA10
ELISA was performed on cell lysate and supernatant by following instructions as
provided in SARS-CoV-2 (2019-nCoV) Spike Detection ELISA Kit (Catalogue no.
KIT40591, Sino Biological, Inc.). The results are shown in figures 4a and 4b.
Transmission Electron Microscopy (TEM)15
Post 48 h of transfection, 1 mL of the spent media is concentrated to 0.5 mL with
ultrafiltration using a 100 kDa concentrator (Catalogue no. UFC210024, Millipore Amicon
Ultra-2 mL) at 3000 g for 3 min. The sample was buffer exchanged 10 times with 1x PBS
using centricon filter. Finally, the sample was further concentrated to 200-250 μL by
ultrafiltration. 10 μL of the sample was placed on a glow-discharged carbon-coated copper20
grid of 400 mesh and incubated for 1 min. The excess sample was removed by careful
soaking with blotting paper. 10 μL of UranyLess staining reagent (Catalogue no. 22409,
Electron Microscopy Sciences) was added and incubated for 1 min. Excess sample was
removed as above. The sample was air-dried by incubating at room temperature for 1 h.
TEM images were captured by Talos™ L120C TEM (ThermoFisher Scientific). The results25
are shown in figure 5.
Analytical or Immunogenicity assays
The testing of multitarget nucleic acid sequence comprises immunizing animals30
(typically mice) with multitarget nucleic acid sequence in appropriate formulation following
prime-boost immunization strategy at pre-determined dosage amounts. The serum is
collected at appropriate intervals and antibody response against the target peptide is
measured by ELISA.
108
The efficacy of the multitarget nucleic acid sequence is evaluated by pseudovirus
neutralization assays well known to persons skilled in the art. The method typically involves
incubating the pseudovirus in the presence of different concentrations of immunised serum
containing the antibody of interest (i.e., antibodies produced against the target peptide) and
adding this mixture to the cells and incubating it further to measure luminescence to5
determine inhibitory or neutralization titre.
Example 2: synthesis of multitarget nucleic sequence (mRNA) – trivalent RBD
construct
Plasmid DNA construction10
The multitarget nucleic acid sequence of example 2 comprising three signal
sequences, three repeats of polynucleotide sequences, each separated by cleavage sequence
was codon optimized for human expression. The multitarget nucleic acid sequence was
synthesized by Twist BioSciences, USA. Each polynucleotide sequence consisted of a
different target sequence. The first polynucleotide sequence consisted of a target sequence15
(encoding receptor binding domain (RBD) of SARS-CoV-2 SBB.1.5 variant), a linker
sequence (glycine serine linker sequence encoding glycine serine linker) and a self-
assembling sequence (ferritin sequence encoding ferritin). The second polynucleotide
sequence consisted of a target sequence (encoding receptor binding domain (RBD) of
SRAS-CoV-1), a linker sequence (glycine serine linker sequence encoding glycine serine20
linker), and a self-assembling sequence (ferritin sequence encoding ferritin). The third
polynucleotide sequence consisted of a target sequence (encoding receptor binding domain
(RBD) of MERS-CoV), a linker sequence (glycine serine linker sequence encoding glycine
serine linker), and a self-assembling (ferritin sequence encoding ferritin). The
polynucleotide sequences were separated by a cleavage sequence encoding two cleavage25
peptides (cleavage peptide-1 and cleavage peptide-2) connected by a linker sequence
(glycine serine linker sequence encoding glycine serine linker). A signal sequence was
included upstream of each of the three polynucleotide sequences. 5’ UTR, cap, and 3’ UTR
sequences were also included in the multitarget nucleic acid sequence. The multitarget
nucleic acid sequence construct was inserted between HindIII and BamHI restriction sites30
of pTwist Kan High Copy (Twist BioScience, USA). Sequence of the multitarget peptide
encoded by the above multitarget nucleic acid sequence of example 2, is provided in Table
2.
109
Table 2: shows sequence of the multitarget peptide encoded by the multitarget
nucleic acid sequence – trivalent RBD construct of example 2.
Signal Peptide MGVKVLFALICIAVAEA (SEQ ID NO: 329)
First
Polypeptid
e
Target Peptide (RBD
of SARS-CoV-2
XBB.1.5)
RVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVA
DYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVS
QIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNY
LYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSY
GFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKC
VNF (SEQ ID NO: 338)
Linker
Peptid
e
Glycine
serine
linker
GGGGSGGGGS (SEQ ID NO: 266)
Self-Assembling
peptide (ferritin
peptide)
LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLF
DHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQK
AYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVL
FKDILDKIELIGNENHGLYLADQYVKGI (SEQ ID NO: 331)
Cleavage
Peptide
Cleavage Peptide-1 RRKRSVS (SEQ ID NO: 300)
Linker Peptide GSG (SEQ ID NO: 350)
Cleavage Peptide-2 ATNFSLLKQAGDVEENPGP (SEQ ID NO: 347)
Signal Peptide MGVKVLFALICIAVAEA (SEQ ID NO: 329)
Second
Polypeptid
e
Target Peptide (RBD
of SARS-CoV-1)
RVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCV
ADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDD
VRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNY
NYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLN
DYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQC
VNF (SEQ ID NO: 339)
Linker
Peptid
e
Glycine
serine
linker
GGGGSGGGGS (SEQ ID NO: 266)
Self-Assembling
peptide (ferritin
peptide)
LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLF
DHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQK
AYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVL
FKDILDKIELIGNENHGLYLADQYVKGI (SEQ ID NO: 331)
Cleavage
Peptide
Cleavage Peptide-1 RRKRSVS (SEQ ID NO: 300)
Linker Peptide GSG (SEQ ID NO: 350)
Cleavage Peptide-2 ATNFSLLKQAGDVEENPGP (SEQ ID NO: 347)
Signal Peptide MGVKVLFALICIAVAEA (SEQ ID NO: 329)
Third
Polypeptid
e
Target Peptide (RBD
of MERS-CoV)
EAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNY
NLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSD
LSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINK
CSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQL
SPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKL
EFANDTKIASQLGNCVEY (SEQ ID NO: 340)
Linker
Peptid
e
Glycine
serine
linker
GGGGSGGGGS (SEQ ID NO: 266)
Self-Assembling
peptide (ferritin
peptide)
LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLF
DHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQK
110
AYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVL
FKDILDKIELIGNENHGLYLADQYVKGIRRKR (SEQ ID NO: 333)
Amino acid sequence of different peptides present in the multitarget peptide encoded
by the multitarget nucleic acid sequence of example 1 is separately identified in:
Complete sequence of the multitarget peptide encoded by the multitarget nucleic acid5
of example 2
MGVKVLFALICIAVAEARVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRI
SNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQT
GNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTE10
IYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCG
PKKSTNLVKNKCVNFGGGGSGGGGSLSKDIIKLLNEQVNKEMNSSNLYMSMSS
WCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEG
LTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKD
ILDKIELIGNENHGLYLADQYVKGIRRKRSVSGSGATNFSLLKQAGDVEENPGP15
MGVKVLFALICIAVAEARVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISN
CVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIAD
YNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGK
PCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCV
NFGGGGSGGGGSLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFL20
FDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESI
NNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQY
VKGIRRKRSVSGSGATNFSLLKQAGDVEENPGPMGVKVLFALICIAVAEA
EAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFS
VNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSN25
PTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSI
VPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGT
DTNSVCPKLEFANDTKIASQLGNCVEYGGGGSGGGGSLSKDIIKLLNEQVNKEMN
SSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHK
FEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILD30
KIELIGNENHGLYLADQYVKGIRRKR (SEQ ID NO: 351)
*The different bolded, underlined, and italicized sequences correspond to the different
sequences in Table 2.
