Multi-target vaccines and therapeutics

Abstract

The present disclosure generally relates to a nucleic acid comprising a plurality of polynucleotide sequences, wherein each of the plurality of polynucleotide sequences comprises 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 of the plurality of polynucleotide sequences is connected to adjacent ones of the plurality of polynucleotide sequences by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the plurality of polynucleotide sequences.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 435,977, filed December 29, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0002] FIELD OF THE INVENTION The present disclosure relates to multi-targeted nucleic acid sequence, multi-targeted peptide, and polypeptide nanoparticles and compositions thereof for vaccine and therapeutic purposes. [Background technology]

[0003] background A wide variety of diseases are caused by infectious agents such as bacteria, viruses, fungi, protozoa, and worms. Despite advances in vaccination and other therapeutic interventions, the economic burden of the eight major diseases alone (HIV / AIDS, malaria, measles, hepatitis, dengue fever, rabies, tuberculosis, and yellow fever) is estimated to reach US$8 trillion, resulting in over 156 million life-years lost in 2016 alone (Armitage, Catherine Nature (2021) 598: S9).

[0004] Many pathogens, such as influenza virus, HIV, human papillomavirus, SARS-CoV-2, Streptococcus pneumoniae, Neisseria meningitidis, Neisseria gonorrhoeae, Trypanosoma brucei, and others, have evolved and adapted to evade currently available vaccines and therapeutics. Such evolutionary changes, resulting in antigenic drift or serotype shift, render current therapeutic interventions ineffective. Targeting multiple facets of disease or multiple pathogens through a single drug or therapeutic has always been a challenge.

[0005] Combination vaccines have traditionally been used to target multiple pathogens or multiple 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).

[0006] To increase the depth and breadth of immune responses to emerging pathogen strains or variants, efforts have been made to present antigens or epitopes on virus-like particles or nanoparticles (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 traditional approaches of recombinantly expressing particles in bacterial, insect, or mammalian expression systems and purifying the particles for use in immunization.

[0007] More recently, mRNA-based vaccines have emerged as a promising alternative to classical approaches to vaccine development. However, current mRNA-based vaccines are still inadequate for tackling emerging strains or variants of pathogens. For example, antibody titers elicited by SARS-CoV-2 vaccines against emerging and antigenically distinct SARS-CoV-2 variants have been found to be lower and to wane over time, suggesting reduced efficacy of 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 enhance coverage (Chalkias, 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 nanoparticles for delivery (Chivukula, Sudha et al. NPJ Vaccines (2021) 6:153; WO2022264109). Such strategies require multiple in vitro transcription (IVT) processes, complicating the manufacturing process and rapid vaccine development during a pandemic. Strategies that simplify manufacturing processes and can induce stronger, more durable, and / or broader immune responses are needed to combat pandemic and evolving pathogens. Thus, it would be advantageous to develop vaccines and therapeutics that provide protection against infection across a wide range of diseases, including multiple facets of a single disease or different strains and / or variations of a pathogen. Summary of the Invention

[0008] Abstract Thus, the present disclosure relates to a nucleic acid comprising a plurality of polynucleotide sequences, wherein some or all of the plurality of polynucleotide sequences 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. In some embodiments, each of the plurality of polynucleotide sequences is connected to adjacent ones of the plurality of polynucleotide sequences by a cleavage sequence. In some embodiments, the nucleic acid further comprises a signal sequence upstream of one or more of the plurality of polynucleotide sequences. In some embodiments, the target 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 present in 5' to 3' order.

[0009] In another aspect, the present disclosure provides a nucleic acid comprising a plurality of polynucleotide sequences, wherein each of the plurality of polynucleotide sequences comprises a target sequence, a linker sequence, and a self-assembling sequence. In some embodiments, each of the plurality of polynucleotide sequences is connected to adjacent ones of the plurality of polynucleotide sequences by a cleavage sequence. In some embodiments, the nucleic acid further comprises a signal sequence upstream of one or more of the plurality of polynucleotide sequences. In some embodiments, the target sequence, the linker sequence, and the self-assembling sequence are present in 5' to 3' order.

[0010] In another aspect, provided herein is a nucleic acid comprising a plurality of polynucleotide sequences, wherein each of the plurality of polynucleotide sequences comprises a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence. In some embodiments, each of the plurality of polynucleotide sequences is connected to adjacent ones of the plurality of polynucleotide sequences by a cleavage sequence. In some embodiments, the nucleic acid further comprises a signal sequence upstream of one or more of the plurality of polynucleotide sequences. In some embodiments, the linker sequence, the target sequence, the linker sequence, and the self-assembling sequence are present in 5' to 3' order.

[0011] In another aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein some or all of the plurality of polypeptides 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. In some embodiments, each of the plurality of polypeptides is connected to an adjacent one of the plurality of polypeptides by a cleavage peptide. In some embodiments, the nucleic acid further encodes a signal peptide at the amino terminus of one or more of the plurality of polypeptides. In some embodiments, the target peptide, linker peptide, and self-assembling peptide, or the linker peptide, target peptide, linker peptide, and self-assembling peptide, are present in order from N-terminus to C-terminus.

[0012] In another aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein each of the plurality of polypeptides comprises a target peptide, a linker peptide, and a self-assembling peptide. In some embodiments, each of the plurality of polypeptides is connected to adjacent polypeptides of the plurality of polypeptides by a cleavage peptide. In some embodiments, the nucleic acid further encodes a signal peptide at the amino terminus of one or more of the plurality of polypeptides. In some embodiments, the target peptide, linker peptide, and self-assembling peptide are present in order from N-terminus to C-terminus.

[0013] In another aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein each of the plurality of polypeptides comprises a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide. In some embodiments, each of the plurality of polypeptides is connected to adjacent polypeptides of the plurality of polypeptides by a cleavage peptide. In some embodiments, the nucleic acid further encodes a signal peptide at the amino terminus of one or more of the plurality of polypeptides. In some embodiments, the linker peptide, target peptide, linker peptide, and self-assembling peptide are present in order from N-terminus to C-terminus.

[0014] In some embodiments, the total number of polynucleotide sequences is 100 or less. In some embodiments, the total number of polynucleotide sequences is 2 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 99. In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is mRNA. In some embodiments, the linker sequence encodes a linker peptide. In some embodiments, the linker peptide is an amino acid linker, a foldon, a 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 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.