Plasmid DNA isolation and linearization, and Invitro transcription (IVT) of the35
multitarget nucleic acid sequence of example 2 has been performed as described in example
1.
Transfection
HEK293T cells (Catalogue no. CRL3216, ATCC) were seeded at 0.05 x106 cells in40
a 24 well plate containing Gibco DMEM medium, high glucose, pyruvate (Catalogue no.
11995065, ThermoFisher Scientific) supplemented with 10% fetal bovine serum and 100
111
units/mL penicillin-streptomycin, and cultured to achieve 70-80% confluency. Transfection
mix containing multitarget nucleic acid sequence of example 2 (mRNA, 0.5-1 ug of example
2) and Lipofectamine™ 2000 (2.5 uL) in 100 uL serum free medium was incubated for 15
min at room temperature and added to the cells and left for incubation for 24 and 48 hours.
Spent media was collected by aspiration, and cells were collected with ice-cold 1x PBS (15
mL) with hard pipetting. The cells were centrifuged at 4000 g for 5 min at 4 °C. Excess PBS
was aspirated. The cells were suspended gently in 100 μL of 1x NETN lysis buffer (100 mM
NaCl, 20 mM Tris-Cl (pH 8.0), 0.5 mM EDTA, 0.5 % (v/v) Nonidet P-40 (NP-40)) with
inhibitors and kept on ice for 20 min. The lysate was centrifuged at 16,000 g for 20 min at 4
°C. The supernatant was aspirated into microtubes.10
Western Blot
A western blot was performed on cell lysate, obtained in the previous step, using Jess
instrument (an automated western blot system Catalogue no. 004-650, ProteinSimple, Bio-
Techne). The cell lysate was diluted and combined with 1 part 5x fluorescent master mix15
(component of Separation Module compatible with Jess, Catalogue no. SM-W001,
ProteinSimple, Bio-Techne) and heated for 5 min at 95 °C. After protein denaturation, the
sample was mixed with luminol-S and peroxide as per manufacturer protocol (Separation
Module, Catalogue no. SM-W001, ProteinSimple, Bio-Techne). The primary antibody
(SARS-CoV-2 spike RBD polyclonal antibody (Catalogue no. E-AB-V-1006 Elab20
Sciences), diluted at a 1:200 ratio along with a secondary-HRP ready to use anti-rabbit
antibody and chemiluminescent substrate (both components of Detection Module
compatible with Jess, Catalogue no. DM-001, ProteinSimple, Bio-Techne), were dispensed
into assigned wells within a provided microplate by the manufacturer. This plate was then
inserted into the Jess instrument where samples were drawn into individual capillaries25
located on a 25-capillary cassette (12-230 kDa Separation Module Catalogue no. SM-W001,
ProteinSimple, Bio-Techne). Electrophoresis and immunodetection were conducted in
automated manner by the Jess instrument. The data was obtained by using the compass
software associated with Jess system. The results are shown in Figure 6.
30
Immunogenicity Assay
Immunogenicity of multitarget nucleic acid encoding multitarget peptide of the
example 2 was assessed by ELISA. Mice were immunized with the multitarget nucleic acid
112
sequence (mRNA) – trivalent RBD construct encapsulated in a lipid nanoparticle and sera
collected on day 28 was used to perform the ELISA to find out titre against each of the target
peptides encoded by the construct of example 2.
Plate preparation: Three ELISA plates were coated, each with S1 subunit of spike
protein either from SARS-CoV-2 (XBB 1.5 variant), SARS-CoV-1, or MERS-CoV at a5
concentration of 100 ng in 100 μL coating buffer (1X PBS, pH 7.4) per well. The ELISA
plates were gently tapped to ensure the S1 subunit of spike protein is evenly coated on the
bottom of every well of the three ELISA plates. The ELISA plates were covered with a plate
sealer and incubated overnight at 4 °C. After overnight incubation, coating buffer was
discarded and the ELISA plates were washed using 300 μL wash buffer (1X PBS10
supplemented with 0.05% (v/v) Tween20) thrice. The ELISA plates were blotted on paper
towel to remove residual liquid from the wells. 200 μL of blocking buffer (1x PBS with 3%
NFDM) was added to each well and the plates were incubated at room temperature for 2 h.
Sample preparation: Mice sera samples were 3-fold serially diluted from 1:100 up to
1:218700 in 1x PBS containing 1% NFDM in a dilution plate.15
Testing: Blocking solution from the ELISA plates was discarded and residual liquid
was removed from the wells using paper towel. ELISA plates were washed thrice using wash
buffer (composition as previously mentioned). 100 μL of the diluted sera sample from
dilution plate was transferred to ELISA plates (in duplicates) as per the predefined plate
layout. ELISA plates were covered with plate sealer and incubated at room temperature for20
1 h. After incubation, sera sample was discarded from the ELISA plates and the plates were
washed thrice using wash buffer (composition as previously mentioned).100 μL of diluted
secondary antibody solution (1:5000 in 1X PBS) was added to each well and the ELISA
plates were covered with plate sealer and incubated at room temperature for 1 h. ELISA
plates were then washed thrice using wash buffer (composition as previously mentioned).25
100 μL of TMB substrate was added to each well and incubated for 15 min. The enzyme
substrate reaction was stopped by adding 50 μL of 1M HCL to each well. The absorbance
was recorded after 5 min using Spark multimode microplate reader (Tecan Trading AG) at
the dual wavelength of 450 nm and 630 nm. End point titre was calculated in the following
manner:30
Cutoff = (Mean of negative control (NC) of all plates) x 4
NC = wells devoid of any sample i.e., containing PBS (phosphate buffered saline).
113
Endpoint Titre = (Respective Dilution/Cutoff value) x Respective Mean Absorbance
Value
Endpoint Titre was considered as the highest sample dilution sample above the
cutoff. The results are shown in figure 7.
5
Pseudovirus Generation and Pseudovirus Neutralization Assay
Lentiviral HIV-1-based pseudoviruses harbouring spike glycoprotein of SARS-CoV-
2 XBB.1.5), SARS-CoV-1 and MERS-CoV were generated by transfecting Lenti-X™ 293T
cells (Catalogue no. 632180, Takara Bio) with 2nd generation packaging plasmid psPAX2
vector (Catalogue no. V010353, Novopro Labs), spike expressing plasmids (Twist10
Biosciences), and lentiviral firefly luciferase reporter plasmid (Catalogue no. LR151, Alstem
Inc.,) with Lipofectamine™ 3000 Transfection Reagent (Catalogue no. L3000150,
Invitrogen). After 12-16 hours of incubation at 37º C and 5% CO2, the transfection media
was replaced with DMEM+10% FBS. The supernatants containing pseudovirus particles
were collected 72 hours post-transfection, centrifuged, filtered, aliquoted, and stored at -8015
ºC.
A pseudovirus neutralization assay was performed to evaluate neutralizing
antibodies in pooled sera from the groups of six mice immunized with multitarget nucleic
acid sequence - trivalent RBD construct of example 2. Briefly, in tissue culture-treated white20
opaque 96 well plates, 1x104 ACE2 stably expressing HEK293 cells (Catalogue no. 79951,
BPS Biosciences) were seeded per well in growth media (DMEM+10% FBS+ 1% Pen-
Strep) and incubated overnight at 37 ºC and 5% CO2. Next, 3-fold serially diluted pooled
mouse sera starting from 1:10 initial dilution was prepared in growth media and mixed in a
1:1 ratio with respective pseudoviruses that yield Relative Luminescence Units (RLU)25
values ranging from 0.5x105 RLU to 5x105 RLU. The mixture is incubated at 37 ºC for one
hour, added to the HEK293-ACE cells, and incubated at 37º C and 5% CO2. After 72 hours,
luminescence activity was measured using Bright-Glo™ Luciferase Assay System
(Catalogue no. E2650, Promega) on Tecan Spark multimode Reader via Spark control
Magellan 3.1 software (Tecan Trading AG). Percentage neutralization was calculated by30
normalizing the test RLU with virus control and cell-only control RLU. The pseudovirus
neutralization reciprocal IC50 ((half-maximal inhibitory concentration) titres were
calculated using a non-linear regression curve fit ‘log(inhibitor) vs. response -- Variable
114
slope (four parameters)’ in GraphPad Prism 10. The results are shown in figures 8a, 8b and
8c.
Example 3: synthesis of multitarget nucleic sequence (mRNA) – pentavalent RBD
construct5
Plasmid DNA construction
The multitarget nucleic acid sequence of example 3 comprising five signal
sequences, five repeats of polynucleotide sequences, each separated by cleavage sequence
was codon optimized for human expression. The multitarget nucleic acid sequence was
synthesized by Twist BioSciences, USA. Each polynucleotide sequence consisted of a10
different target sequence. The first polynucleotide sequence consisted of a target sequence
(encoding receptor binding domain (RBD) of SARS-CoV-2 SBB.1.5 variant), a linker
sequence (glycine serine linker sequence encoding glycine serine linker) and a self-
assembling sequence (ferritin sequence encoding ferritin). The second polynucleotide
sequence consisted of a target sequence (encoding receptor binding domain (RBD) of15
SRAS-CoV-1), a linker sequence (glycine serine linker sequence encoding glycine serine
linker), and a self-assembling sequence (ferritin sequence encoding ferritin). The third
polynucleotide sequence consisted of a target sequence (encoding receptor binding domain
(RBD) of MERS-CoV), a linker sequence (glycine serine linker sequence encoding glycine
serine linker), and a self-assembling sequence (ferritin sequence encoding ferritin). The20
fourth polynucleotide sequence consisted of a target sequence (encoding receptor binding
domain (RBD) of OC43), a linker sequence (glycine serine linker sequence encoding glycine
serine linker), and a self-assembling sequence (ferritin sequence encoding ferritin). The fifth
polynucleotide sequence consisted of a target sequence (encoding receptor binding domain
(RBD) of HKU1), a linker sequence (glycine serine linker sequence encoding glycine serine25
linker), a self-assembling sequence (ferritin sequence encoding ferritin). The polynucleotide
sequences were separated by a cleavage sequence encoding two cleavage peptides (cleavage
peptide-1 and cleavage peptide-2) connected by a linker sequence (glycine serine linker
sequence encoding glycine serine linker). A signal sequence was included upstream of each
of the five polynucleotide sequences. 5’ UTR, cap, and 3’ UTR sequences were also included30
in the multitarget nucleic acid sequence. The multitarget nucleic acid sequence construct
was inserted between HindIII and BamHI restriction sites of pTwist Kan High Copy (Twist
115
BioScience, USA). Sequence of the multitarget peptide encoded by the multitarget nucleic
acid sequence – pentavalent RBD construct of example 3, is provided in Table 3.