[0015] 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 a species of the genus Acwifex, 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 codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof. In some embodiments, the ferritin is composed of ferritin subunits or ferritin peptides. In some embodiments, the ferritin peptide is derived from Helicobacter pylori ferritin. In some embodiments, the self-assembling peptide has an amino acid sequence of any one of SEQ ID NOs: 254-261, 331, and 333.

[0016] In some embodiments, the cleavage sequence encodes one or more cleavage sequences. In some embodiments, the one or more cleavage sequences are optionally connected to each other by a linker. In some embodiments, the cleavage peptide is a Golgi-specific cleavage peptide or a self-cleaving peptide. In some embodiments, the cleavage peptide has the amino acid sequence of any one of SEQ ID NOs: 300-311 and 347-349.

[0017] In some embodiments, the signal sequence encodes a signal peptide. In some embodiments, the signal peptide is present at the amino terminus of the first polypeptide. In some embodiments, the nucleic acid further encodes a second signal peptide at the amino terminus of all or some of the polypeptides. In some embodiments, the signal peptide has the amino acid sequence of any one of SEQ ID NOs: 312-329.

[0018] 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, or a fragment, mutant, or variant 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 a family comprising Picornaviridae, Caliciviridae, 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. In some embodiments, the bacterium is selected from a genus including 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 some embodiments, the virus is selected from a family consisting of Coronaviridae, Herpesviridae, Poxviridae, Flaviviridae, Togaviridae, Retroviridae, Paramyxoviridae, or a combination thereof. In some embodiments, the virus is an alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus, torovirus, or a combination thereof.

[0019] In some embodiments, the beta coronavirus is SARS-CoV-1, SARS-CoV-2, MERS-CoV, OC43, HKU1, bat coronavirus, other beta coronavirus, or a combination thereof. In some embodiments, the target peptide is a coronavirus spike protein, membrane protein, envelope protein, or nucleocapsid protein. In some embodiments, the target peptide is a receptor-binding domain, fusion peptide, or stem helix of the spike protein, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment thereof, a mutant, or a variant thereof. In some embodiments, the target peptide is a receptor-binding domain obtained or derived from a beta coronavirus, including SARS-CoV-1, SARS-CoV-2, MERS-CoV, OC43, HKU1, bat coronavirus, other beta coronavirus, or a combination thereof. In some embodiments, the target peptide is glycoprotein B, glycoprotein C, glycoprotein D, glycoprotein E, glycoprotein K, glycoprotein L, and glycoprotein M of herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0020] In some embodiments, the target peptide is glycoprotein B, glycoprotein H, glycoprotein L, glycoprotein M, or glycoprotein N of human cytomegalovirus (HCMV), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant 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 a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant 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 a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof. In some embodiments, the target peptide is a poxvirus F9 membrane protein, a poxvirus H3L protein, a poxvirus A4 protein, a poxvirus A27 protein, a poxvirus A33 protein, a poxvirus A56 protein, a poxvirus B5 protein, or a poxvirus L1 protein, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, a mutant, or a variant thereof.

[0021] In some embodiments, the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a flavivirus or hepacivirus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof. In some embodiments, the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a Japanese encephalitis virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof. In some embodiments, the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a Zika virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof. In some embodiments, the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Yellow Fever Virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof. In some embodiments, the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of West Nile Virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.In some embodiments, the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Hepatitis C virus, or a combination thereof including a codon-optimized nucleic acid sequence thereof, a fragment, a mutant, or a variant thereof. In some embodiments, the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Dengue virus, or a combination thereof including a codon-optimized nucleic acid sequence thereof, a fragment, a mutant, or a variant thereof.

[0022] In some embodiments, the target peptide is an alphavirus capsid protein or envelope protein such as E1, E2, and E3 proteins, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment thereof, a mutant, or a variant thereof. In some embodiments, the target peptide is an alphavirus E2 protein domain A, domain B, or domain C, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment thereof, a mutant, or a variant thereof. In some embodiments, the target peptide is a chikungunya virus capsid protein or envelope protein such as E1, E2, and E3 proteins, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment thereof, a mutant, or a variant thereof. In some embodiments, the target peptide is a retrovirus gag, pol, and env protein, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment thereof, a mutant, or a variant thereof.

[0023] In some embodiments, the target peptide is a lentiviral-derived p17 Gag , p24 Gag , p7 Gag , p6 Gag, gp12 Env , gp41 Env In some embodiments, the target peptide is p17 from human immunodeficiency virus (HIV), or pol protein, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment thereof, a mutant or variant thereof. Gag , p24 Gag , p7 Gag , p6 Gag , gp12 Env , gp41 Env or pol protein, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0024] In some embodiments, the target peptide is the 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 virus (MuV), parainfluenza virus type 5 (PIV5), human parainfluenza virus types 2, 4a, and 4b (HPIV2 / 4a / 4b), Newcastle disease virus (NDV), human parainfluenza virus types 1 and 3 (HPIV1 / 3), Nipah virus (NiV), measles virus (MeV), human respiratory syncytial virus (HRSV) A2, B1, S2, or human metapneumovirus (HMPV), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof. In some embodiments, the target peptide is a 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 (HRSV) A2, B1, or S2, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment thereof, a mutant, or a variant thereof. In some embodiments, the target peptide is a human papillomavirus protein, for example, an early protein (E1, E2, E4, E5, E6, or E7), or a late protein (L1 (major capsid protein) or L2 (minor capsid protein), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment thereof, a mutant, or a variant thereof. In some embodiments, the target peptide is a major capsid protein (L1) of human papillomavirus that has lost the ability to self-assemble, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof.In some embodiments, the target peptide is the minor capsid protein (L2) of human papillomavirus, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof.

[0025] In some embodiments, the target peptide has the amino acid sequence of any one of SEQ ID NOs: 1-253, 334-337, 338-346, and 353-388.

[0026] In another aspect, provided herein is a lipid nanoparticle composition comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and a 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 an amount of 25 mol percent to 70 mol percent. In some embodiments, the phospholipid is present in an amount of 2 mol percent to about 30 mol percent. In some embodiments, the sterol is present in an amount of 30 mol percent to about 65 mol percent. In some embodiments, the PEG-lipid is present in an amount of 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 of 1 mol percent to 25 mol percent. In some embodiments, the ionizable polymer is selected from the group consisting of chitosan, cellulose derivatives, poly-L-lysine, poly-L-glutamic acid, and / or derivatives thereof or combinations thereof.