Table 3: shows sequence of the multitarget peptide encoded by the multitarget
nucleic acid sequence – pentavalent RBD construct of example 3.5
Signal Peptide MGVKVLFALICIAVAEA (SEQ ID NO: 329)
First
Polypeptide
Target Peptide
(RBD of SARS-CoV-
2 XBB.1.5)
RVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCV
ADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNE
VSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGN
YNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSP
LQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLV
KNKCVNF (SEQ ID NO: 338)
Linker
Peptide
Glycine
serine
linker
GGGGSGGGGS (SEQ ID NO: 266)
Self-Assembling
peptide (ferritin
peptide)
LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFL
FDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQ
KAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEE
VLFKDILDKIELIGNENHGLYLADQYVKGI (SEQ ID NO: 331)
Cleavage
Peptide
Cleavage Peptide-1 RRKRSVS (SEQ ID NO: 300)
Linker Peptide GSG (SEQ ID NO: 350)
Cleavage Peptide-
2 ATNFSLLKQAGDVEENPGP (SEQ ID NO: 347)
Signal Peptide MGVKVLFALICIAVAEA (SEQ ID NO: 329)
Second
Polypeptide
Target Peptide
(RBD of SARS-CoV-
1)
RVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNC
VADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKG
DDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATST
GNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCY
WPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTD
LIKNQCVNF (SEQ ID NO: 339)
Linker
Peptide
Glycine
serine
linker
GGGGSGGGGS (SEQ ID NO: 266)
Self-Assembling
peptide (ferritin
peptide)
LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFL
FDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQ
KAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEE
VLFKDILDKIELIGNENHGLYLADQYVKGI (SEQ ID NO: 331)
Cleavage
Peptide
Cleavage Peptide-1 RRKRSVS (SEQ ID NO: 300)
Linker Peptide GSG (SEQ ID NO: 350)
Cleavage Peptide-
2 ATNFSLLKQAGDVEENPGP (SEQ ID NO: 347)
Signal Peptide MGVKVLFALICIAVAEA (SEQ ID NO: 329)
Third
Polypeptide
Target Peptide
(RBD of MERS-CoV)
EAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCN
YNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMK
SDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYS
YINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDY
YRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTN
SVCPKLEFANDTKIASQLGNCVEY (SEQ ID NO: 340)
116
Linker
Peptide
Glycine
serine
linker
GGGGSGGGGS (SEQ ID NO: 266)
Self-Assembling
peptide (ferritin
peptide)
LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFL
FDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQ
KAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEE
VLFKDILDKIELIGNENHGLYLADQYVKGI (SEQ ID NO: 331)
Cleavage
peptide
Cleavage Peptide-1 RRKRSVS (SEQ ID NO: 300)
Linker Peptide GSG (SEQ ID NO: 350)
Cleavage Peptide-
2 ATNFSLLKQAGDVEENPGP (SEQ ID NO: 347)
Signal Peptide MGVKVLFALICIAVAEA (SEQ ID NO: 329)
Fourth
Polypeptide
Target Peptide
(RBD of OC43)
RADVYRRKPDLPNCNIEAWLNDKSVPSPLNWERKTFSNCNF
NMSSLMSFIQADSFTCNNIDAAKIYGMCFSSITIDKFAIPNGRK
VDLQLGNLGYLQSFNYRIDTTATSCQLYYNLPAANVSVSRFN
PSTWNKRFGFIEDSVFKPQPAGVFTDHDVVYAQHCFKAPKN
FCPCKSNSSLCVGSGPGKNNGIGTCPAGTNYLTCHNLCNP
DPITFTGPYKCPQTKSLVGIGEHCSGLAVKSDHCGGNPCTC
QPQAFLGWSADSCVQGDKCNIFANLILHDVNSGL (SEQ ID
NO: 341)
Linker
Peptide
Glycine
serine
linker
GGGGSGGGGS (SEQ ID NO: 266)
Self-Assembling
peptide (ferritin
peptide)
LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFL
FDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQ
KAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEE
VLFKDILDKIELIGNENHGLYLADQYVKGI (SEQ ID NO: 331)
Cleavage
Peptide
Cleavage Peptide-1 RRKRSVS (SEQ ID NO: 300)
Linker Peptide GSG (SEQ ID NO: 350)
Cleavage Peptide-
2 ATNFSLLKQAGDVEENPGP (SEQ ID NO: 347)
Signal Peptide MGVKVLFALICIAVAEA SEQ ID NO: 329)
Fifth
Polypeptide
Target Peptide
(RBD of HKU1)
RIPDLPDCDIDKWLNNFNVPSPLNWERKIFSNCNFNLSTLLR
LVHTDSFSCNNFDESKIYGSCFKSIVLDKFAIPNSRRSDLQLG
SSGFLQSSNYKIDTTSSSCQLYYSLPAINVTINNYNPSSWNRR
YGFNNFNLSSHSVVYSRYCFSVNNTFCPCAKPSFASSCKSHK
PPSASCPIGTNYRSCESTTVLDHTDWCRCSCLPDPITAYDPR
SCSQKKSLVGVGEHCAGFGVDEEKCGVLDGSYNVSCLCST
DAFLGWSYDTCVSNNRCNIFSNFILNGINSGTTCSND (SEQ
ID NO: 343)
Linker
Peptide
Glycine
serine
linker
GGGGSGGGGS (SEQ ID NO: 266)
Self-Assembling
peptide (ferritin
peptide)
LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFL
FDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQ
KAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEE
VLFKDILDKIELIGNENHGLYLADQYVKGIRRKR (SEQ ID NO:
333)
117
Amino acid sequence of different peptides present in the multitarget peptide encoded
by the multitarget nucleic acid sequence – pentavalent RBD construct of example 3 is
separately identified in:
Complete sequence of the multitarget peptide encoded by the multitarget nucleic acid5
of example 3
MGVKVLFALICIAVAEARVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRI
SNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQT
GNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTE10
IYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCG
PKKSTNLVKNKCVNFGGGGSGGGGSLSKDIIKLLNEQVNKEMNSSNLYMSMSS
WCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEG
LTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKD
ILDKIELIGNENHGLYLADQYVKGIRRKRSVSGSGATNFSLLKQAGDVEENPGP15
MGVKVLFALICIAVAEARVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISN
CVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIAD
YNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGK
PCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCV
NFGGGGSGGGGSLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFL20
FDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESI
NNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQY
VKGIRRKRSVSGSGATNFSLLKQAGDVEENPGPMGVKVLFALICIAVAEAEAKPSGSV
VEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPA
AIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLT25
TITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQL
SPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQL
GNCVEYGGGGSGGGGSLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLF
LFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINN
IVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIR30
RKRSVSGSGATNFSLLKQAGDVEENPGPMGVKVLFALICIAVAEARADVYRRKPD
LPNCNIEAWLNDKSVPSPLNWERKTFSNCNFNMSSLMSFIQADSFTCNNIDAAKIY
GMCFSSITIDKFAIPNGRKVDLQLGNLGYLQSFNYRIDTTATSCQLYYNLPAANVSV
SRFNPSTWNKRFGFIEDSVFKPQPAGVFTDHDVVYAQHCFKAPKNFCPCKSNSSLC
VGSGPGKNNGIGTCPAGTNYLTCHNLCNPDPITFTGPYKCPQTKSLVGIGEHCSGL35
AVKSDHCGGNPCTCQPQAFLGWSADSCVQGDKCNIFANLILHDVNSGLGGGGSG
GGGSLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAE
EYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNI
VDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQY
VKGIRRKRSVSGSGATNFSLLKQAGDVEENPGPMGVKVLFALICIAVAEARIPDLP40
DCDIDKWLNNFNVPSPLNWERKIFSNCNFNLSTLLRLVHTDSFSCNNFDESKIYGSCFK
SIVLDKFAIPNSRRSDLQLGSSGFLQSSNYKIDTTSSSCQLYYSLPAINVTINNYNPSSWNRR
YGFNNFNLSSHSVVYSRYCFSVNNTFCPCAKPSFASSCKSHKPPSASCPIGTNYRSCESTT
VLDHTDWCRCSCLPDPITAYDPRSCSQKKSLVGVGEHCAGFGVDEEKCGVLDGSYNVS
CLCSTDAFLGWSYDTCVSNNRCNIFSNFILNGINSGTTCSNDGGGGSGGGGSLSKDII45
118
KLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLN
ENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFL
QWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIRRKR (SEQ ID NO:
352)
*The different bolded, underlined, and italicized sequences correspond to the different5
sequences in Table 3.
Plasmid DNA isolation and linearization, and Invitro transcription (IVT) of the
multitarget nucleic acid sequence of example 3 has been done as described in example 1.
Transfection10
HEK293T cells (Catalogue no. CRL3216, ATCC) were seeded at 0.05 x106 cells in
a 24 well plate containing Gibco DMEM medium, high glucose, pyruvate (Catalogue no.
11995065, ThermoFisher Scientific) supplemented with 10% fetal bovine serum and 100
units/mL penicillin-streptomycin, and cultured to achieve 70-80% confluency. Transfection
mix containing multitarget nucleic acid of example 3 (mRNA, 0.5-1 ug of example 3) and15
Lipofectamine™ 2000 (2.5 uL) in 100 uL serum free medium was incubated for 15 min at
room temperature and added to the cells and left for incubation for 24 and 48 hours. Spent
media was collected by aspiration, and cells were collected with ice-cold 1x PBS (1 mL)
with hard pipetting. The cells were centrifuged at 4000 g for 5 min at 4 °C. Excess PBS was
aspirated. The cells were suspended gently in 100 μL of 1x NETN lysis buffer (100 mM20
NaCl, 20 mM Tris-Cl (pH 8.0), 0.5 mM EDTA, 0.5 % (v/v) Nonidet P-40 (NP-40)) with
inhibitors and kept on ice for 20 min. The lysate was centrifuged at 16,000 g for 20 min at 4
°C. The supernatant was aspirated into microtubes.
Western Blot25
A western blot was performed on cell lysate, obtained in the previous step, using Jess
instrument (an automated western blot system Catalogue no. 004-650, ProteinSimple, Bio-
Techne). The cell lysate was diluted and combined with 1 part 5x fluorescent master mix
(component of Separation Module compatible with Jess, Catalogue no. SM-W001,
ProteinSimple, Bio-Techne) and heated for 5 min at 95 °C. After protein denaturation, the30
sample was mixed with luminol-S and peroxide as per manufacturer protocol (Separation
Module, Catalogue no. SM-W001, ProteinSimple, Bio-Techne). The primary antibody
(SARS-CoV-2 spike RBD polyclonal antibody (Catalogue no. E-AB-V-1006 Elab
Sciences), diluted at 1:200 ratio along with a secondary-HRP ready to use anti-rabbit
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antibody and chemiluminescent substrate (both components of Detection Module
compatible with Jess, Catalogue no. DM-001, ProteinSimple, Bio-Techne), were dispensed
into assigned wells within a provided microplate by the manufacturer. This plate was then
inserted into the Jess instrument where samples were drawn into individual capillaries
located on a 25-capillary cassette (12-230 kDa Separation Module Catalogue no. SM-W001,5
ProteinSimple, Bio-Techne). Electrophoresis and immunodetection were conducted in
automated manner by the Jess instrument. The data was obtained by using the compass
software associated with Jess system. The results are shown in figure 9.
INCORPORATION BY REFERENCE10
Each of the patents, published patent applications, and non-patent references cited
herein are hereby incorporated by reference in their entirety.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than
routine experimentation, many equivalents to the specific embodiments of the invention15
described herein. Such equivalents are intended to be encompassed by the following claims.