[0027] In another aspect, provided herein is a method of treating or preventing a disease, the method comprising administering a nucleic acid disclosed herein to a subject in need thereof. In another aspect, provided herein is a method of treating or preventing a disease, the method comprising administering a multi-target peptide disclosed herein to a subject in need thereof. In another aspect, provided herein is a method of treating or preventing a disease, the method comprising administering a lipid nanoparticle composition disclosed herein to a subject in need thereof.

[0028] In another aspect, provided herein is the use of a nucleic acid sequence disclosed herein in the manufacture of a medicament for the treatment or prevention of a disease in a subject. In another aspect, provided herein is the 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. In another aspect, provided herein are multi-target peptides encoded by the nucleic acids disclosed herein.

[0029] In another aspect, provided herein is a multi-target peptide comprising two or more polypeptides, wherein some or all of the polypeptides 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, wherein one polypeptide is connected to another polypeptide by a cleavage peptide, and wherein the multi-target peptide includes a signal peptide upstream (amino-terminal) of one or more of the polypeptides. In some embodiments, the signal peptide may be present at the amino-terminal side of the first polypeptide. In some embodiments, the signal peptide may be present at the amino-terminal side of some or each of the polypeptides.

[0030] In another aspect, provided herein is a polypeptide nanoparticle, comprising at least two or up to 100 polypeptides disclosed herein.In some embodiments, the polypeptide is a homologous polypeptide, a heterologous polypeptide, an oligomeric complex, or a combination thereof.In some embodiments, the polypeptide nanoparticle is icosahedral, helical, spherical, rod-shaped, or a combination thereof.

[0031] In another aspect, provided herein is a nucleic acid sequence comprising a multi-target nucleic acid sequence comprising two or more polynucleotide sequences, wherein some or all of the polynucleotide sequences 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, wherein one polynucleotide sequence is connected to another polynucleotide sequence by a cleavage sequence, and wherein the multi-target nucleic acid sequence includes a signal sequence upstream of one or more of the polynucleotide sequences. In some embodiments, the linker sequence connects the signal sequence to 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, the signal sequence is present upstream of the first polynucleotide sequence. In some embodiments, the signal sequence is present upstream of all of the polynucleotide sequences. In some embodiments, the linker sequence connects the target sequence to the self-assembling sequence in the polynucleotide sequences. In some embodiments, one linker sequence connects the cleavage sequence to the target sequence, and another linker sequence connects the target sequence to the self-assembling sequence in the polynucleotide sequence. In some embodiments, the multi-target nucleic acid sequence is DNA or RNA. In some embodiments, the multi-target nucleic acid sequence is mRNA. In some embodiments, the multi-target nucleic acid sequence encodes a multi-target peptide. In some embodiments, the multi-target nucleic acid is encapsulated in a lipid nanoparticle composition. In some embodiments, the multi-target nucleic acid sequence is synthesized through a single in vitro transcription (IVT) process.

[0032] In some aspects, the present disclosure relates to nucleic acid sequences encoding the multi-target peptides described herein.

[0033] In some embodiments, the present disclosure relates to a nucleic acid sequence encoding a multi-target peptide comprising two or more polypeptides, wherein some or all of the polypeptides 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, wherein one polypeptide is connected to another polypeptide by a cleavage peptide, and wherein the multi-target peptide includes a signal peptide upstream of one or more of the polypeptides. In some embodiments, the signal peptide may be present upstream of all or some of the polypeptides. In some embodiments, the signal peptide may be present upstream of the first polypeptide. In some embodiments, the signal peptide may be present upstream of all of the polypeptides.

[0034] In some embodiments, the present disclosure also relates to a multi-target peptide comprising two or more polypeptides, wherein some or all of the polypeptides 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, wherein one polypeptide is connected to another polypeptide by a cleavage peptide, and wherein the multi-target peptide includes a signal peptide upstream (amino-terminal side) of one or more of the polypeptides. In some embodiments, the signal peptide may be upstream (amino-terminal side) of all or some of the polypeptides. In some embodiments, the signal peptide may be upstream (amino-terminal side) of the first polypeptide. In some embodiments, the signal peptide may be upstream (amino-terminal side) of all of the polypeptides.

[0035] In some embodiments, a linker peptide connects a signal peptide to a first polypeptide in the multi-targeting peptide. In some embodiments, a linker peptide connects a targeting peptide to a self-assembling peptide in the polypeptide. In some embodiments, one linker peptide connects a cleavage peptide to a targeting peptide, and another linker peptide connects a targeting peptide to a self-assembling peptide in the polypeptide. In some embodiments, the multi-targeting peptide comprises a homologous polypeptide. In some other embodiments, the multi-targeting peptide comprises a heterologous polypeptide. In some embodiments, the multi-targeting peptide comprises a homologous polypeptide or a heterologous polypeptide. In some embodiments, the present disclosure relates to polypeptide nanoparticles comprising one or more homologous polypeptides, one or more heterologous polypeptides, one or more oligomeric complexes, or a combination thereof. In some embodiments, the homologous polypeptide, heterologous polypeptide, or oligomeric complex may comprise a targeting peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a targeting peptide, a linker peptide, and a self-assembling peptide, or a combination thereof. In some embodiments, the polypeptide in the polypeptide nanoparticle may also comprise some residues of a cleavage peptide.

[0036] In some embodiments, the present disclosure relates to polypeptide nanoparticles formed by the self-assembly of two or more polypeptides, wherein some or all of the polypeptides comprise a targeting peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a targeting peptide, a linker peptide, and a self-assembling peptide, or a combination thereof. In some embodiments, the polypeptide in the polypeptide nanoparticle may also have some residues of a truncated peptide. In some embodiments, the polypeptide nanoparticle is composed of a homologous polypeptide, a heterologous polypeptide, an oligomeric complex, or a combination thereof.

[0037] In some aspects, provided herein is a multi-target nucleic acid sequence as 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 multi-target nucleic acid sequence as described herein.

[0038] In some other aspects, the lipid nanoparticle composition comprises an ionizable polymer, a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and a multi-targeting nucleic acid sequence described herein. In some aspects, provided herein are methods of treating or preventing a disease, the methods comprising administering a multi-target nucleic acid sequence as described herein to a subject in need thereof.

[0039] In some aspects, provided herein are methods of treating or preventing a disease, the methods comprising administering to a subject in need thereof a lipid nanoparticle composition comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and a multi-targeting nucleic acid sequence as described herein.

[0040] In some aspects, provided herein are methods of treating or preventing disease, the methods comprising administering to a subject in need thereof a lipid nanoparticle composition comprising an ionizable polymer, a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and a multi-targeting nucleic acid sequence as described herein.

[0041] In some aspects, the present disclosure relates to the use of a lipid nanoparticle composition comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and a multi-targeting nucleic acid sequence as described herein in the manufacture of a medicament for the treatment or prevention of a disease in a subject.

[0042] In some embodiments, the target sequence is obtained from prokaryotes or eukaryotes, unicellular or multicellular organisms, viruses, bacteria, fungi, protozoa, parasites, mycoplasma, animals, humans, or combinations thereof, including codon-optimized sequences, fragments, variants, or mutants of such target sequences.In some embodiments, the target sequence can be modified or unmodified.In some embodiments, the target sequence is obtained from viruses belonging to the following families, such as Picornaviridae, Caliciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Arteriviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Bornaviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Retroviridae, Polyomaviridae, Herpesviridae, Poxviridae, Papillomaviridae, Hepadnaviridae, Adenoviridae, Parvoviridae, Hepeviridae, Circoviridae, or combinations thereof. In some embodiments, the target sequence is obtained from bacteria belonging to the following genera, such as 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 some embodiments, the target sequence encodes a target peptide. In some embodiments, the target peptide is an antigen, a fragment thereof, or a variant thereof. In some embodiments, the target sequence or target peptide may be modified or unmodified. In some embodiments, the target peptide controls or modifies a cellular function. In some embodiments, the target peptide may have an immunostimulatory or immunomodulatory effect.

[0043] In some embodiments, the self-assembling sequence encodes a self-assembling peptide. In some embodiments, the self-assembling peptide includes, but is not limited to, lumazine synthase from Acwifex 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) from influenza A virus, ferritin peptide, or a combination thereof, including a fragment, mutant, or variant thereof. In some embodiments, the ferritin peptide is Helicobacter pylori ferritin or a fragment, mutant, or variant thereof.

[0044] In some embodiments, the linker sequence encodes a linker peptide. In some embodiments, the linker peptide connects the target peptide to the self-assembling peptide in the polypeptide. In some embodiments, the linker peptide connects the signal peptide to the first polypeptide. In some embodiments, one linker peptide connects the cleavage peptide to the target peptide, and another linker peptide connects the target peptide to the self-assembling peptide in the polypeptide. The linker peptide may be an amino acid linker, a foldon, a scaffold, or a combination thereof.

[0045] In some embodiments, the cleavage sequence encodes a cleavage peptide. The cleavage peptide connects one polypeptide to another polypeptide, e.g., an adjacent polypeptide. The cleavage peptide carries a cleavage site. In some embodiments, the cleavage peptide facilitates the action of cellular proteases to cleave the multi-target 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 (e.g., cleavage peptide-1, cleavage peptide-2, etc.), optionally connected via a linker. In some embodiments, the cleavage peptide may self-cleave into individual polypeptides or may be cleaved by the action of cellular proteases. In some embodiments, the cleavage peptide is a substrate for a Golgi-specific protease.

[0046] In some embodiments, the signal sequence encodes a signal peptide. The signal peptide is present upstream (amino terminal) of one or more polypeptides in the multi-target peptide. In some embodiments, the signal peptide is present upstream (amino terminal) of the first polypeptide. In some embodiments, the signal peptide is present upstream (amino terminal) of several polypeptides. In some embodiments, the signal peptide is present upstream (amino terminal) of all polypeptides. In some embodiments, the signal peptide transports the multi-target peptide to an organelle. In some embodiments, the signal peptide transports the multi-target peptide to the Golgi apparatus or Golgi body. In some embodiments, the signal peptide is a Golgi targeting signal peptide.

[0047] In some aspects, the present disclosure also includes methods of transforming a cell with a multi-target nucleic acid sequence as described herein. [Brief explanation of the drawings]

[0048] [Figure 1]FIG. 1 shows a representative schematic diagram of a multi-target nucleic acid sequence, in which each polynucleotide sequence (PS) includes 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). Multiple polynucleotide sequences are connected through cleavage sequences (CS) such that a cleavage sequence exists between any two polynucleotide sequences. The multi-target nucleic acid sequence has a signal sequence (SS) upstream of the first polynucleotide sequence. The letter "n" in FIG. 1 represents any number from 1 to 98. The multi-target nucleic acid sequence may additionally have a 5' cap and a 3' poly(A) tail. This multi-target nucleic acid sequence encodes the corresponding multi-target peptide depicted in FIG. 2.

[0049] [Figure 2] Figure 2 shows a representative schematic diagram of a multi-target peptide, in which each polypeptide (PP) comprises a target peptide (TP), a linker peptide (LP), and a self-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 cleavage peptides (CP) such that a cleavage peptide exists between any two polypeptides. The multi-target peptide has a signal peptide (SP) at the N-terminus of the first polypeptide. The letter "n" represents any number from 1 to 98.

[0050] [Figure 3] Figure 3a shows a Western blot of cell lysates (lanes 4 and 5) using a polyclonal anti-spike antibody against SARS-CoV-2 demonstrating expression of the multi-target peptide. Figure 3b shows a Western blot of supernatants (lane 5) using a polyclonal anti-spike RBD antibody against SARS-CoV-2 confirming cleavage of the multi-target peptide.

[0051] [Figure 4] Figure 4 shows the estimation of polypeptide nanoparticles in the cell lysate and supernatant by ELISA. There is a decrease in protein concentration in the cell lysate at 48 hours and a corresponding increase in protein concentration in the supernatant at 48 hours. These results indicate an increase in the amount of polypeptide nanoparticles in the supernatant over time.

[0052] [Figure 5] FIG. 5 shows TEM images demonstrating the formation of polypeptide nanoparticles.

[0053] [Figure 6] Figure 6 shows a Western blot (lane 2) of cell lysate using a polyclonal anti-spike antibody against SARS-CoV-2, demonstrating target peptide expression by the multi-target nucleic acid sequence-trivalent RBD construct of Example 2. Lanes 1 and 3 are a protein ladder and a negative control, respectively.

[0054] [Figure 7a-7b] 7a, 7b, and 7c show ELISA data demonstrating the production of antibodies (IgG) against each target peptide encoded by the multi-target nucleic acid sequence-trivalent RBD construct of Example 2. [Figure 7c] 7a, 7b, and 7c show ELISA data demonstrating the production of antibodies (IgG) against each target peptide encoded by the multi-target nucleic acid sequence-trivalent RBD construct of Example 2.