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We claim:
1. A nucleic acid comprising a plurality of polynucleotide sequences, wherein
some or all polynucleotide sequence of the plurality comprises either a target
sequence, a linker sequence, and a self-assembling sequence or a linker sequence,
a target sequence, a linker sequence and a self-assembling sequence or a
combination thereof, wherein each polynucleotide sequence of the plurality is
connected to an adjacent polynucleotide sequence of the plurality by a cleavage
sequence, and wherein the nucleic acid further comprises a signal sequence
upstream of one or more of the polynucleotide sequences of the plurality.
2. A nucleic acid comprising a plurality of polynucleotide sequences, wherein
each polynucleotide sequence of the plurality comprises a target sequence, a linker
sequence, and a self-assembling sequence, wherein each polynucleotide sequence
of the plurality is connected to an adjacent polynucleotide sequence of the plurality
by a cleavage sequence, and wherein the nucleic acid further comprises a signal
sequence upstream of one or more of the polynucleotide sequences of the plurality.
3. A nucleic acid comprising a plurality of polynucleotide sequences, wherein
each polynucleotide sequence of the plurality comprises a linker sequence, a target
sequence, a linker sequence, and a self-assembling sequence, wherein each
polynucleotide sequence of the plurality is connected to an adjacent polynucleotide
sequence of the plurality by a cleavage sequence, and wherein the nucleic acid
further comprises a signal sequence upstream of one or more of the polynucleotide
sequences of the plurality.
4. A nucleic acid encoding a plurality of polypeptides, wherein some or all
polypeptides of the plurality comprises either a target peptide, a linker peptide, and
a self-assembling peptide or a linker peptide, a target peptide, a linker peptide and
a self-assembling peptide or a combination thereof, wherein each polypeptide of
the plurality is connected to an adjacent polypeptide of the plurality by a cleavage
peptide, and wherein the nucleic acid further encodes a signal peptide on the amino-
terminus of one or more of the polypeptides of the plurality.
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5. A nucleic acid encoding a plurality of polypeptides, wherein each
polypeptide of the plurality comprises a target peptide, a linker peptide, and a self-
assembling peptide, wherein each polypeptide of the plurality is connected to an
adjacent polypeptide of the plurality by a cleavage peptide, and wherein the nucleic
acid further encodes a signal peptide on the amino-terminus of one or more of the
polypeptides of the plurality.
6. A nucleic acid encoding a plurality of polypeptides, wherein each
polypeptide of the plurality comprises a linker peptide, a target peptide, a linker
peptide, and a self-assembling peptide, wherein each polypeptide of the plurality is
connected to an adjacent polypeptide of the plurality by a cleavage peptide, and
wherein the nucleic acid further encodes a signal peptide on the amino-terminus of
one or more of the polypeptides of the plurality.
7. The nucleic acid according to any of the preceding claims, wherein total
number of the polynucleotide sequences is not more than 100.
8. The nucleic acid according to any of the preceding claims, wherein total
number of the polynucleotide sequences is between 2-10, 10-20, 20-30, 30-40, 40-
50, 50-60, 60-70, 70-80, 80-90, or 90-99.
9. The nucleic acid according to any of the preceding claims, wherein the
nucleic acid is a DNA or an RNA.
10. The nucleic acid according to claim 9, wherein the RNA is an mRNA.
11. The nucleic acid according to any of the preceding claims wherein the linker
sequence encodes a linker peptide.
12. The nucleic acid according to claim 11, wherein the linker peptide is an
amino acid linker, a foldon, a scaffold or a combination thereof.
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13. The nucleic acid according to claim 12, wherein the amino acid linker
comprises 2 to 49 amino acids.
14. The nucleic acid according to claim 13, wherein the amino acid linker is a
glycine serine linker, a glycine proline linker, a glycine threonine linker, an alanine
serine linker, any combination of two amino acids, or a combination thereof.
15. The nucleic acid according to any of the preceding claims, wherein the
linker peptide has an amino acid sequence of any one of SEQ ID NOs: 262-299,
330, and 350.
16. The nucleic acid according to any of the preceding claims, wherein the self-
assembling sequence encodes a self-assembling peptide.
17. The nucleic acid according to claim 16, wherein the self-assembling peptide
is a lumazine synthase from Aquifex species, a hepatitis B surface antigen (HBsAg)
from Hepatitis B Virus, a hepatitis B core antigen (HBcAg) from Hepatitis B virus,
a human papillomavirus L1 (HPV L1) protein, a matrix protein M1 from influenza
A virus, a ferritin, a riboflavin synthase, or a combination thereof, including their
codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
18. The nucleic acid according to claim 17, wherein the ferritin comprises of a
ferritin subunit or a ferritin peptide.
19. The nucleic acid according to claim 18, wherein the ferritin peptide is
derived from Helicobacter pylori ferritin.
20. The nucleic acid according to any of the preceding claims, wherein the self-
assembling peptide has an amino acid sequence of any one of SEQ ID NOs: 254-
261, 331 and 333.
21. The nucleic acid according to any of the preceding claims, wherein the
cleavage sequence encodes one or more cleavage peptide.
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22. The nucleic acid according to claim 21, wherein the one or more cleavage
peptides are optionally connected to each other by a linker peptide.
23. The nucleic acid according to claim 22, wherein the cleavage peptide is a
golgi specific cleavage peptide or a self-cleaving peptide.
24. The nucleic acid according to any of the preceding claims, wherein the
cleavage peptide has an amino acid sequence of any one of SEQ ID NOs: 300-311
and 347-349.
25. The nucleic acid according to any of the preceding claims, wherein the
signal sequence encodes a signal peptide.
26. The nucleic acid according to claim 25, wherein the signal peptide is present
on the amino-terminus of one or more of the polypeptides of the plurality.
27. The nucleic acid according to claim 26, wherein the nucleic acid further
encodes a second signal peptide on the amino-terminus of all or some polypeptides
of the plurality.
28. The nucleic acid according to any of the preceding claims, wherein the
signal peptide has an amino acid sequence of any one of SEQ ID NOs: 312-329.
29. The nucleic acid according to any of the preceding claims, wherein the
target sequence encodes a target peptide.