[0055] [Figure 8a] 8a, 8b, and 8c show pseudovirus neutralization of antibodies generated against each target peptide encoded by the multi-target nucleic acid sequence-trivalent RBD construct of Example 2. [Figure 8b]8a, 8b, and 8c show pseudovirus neutralization of antibodies generated against each target peptide encoded by the multi-target nucleic acid sequence-trivalent RBD construct of Example 2. [Figure 8c] 8a, 8b, and 8c show pseudovirus neutralization of antibodies generated against each target peptide encoded by the multi-target nucleic acid sequence-trivalent RBD construct of Example 2.

[0056] [Figure 9] Figure 9 shows a Western blot (lane 3) of cell lysates using a polyclonal anti-spike antibody against SARS-CoV-2 showing expression of target peptides encoded by the multi-target nucleic acid sequence-pentavalent RBD construct of Example 3. Lanes 1 and 2 are a protein ladder and a negative control, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0057] explanation Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Some of the terms are briefly defined herein below; the definitions should not be construed in a limiting sense.

[0058] The singular forms "a," "an," and "the," when used in the specification, also include plural aspects unless the context dictates otherwise. Likewise, any singular term used in the specification also refers to the plural, and vice versa, unless the context dictates otherwise. As used herein and in the claim(s), when used in conjunction with the word "comprising," the word "a" or "an" may mean one or more than one. As used herein, "another" may mean at least a second, or third or more.

[0059] It should be noted that the word "comprising" or any of its forms, e.g., "comprise" or "comprises," "having" or any of its forms, e.g., "have" or "has," "including" or any of its forms, e.g., "include" or "includes," or "containing" or any of its forms, e.g., "contains" or "contains," are open-ended and do not exclude additional, unrecited elements or method steps.

[0060] Whenever any amount or range is described, one of ordinary skill in the art will recognize that amounts or ranges within 10 or 20 percent of the described value would also be expected to be appropriate and reasonable and would fall within the scope of the invention.

[0061] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Generally, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, proteins, adjuvants, pharmaceutical biotechnology, and biologics manufacturing described herein, and the techniques thereof, are well known and commonly used in the art. The methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification.

[0062] The terms "composition" and "formulation" are used interchangeably and refer to a lipid nanoparticle composition comprising a multi-target nucleic acid sequence and lipid components such as cationic lipids, phospholipids, sterols, and PEG-lipids. The composition may optionally contain an ionizable polymer. The composition may additionally contain a pharmaceutical carrier or excipient, such as, but not limited to, a buffering agent, a stabilizer, an osmotic pressure adjusting agent, a surfactant, a chelating agent, a salt, an antioxidant, a diluent, and / or a preservative, or a combination thereof.

[0063] The terms "therapeutic," "therapeutic agent," "prophylactic," "prophylactic agent," or drug are used interchangeably and refer to a compound (such as a multi-target nucleic acid sequence) or composition (such as the lipid nanoparticle compositions described herein) that has a biological effect or combination of biological effects that prevents, inhibits, eliminates, or prevents the progression of a disease or other abnormal biological process in a subject, e.g., an animal or human.

[0064] The term "preventing" is art-recognized and, when used in reference to a condition such as an infection, is well understood in the art and includes administration of a composition that reduces the frequency or severity of, or delays the onset of, one or more symptoms of a medical condition in a subject compared to subjects not receiving the composition. Thus, prevention of a condition such as an infection includes reducing the frequency or severity of, one or more symptoms of a medical condition in a population of patients, e.g., by a statistically and / or clinically significant amount, compared to a control population not receiving the treatment. Similarly, prevention of an infection includes reducing the likelihood that a patient receiving the treatment will develop the infection or associated symptoms, compared to patients not receiving the treatment.

[0065] The terms "molar ratio," "mol ratio," "mol percent," "mol percent," "mol%," or "mol%" are used interchangeably and refer to the average moles of components expressed as a percentage of the total moles of all lipid components (such as cationic lipids, phospholipids, sterols, and PEG-lipids) and, if present, ionizable polymer component(s) present in the lipid nanoparticle composition described herein. For example, 50 mol% cationic lipid means that 50 mol% cationic lipid is present in the lipid nanoparticle composition, and other lipid components make up the remaining 50%, so that the total amount of all lipid components together constitutes 100 mol%. Alternatively, 50 mol% cationic lipid also means that 50 mol% cationic lipid is present in the lipid nanoparticle composition, and other lipid components and ionizable polymer components together constitute the remaining 50 mol%, so that the total amount of all lipid components and ionizable polymer components together constitutes 100 mol%.

[0066] The terms "antibody" and "antibodies" are used interchangeably herein and refer to any antibody or antibody fragment (whether naturally produced or recombinantly produced) that retains antigen-binding activity. This includes monoclonal or polyclonal antibodies, single-chain antibodies, Fab fragments of monoclonal or polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, multispecific antibodies, or nanobodies.

[0067] The term "buffer," as used herein, means an agent that maintains the pH of a solution within a desired range. The term "cell" as used herein means a single cell, or a population of cells, or a plurality of cells.

[0068] The terms "biologically effective amount" or "therapeutically effective amount," as used herein, refer to an amount of an agent, e.g., a treatment, drug, therapeutic agent, prophylactic agent, diagnostic agent, composition, etc., that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, prevent, diagnose, ameliorate symptoms of, and / or delay the onset of, the infection, disease, disorder, and / or condition. The therapeutically effective amount herein may vary depending on factors such as the stage of the disease, the age, sex, and weight of the patient.

[0069] As used herein, the term "treat" or "treatment" includes reducing, arresting, or reversing the symptoms, clinical signs, or underlying pathology of a condition, stabilizing or improving the subject's condition, or reducing the likelihood that the subject's condition would worsen to the same extent as if the subject had not received treatment. Treatment may be administered to subjects who do not show signs of disease and / or who show only early signs of disease, with the aim of reducing the risk of developing disease-related conditions.

[0070] The term "subject," as used herein, refers to a living mammal and may be used interchangeably with the term "patient." Examples of mammals include, but are not limited to, any member of the class Mammalia: non-human primates such as humans, chimpanzees, and other ape and monkey species; livestock animals such as cows, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; research animals, including rodents such as rats, mice, and guinea pigs; and the like. The term does not denote a particular age or sex.

[0071] As used herein, an individual who is "at risk" of developing a particular disease, disorder or condition may or may not have detectable disease or disease symptoms, and may or may not have detectable disease or disease symptoms present before the treatment method described herein. "At risk" refers to an individual having one or more risk factors, which are measurable parameters known in the art to be correlated with the development of a particular disease, disorder or condition.An individual who has one or more of these risk factors is more likely to develop a particular disease, disorder or condition than an individual who does not have one or more of these risk factors.

[0072] The term "disease," as used herein, means an interruption, cessation, or disorder of a bodily function, system, or organ. Non-limiting examples of diseases include malignant diseases, autoimmune diseases, genetic diseases, metabolic diseases, or infectious diseases.

[0073] As used herein, administration "conjointly" with another compound or composition includes simultaneous administration and / or administration at different times. Conjoint administration also includes administration as a co-formulation or as separate compositions, including at different administration frequencies or intervals, and using the same or different routes of administration.

[0074] The terms "multitarget nucleic acid sequence" or "multitargeted nucleic acid sequence" are used interchangeably and refer to two or more polynucleotide sequences, wherein some or all of the polynucleotide sequences 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, wherein one polynucleotide sequence is connected to another polynucleotide sequence by a cleavage sequence, and wherein the multitarget nucleic acid sequence includes a signal sequence upstream of one or more of the polynucleotide sequences. In some embodiments, the signal sequence is present upstream of the first polynucleotide sequence. In some embodiments, the signal sequence is present upstream of some of the 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 may comprise a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence. Thus, in some embodiments, one linker sequence connects the cleavage sequence to the target sequence, and another linker sequence connects the target sequence to the self-assembling sequence in the polynucleotide sequence. In some embodiments, the linker sequence connects the signal sequence to the polynucleotide sequence. As shown in Figure 1, the multi-target nucleic acid sequence may contain multiple repeats of a polynucleotide sequence, where each polynucleotide sequence may contain 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, such that the total number of polynucleotide sequences in the multi-target nucleic acid sequence is 100 or less. In some embodiments, the linker sequence connects the signal sequence to the first polynucleotide sequence. In some embodiments, the signal sequence is present upstream of some or all of the polynucleotide sequences. The multi-target nucleic acid sequence encodes a multi-target peptide.

[0075] The terms "multi-target peptide" or "multi-targeting peptide" are used interchangeably and refer to two or more polypeptides, wherein some or all of the polypeptides 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, wherein one polypeptide is connected to another polypeptide by a cleavage peptide, and wherein the multi-target peptide includes a signal peptide upstream (amino terminal) of one or more of the polypeptides. In some embodiments, the signal peptide is present at the amino terminal end of the first polypeptide. In some embodiments, the signal peptide is present at the amino terminal end of some of the polypeptides. In some embodiments, the signal peptide is present amino terminally 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 the cleavage sequence to the target sequence, and another linker peptide connects the target peptide to the self-assembling peptide in the multi-target peptide. In some embodiments, a multi-target peptide may 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 the multi-target peptide is 100 or less. In some embodiments, a linker peptide connects a signal peptide to the polypeptides. In some embodiments, a signal peptide is present on the amino terminal side of each of some or all of the polypeptides. A multi-target peptide may comprise homologous or heterologous polypeptides.

[0076] The term "polynucleotide sequence" as used herein means a sequence of nucleotides that encodes a polypeptide. The terms "protein" or "peptide" are used interchangeably herein and refer to a polymer of amino acids linked through peptide bonds, but do not refer to any particular length. The terms also encompass fusion proteins, muteins, analogs, or modified forms.

[0077] The term "polypeptide" as used herein refers to a sequence of amino acids comprising 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, a polypeptide comprises a target peptide, a linker peptide, and a self-assembling peptide. In some embodiments, a polypeptide comprises a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide. In some embodiments, a polypeptide has several residues (amino acids) of a cleavage peptide. In some embodiments, a polypeptide has a signal peptide.

[0078] The term "target sequence," as used herein, means a sequence of nucleotides that encodes a target peptide.

[0079] The term "target peptide" as used herein refers to a sequence of amino acids that has an immunostimulatory or immunomodulatory effect. Target peptide also refers to a peptide of interest. In some embodiments, the target peptide is an antigen. In some embodiments, the target peptides in two or more polypeptides may be the same, i.e., homologous polypeptides. In some other embodiments, the target peptides in two or more polypeptides may be different, i.e., heterologous polypeptides.

[0080] The term "signal sequence," as used herein, means a sequence of nucleotides that encodes a signal peptide.

[0081] The term "signal peptide," as used herein, refers to a sequence of amino acids that transports a multi-target peptide to a specific organelle. In some embodiments, the signal peptide transports the multi-target peptide to the Golgi apparatus or Golgi body. The signal peptide is present at the N-terminus (amino-terminus) of one or more polypeptides. In some embodiments, the signal peptide is present at the N-terminus of some or all polypeptides. In some embodiments, the signal peptide is present at the N-terminus of a first polypeptide. In some embodiments, the signal peptide is present at the N-terminus of some polypeptides. In some embodiments, the signal peptide is present at the N-terminus of all polypeptides. In some embodiments, the signal peptide is encoded by a signal sequence. In some embodiments, the signal peptide is a Golgi-targeting signal peptide.

[0082] The term "cleavage sequence" as used herein means a sequence of nucleotides that encodes a cleavage peptide.

[0083] The term "cleavage peptide," as used herein, refers to a sequence of amino acids that promotes cellular proteases to cleave a multi-target peptide into individual polypeptides or to self-cleave the multi-target peptide into individual polypeptides. A cleavage peptide exists between any two polypeptides. It connects one polypeptide to another polypeptide, for example, an adjacent polypeptide. A cleavage peptide carries one or more cleavage sites. In some embodiments, a cleavage peptide is a substrate for a protease. In some embodiments, a cleavage peptide undergoes self-cleavage to generate individual polypeptides. In some embodiments, a cleavage peptide is a substrate for a Golgi-specific protease. In some embodiments, a cleavage peptide contains one or more cleavage sequences, such as cleavage peptide-1, cleavage peptide-2, etc. In some embodiments, a cleavage peptide optionally contains a linker peptide between two cleavage peptides. In some embodiments, a cleavage peptide may self-cleave into individual polypeptides or may be cleaved by the action of a cellular protease.

[0084] The term "linker sequence" as used herein means a sequence of nucleotides that encodes a linker peptide.