30. The nucleic acid sequence according to claim 29, wherein the target peptide
is encoded by a codon optimized nucleic acid sequence, or fragments, mutants, or
variants thereof.
31. The nucleic acid according to claim 30, wherein the target peptide is
obtained from a prokaryote, a eukaryote, a unicellular organism, a multicellular
organism, a virus, a bacterium, a fungus, a protozoan, a worm, a mycoplasma, an
animal, a human or combination thereof.
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32. The nucleic acid according to claim 31, wherein the virus is selected from
the family comprising picornaviride, calciviridae, astroviridae, togaviridae,
flaviviridae, coronaviridae, arteriviridae, rhabndoviridae, filoviridae,
paramyxoviridae, bornaviridae, orthomyxoviridae, bunyaviridae, arenaviridae,
reoviridae, retroviridae, polyomaviridae, herpesviridae, poxviridae,
papillomaviridae, hepadnaviridae, adenoviridae, parvoviridae, hepeviridae,
circoviridae or a combination thereof.
33. The nucleic acid according to claim 31, wherein the bacterium is selected
from the genus comprising Bacillus, Bordetella, Borrelia, Brucella, Campylobacter,
Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia,
Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium,
Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella,
Staphylococcus, Streptococcus, Vibrio, Yersinia, or a combination thereof.
34. The nucleic acid according to claim 32, wherein the virus is selected from
the family consisting of coronaviridae, herpesviridae, poxviridae, flaviviridae,
togaviridae, retroviridae, paramyxoviridae, and a combination thereof.
35. The nucleic acid according to claim 31, wherein the virus is
alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus, torovirus
or a combination thereof.
36. The nucleic acid according to claim 35, wherein the betacoronavirus is a
SARS-CoV-1, a SARS-CoV-2, a MERS-CoV, an OC43, a HKU1, a bat coronavirus,
other betacoronavirus, or a combination thereof.
37. The nucleic acid according to claim 30, wherein the target peptide is a spike
protein, a membrane protein, an envelope protein or a nucleocapsid protein of
coronaviruses.
38. The nucleic acid according to claim 30, wherein the target peptide is a
glycoprotein B, a glycoprotein C, a glycoprotein D, a glycoprotein E, a glycoprotein
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K, a glycoprotein L, or a glycoprotein M of a herpes simplex virus 1 (HSV-1) or a
herpes simplex virus 2 (HSV-2), or a combination thereof, including their codon
optimized nucleic acid sequences, fragments, mutants or variants thereof.
39. The nucleic acid according to claim 30, wherein the target peptide is a
glycoprotein B, a glycoprotein H, a glycoprotein L, a glycoprotein M, or a
glycoprotein N of a human cytomegalovirus (HCMV), or a combination thereof,
including their codon optimized nucleic acid sequences, fragments, mutants, or
variants thereof.
40. The nucleic acid according to claim 30, wherein the target peptide is a
glycoprotein B, a glycoprotein C, a glycoprotein H, or a glycoprotein L of a
varicella-zoster virus (VZV), or a combination thereof, including their codon
optimized nucleic acid sequences, fragments, mutants, or variants thereof.
41. The nucleic acid according to claim 30, wherein the target peptide is a
glycoprotein B, a glycoprotein H, a glycoprotein L, a glycoprotein M, a
glycoprotein N, a glycoprotein 42, a glycoprotein 350 of an Epstein-Barr virus
(EBV), or a combination thereof, including their codon optimized nucleic acid
sequences, fragments, mutants, or variants thereof.
42. The nucleic acid according to claim 30, wherein the target peptide is a F9
membrane protein of a poxvirus, a H3L protein of a poxvirus, an A4 protein of a
poxvirus, an A27 protein of poxvirus, an A33 protein of a poxvirus, an A56 protein
of a poxvirus, a B5 protein of a poxvirus, or a L1 protein of a poxvirus, or a
combination thereof, including their codon optimized nucleic acid sequences,
fragments, mutants, or variants thereof.
43. The nucleic acid according to claim 30, wherein the target peptide is a capsid
protein, a membrane protein, an envelope protein, or a non-structural protein (such
as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of flaviviruses or hepaciviruses,
or a combination thereof, including their codon optimized nucleic acid sequences,
fragments, mutants, or variants thereof.
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44. The nucleic acid according to claim 30, wherein the target peptide is a capsid
protein, a membrane protein, an envelope protein, or a non-structural protein (such
as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a Japanese encephalitis virus,
or a combination thereof, including their codon optimized nucleic acid sequences,
fragments, mutants, or variants thereof.
45. The nucleic acid according to claim 30, wherein the target peptide is a capsid
protein, a membrane protein, an envelope protein, or a non-structural protein (such
as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a zika virus, or a combination
thereof, including their codon optimized nucleic acid sequences, fragments,
mutants, or variants thereof.
46. The nucleic acid according to claim 30, wherein the target peptide is a capsid
protein, a membrane protein, an envelope protein, or a non-structural protein (such
as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a yellow fever virus, or a
combination thereof, including their codon optimized nucleic acid sequences,
fragments, mutants, or variants thereof.
47. The nucleic acid according to claim 30, wherein the target peptide is a capsid
protein, a membrane protein, an envelope protein, or a non-structural protein (such
as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a west nile virus, or a
combination thereof, including their codon optimized nucleic acid sequences,
fragments, mutants, or variants thereof.
48. The nucleic acid according to claim 30, wherein the target peptide is a capsid
protein, a membrane protein, an envelope protein, or a non-structural protein (such
as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a hepatitis C virus, or a
combination thereof, including their codon optimized nucleic acid sequences,
fragments, mutants, or variants thereof.
49. The nucleic acid according to claim 30, wherein the target peptide is a capsid
protein, a membrane protein, an envelope protein, or a non-structural protein (such
as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a dengue virus, or a
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combination thereof, including their codon optimized nucleic acid sequences,
fragments, mutants, or variants thereof.
50. The nucleic acid according to claim 30, wherein the target peptide is a capsid
protein, or an envelope protein such as E1, E2 and E3 protein of alphaviruses, or a
combination thereof, including their codon optimized nucleic acid sequences,
fragments, mutants, or variants thereof.