[0085] The terms "linker peptide" or "peptide linker" are used interchangeably and refer to a sequence of amino acids that connects a target peptide to a self-assembling peptide, a signal peptide to a target peptide, a cleavage peptide to a target peptide, a signal peptide to a polypeptide, or two cleavage peptides. In some embodiments, a linker peptide connects a signal peptide to a polypeptide. In some embodiments, a linker peptide connects a target peptide to a self-assembling peptide in a polypeptide. In some embodiments, one linker peptide connects a cleavage peptide to a target peptide and another linker peptide connects a target peptide to a self-assembling peptide in a polypeptide. In some embodiments, one linker peptide connects a cleavage peptide to a target peptide and another linker peptide connects a target peptide to a self-assembling peptide. In some embodiments, one linker peptide connects a cleavage peptide to a target peptide and another linker peptide connects a target peptide to a self-assembling peptide. In some embodiments, one linker peptide connects a cleavage peptide to a target peptide and another linker peptide connects a signal peptide to a target peptide. In some embodiments, a 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.

[0086] The term "amino acid linker sequence" as used herein means a sequence of nucleotides that encodes an amino acid linker.

[0087] The term "amino acid linker," as used herein, refers to a sequence of amino acids that provides structural integrity to a polypeptide, ensuring that its components remain in their native or stable conformation as much as possible. In some embodiments, the amino acid linker also assists in orienting the polypeptide so that, in the absence of a foldon or scaffold, a domain or epitope on the target peptide is exposed or presented for interaction or communication with cells, biomolecules, or the immune system. In some embodiments, the amino acid linker connects two cleavage peptides. Some non-limiting examples of amino acid linkers include 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. In some embodiments, the amino acid linker is approximately 2 to 49 amino acids in length.

[0088] 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, refers to a sequence of amino acids that includes one or more glycines (G) and serines (S), in any combination, without any preference for order or limitation on the number of occurrences of either glycine or serine. In some embodiments, the glycine serine linker is a few to several amino acids long.

[0089] The term "foldon sequence" as used herein means a sequence of nucleotides that encodes a foldon. As used herein, the term " foldon " refers to the amino acid sequence that allows two or more homologous polypeptides to assemble and form oligomeric complexes.In some embodiments, foldon also assists in the orientation of polypeptide, so that the domain or epitope on target peptide is exposed or presented for interaction or communication with cell or biomolecule or immune system.

[0090] 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 a target peptide and helps orient the target peptide so that domains or epitopes of the target peptide are exposed or presented for interaction or communication with cells or biomolecules or the immune system.

[0091] The term "oligomeric complex," as used herein, refers to 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 four homologous polypeptides, at least five homologous polypeptides, or at least six homologous polypeptides, etc.

[0092] 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 allows a polypeptide to self-assemble into a polypeptide nanoparticle.

[0093] The terms "self-assembly" or "self-assemble" or "self-assembling" are used interchangeably and refer to the ability of a polypeptide to undergo multimerization to form a polypeptide nanoparticle. In some embodiments, a polypeptide nanoparticle may have at least two polypeptides (dimers or 2-mers), at least three polypeptides (trimers or 3-mers), at least four polypeptides (tetramers or 4-mers), at least five polypeptides (pentamers or 5-mers), at least six polypeptides (hexamers or 6-mers), at least seven polypeptides (heptamers or 7-mers), at least eight polypeptides (octamers or 8-mers), etc. In some embodiments, the polypeptide nanoparticles are up to 100-mers. In some embodiments, a combination of hydrogen bonds, disulfide bonds, hydrophobic interactions, electrostatic interactions, and / or van der Waals forces maintain the self-assembled structure.

[0094] The term "multimerization" as used herein means the association of two or more units of homologous or heterologous polypeptides, or oligomeric complexes, or combinations thereof. The term "polypeptide nanoparticles" as used herein refers to nanoparticles formed by the self-assembly of polypeptides. In some embodiments, polypeptide nanoparticles are composed of two or more homologous polypeptides, or two or more heterologous polypeptides, or one or more oligomeric complexes, or combinations thereof.

[0095] The term "homologous polypeptides" as used herein refers to polypeptides in a multi-target peptide that have the same target peptide. For example, if two polypeptides in a multi-target peptide have the same target peptide, they are considered to be homologous polypeptides.

[0096] The term "heterologous polypeptide" as used herein refers to a polypeptide in a multi-target peptide that has different target peptides. For example, if two polypeptides in a multi-target peptide have different or non-identical target peptides, they are considered to be heterologous polypeptides.

[0097] The term "fragment," as used herein, whether in reference to a nucleic acid, nucleotide, protein, polypeptide, or peptide, means a nucleic acid, protein, polypeptide, or peptide sequence of any length other than the full length of the respective nucleic acid, protein, polypeptide, or peptide sequence.

[0098] The term "variant," as used herein, whether in reference to a nucleic acid, nucleotide, protein, polypeptide, or peptide sequence, refers to a homolog, ortholog, paralog, mutant, or analog of the respective nucleic acid, protein, polypeptide, or peptide sequence.

[0099] The term "mutant," as used herein, refers to a sequence that is not a wild-type sequence, whether it relates to a nucleic acid, nucleotide, protein, polypeptide, or peptide sequence. Mutants are also understood to refer to nucleic acid, nucleotide, protein, polypeptide, or peptide sequences that carry a mutation. The term "mutation," as used herein, refers to a change or modification in a nucleic acid or amino acid sequence compared to a reference sequence, and includes insertions, deletions, substitutions, or combinations thereof.

[0100] Multi-target nucleic acid sequences and multi-target peptides In the present disclosure, a multi-target nucleic acid sequence refers to two or more polynucleotide sequences, wherein some or all of the polynucleotide sequences comprise 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, wherein one polynucleotide sequence is connected to another polynucleotide sequence by a cleavage sequence, and wherein the multi-target nucleic acid sequence includes a signal sequence upstream of one or more of the polynucleotide sequences. In some embodiments, the signal sequence may be present upstream of all or some of the polynucleotide sequences. In some embodiments, the signal sequence is present upstream of the first polynucleotide sequence. In some embodiments, the signal sequence is present upstream of some of the 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 comprises a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence. In some embodiments, a multi-target 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 some embodiments, a multi-target nucleic acid sequence may comprise 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, such that the total number of polynucleotide sequences in the multi-target nucleic acid sequence is 100 or less. In some embodiments, a linker sequence connects a signal sequence to a polynucleotide sequence. In some embodiments, one linker sequence connects a cleavage sequence to a target sequence, and another linker sequence connects a target sequence to a self-assembling sequence in a polynucleotide sequence. Some exemplary descriptions of multi-target nucleic acid sequences are provided in Figure 1, and their representative encoded multi-target peptides are provided in Figure 2.

[0101] The term "nucleic acid" as used herein refers to a polymer containing two or more nucleotides, such as deoxyribonucleotides or ribonucleotides, in unmodified or modified form. Nucleic acids can be single-stranded or double-stranded, linear or circular. The term nucleic acid also encompasses fragments, variants, mutants, or codon-optimized sequences of deoxyribonucleotides or ribonucleotides.