51. The nucleic acid according to claim 30, wherein the target peptide is a
domain A, a domain B, or a domain C of the E2 protein of alphaviruses, or a
combination thereof, including their codon optimized nucleic acid sequences,
fragments, mutants, or variants thereof.
52. The nucleic acid according to claim 30, wherein the target peptide is a capsid
protein, or an envelope protein such as E1, E2 and E3 protein of a chikungunya
virus, or a combination thereof, including their codon optimized nucleic acid
sequences, fragments, mutants, or variants thereof.
53. The nucleic acid according to claim 30, wherein the target peptide is a gag,
a pol and an env proteins of retroviruses, or a combination thereof, including their
codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
54. The nucleic acid according to claim 30, wherein the target peptide is a
p17Gag, a p24Gag, a p7Gag, a p6Gag, a gp12Env, a gp41Env, or a pol protein from
lentiviruses, or a combination thereof, including their codon optimized nucleic acid
sequences, fragments, mutants, or variants thereof.
55. The nucleic acid according to claim 30, wherein the target peptide is a
p17Gag, a p24Gag, a p7Gag, a p6Gag, a gp12Env, a gp41Env, or a pol protein from a human
immunodeficiency virus (HIV), or a combination thereof, including their codon
optimized nucleic acid sequences, fragments, mutants, or variants thereof.
56. The nucleic acid according to claim 30, wherein the target peptide is a
nucleocapsid protein, a P protein, a V protein, a W protein, a D protein, an I protein,
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a C protein, a L protein, a M protein, a H (hemagglutinin) protein, a HN
(hemagglutinin-neuraminidase) protein, a G protein or a F protein of a Mumps virus
(MuV), a Parainfluenza virus type 5 (PIV5), a Human parainfluenza virus type 2,
types 4a and 4b (HPIV2/4a/4b), a Newcastle disease virus (NDV), a Human
parainfluenza virus type 1 and type 3 (HPIV1/3), a Nipah virus (NiV), a Measles
virus (MeV), a Human respiratory syncytial virus A2, B1, S2, (HRSV), or a Human
metapneumovirus (HMPV), or a combination thereof, including their codon
optimized nucleic acid sequences, fragments, mutants, or variants thereof.
57. The nucleic acid according to claim 30, wherein the target peptide is a
nucleocapsid protein, a P protein, a V protein, a W protein, a D protein, an I protein,
a C protein, a L protein, a M protein, a H (hemagglutinin) protein, a HN
(hemagglutinin-neuraminidase) protein, a G protein or a F protein of a human
respiratory syncytial virus A2, B1, S2 (HRSV), or a combination thereof, including
their codon optimized nucleic acid sequences, fragments, mutants, or variants
thereof.
58. The nucleic acid according to claim 30, wherein the target peptide is an aE1,
an E2, an E4, an aE5, an E6, an E7, a L1, or a L2 protein of papillomavirus,
preferably a human papillomavirus, or a combination thereof, including their codon
optimized nucleic acid sequences, fragments, mutants, or variants thereof.
59. The nucleic acid according to any of the preceding claims, wherein the
target peptide has an amino acid sequence of any one of SEQ ID NOs: 1-253, 334-
337, 338-346, and 353-388.
60. A lipid nanoparticle composition comprising cationic lipid, a phospholipid,
a sterol, a PEG-lipid, and the nucleic acid according to any of the preceding claims.
61. The lipid nanoparticle composition according to claim 60, wherein the
cationic lipid comprises an ionizable lipid.
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62. The lipid nanoparticle composition according to claim 61, wherein the
ionizable lipid is present in an amount from 25 mol percent to 70 mol percent.
63. The lipid nanoparticle composition according to claim 60, wherein the
phospholipid is present in an amount from 2 mol percent to about 30 mol percent.
64. The lipid nanoparticle composition according to claim 60, wherein the sterol
is present in an amount from 30 mol percent to about 65 mol percent.
65. The lipid nanoparticle composition according to claim 60, wherein the PEG-
lipid is present in an amount from 0.2 mol percent to about 2.0 mol percent.
66. The lipid nanoparticle composition according to claim 60, additionally
comprising an ionizable polymer.
67. The lipid nanoparticle composition according to claim 66, wherein the
ionizable polymer is present in an amount from 1 mol percent to 25 mol percent.
68. The lipid nanoparticle composition according to claim 66, wherein the
ionizable polymer is selected from the group comprising a chitosan, a cellulose
derivative, a poly-L-lysine, a poly-L-glutamic acid, and/or their derivatives or a
combination thereof.
69. A method of treating or preventing a disease, comprising administrating to
a subject in need thereof the nucleic acid according to any one of claims 1-59.
70. A method of treating of preventing a disease, comprising administrating to
a subject in need thereof the lipid nanoparticle composition according to any one of
claims 60-68.
71. Use of the nucleic acid sequence according to any one of claims 1-59, in the
manufacture of a medicament for the treatment or prevention of a disease in a
subject.
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72. Use of a lipid nanoparticle composition according to any one of claims 60-
68 in the manufacture of a medicament for the treatment or prevention of a disease
in a subject.
73. A multitarget peptide encoded by the nucleic acid of any one of claims 1-
59.
74. A multitarget peptide comprising two or more polypeptides, wherein some
or all polypeptides comprises either a target peptide, a linker peptide, and a self-
assembling peptide, or a linker peptide, a target peptide, a linker peptide, and a self-
assembling peptide, or combination thereof, wherein one polypeptide is connected
to another polypeptide by a cleavage peptide, wherein the multitarget peptide
includes a signal peptide upstream of one or more of the polypeptides.
75. A polypeptide nanoparticle comprising at least 2 or up to 100 polypeptides
according to any one of claims 1 to 59.
76. The polypeptide nanoparticle according to claim 75, wherein the
polypeptides are homologous polypeptides, heterologous polypeptides, oligomeric
complex or a combination thereof.
77. The polypeptide nanoparticle according to claim 76, wherein the
polypeptide nanoparticle is icosahedral, helical, spherical, rod-like or a combination
thereof.