[0102] The term "nucleotide" as used herein means a ribonucleotide or a deoxyribonucleotide. When the term nucleotide is used in the context of RNA, it refers to a ribonucleotide, and when it is used in the context of DNA, it refers to a deoxyribonucleotide.

[0103] In some embodiments, the multi-target nucleic acid sequence is DNA, RNA, or mRNA. The multi-target nucleic acid sequence can be from a few nucleotides to several thousand nucleotides in length.

[0104] Deoxyribonucleic acid (DNA) The terms "deoxyribonucleic acid" or "DNA" are used interchangeably herein and refer to a polymer of deoxyribonucleotides. DNA can be single- or double-stranded, linear, or circular. In some embodiments, the multi-target nucleic acid sequence is DNA. In some embodiments, the DNA encodes a multi-target peptide described herein.

[0105] Ribonucleic acid (RNA) The terms "ribonucleic acid" or "RNA" are used interchangeably herein and refer to a polymer of ribonucleotides. RNA can be single-stranded or double-stranded, linear or circular. The term RNA also encompasses messenger RNA (mRNA). In some embodiments, the multi-target nucleic acid sequence is mRNA.

[0106] In some embodiments, the mRNA encodes a multi-target peptide as described herein. In some embodiments, the mRNA may be unmodified, modified, or a combination of both. The modification may be in the nucleobase of the nucleotide, or in the sugar moiety of the nucleotide, or in the phosphate of the nucleotide.

[0107] In some embodiments, the mRNA is produced using a recombinant expression system, or is chemically synthesized, or is obtained through in vitro transcription. In some embodiments, the mRNA is obtained through a simple in vitro transcription (IVT) process. In some embodiments, the mRNA is circular. In other embodiments, the mRNA is linear.

[0108] In some embodiments, mRNA is self-amplifying or self-replicating.Self-amplifying or self-replicating mRNA as used herein refers to mRNA that self-replicates after being delivered into cells.This mRNA typically contains a replicase sequence, usually derived from alphavirus, which allows the original strand of mRNA that encodes the target protein to be amplified after being delivered into cells (Beissert, Tim et al. Molecular Therapy (2020) 28:119-128).

[0109] Target Sequences and Target Peptides The multi-target nucleic acid sequence and the multi-target peptide include target sequences and target peptides, respectively. In some embodiments, the target sequence is DNA or RNA. In other embodiments, the target sequence is mRNA. The target sequence may be modified or unmodified. The target sequence may include a codon-optimized sequence, a fragment, a mutant, a variant, or a combination thereof. The target sequence may be obtained from prokaryotes or eukaryotes, unicellular or multicellular organisms, viruses, bacteria, fungi, protozoans, parasites, mycoplasma, animals, humans, or a combination thereof.

[0110] In some embodiments, the target sequence is a sequence of nucleotides that encodes a target peptide. In some embodiments, the target peptide comprises a sequence of amino acids that controls or regulates cellular function. The term "cellular function" as used herein refers to various cellular or biological processes, such as, but not limited to, biosynthesis, cell division, cell cycle control, cell metabolism, ion transport, absorption, secretion, homeostasis, replication, transcription, translation, cell signaling, endocytosis, exocytosis, phagocytosis, trogocytosis, pyroptosis, apoptosis, DNA replication, DNA repair, protein synthesis, gene regulation, cell repair, cell growth, cell differentiation, cell trafficking, cell proliferation, metabolic pathways, etc.

[0111] The terms "regulate" or "modulate" or "regulation" or "modulation" are used interchangeably herein and refer to the act of controlling a cellular or biological process or having a modifying or controlling influence on a cellular or biological process.

[0112] In some aspects, the target peptide can perform one or more functions, such as, but not limited to, immunostimulation, immunomodulation, etc. In some embodiments, the target peptide refers to a peptide of interest. In some embodiments, the target peptide is an antigen. In some embodiments, the target peptide is identical in two or more polypeptides, e.g., homologous polypeptides. In some embodiments, the target peptide is different in two or more polypeptides, e.g., in heterologous polypeptides.

[0113] In some embodiments, the target peptide may be from a few amino acids to several hundred amino acids in length.

[0114] In some embodiments, the antigen is selected from the group consisting of, but not limited to, 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 D1, Ep-CAM, Eph derived from A2, EphA3, ELF-2, FGF-5, G250, gonadotropin-releasing hormone, HER-2, intestinal carboxylesterase (iCE), IL13Ralpha2, 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;Cowpox virus, vaccinia virus, pseudocowpox virus, human herpesvirus 1, human herpesvirus 2, cytomegalovirus, human adenoviruses A-F, polyomavirus, human papillomavirus, parvovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, orthoreovirus, rotavirus, Ebola virus, parainfluenza virus, influenza viruses (e.g., H5N1 influenza virus, influenza A virus, influenza B virus, influenza C virus), measles virus, mumps virus those derived from viruses such as rubella virus, pneumovirus, human respiratory syncytial virus, rabies virus, California encephalitis virus, Japanese encephalitis virus, Hantaan virus, lymphocytic choriomeningitis virus, Epstein-Barr virus (EBV), coronaviruses (e.g., MERS-CoV, SARS-CoV-1, SARS-CoV-2, OC43, HKU1, bat coronaviruses, other betacoronaviruses), enteroviruses, rhinoviruses, polioviruses, noroviruses, flaviviruses, dengue virus, West Nile virus, yellow fever virus, and chickenpox;Bacillus anthracis, Brucella spp., Bordetella pertussis, Candida spp., Chlamydia pneumoniae, Chlamydia psittaci, Cholera, Clostridium botulinum, Coccidioides immitis, Cryptococcus spp., Diphtheria, Escherichia coli 0151: H7, Enterohemorrhagic Escherichia coli, Enterotoxigenic Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Legionella spp., Leptospira spp., Listeria spp., Neisseria meningitidis, Mycoplasma pneumoniae pneumoniae, Mycobacterium, whooping cough, pneumonia, Salmonella, Shigella, Staphylococcus, Streptococcus pneumoniae and Yersinia enterocolitica, or protozoa, such as Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi, or a combination thereof;

[0115] Antigens may be, without limitation, allergens derived from cells, cell extracts, proteins, polypeptides, peptides, peptidomimetics of other molecules (such as polysaccharides and small molecules, lipids, glycolipids, and carbohydrates), of plants, animals, fungi, insects, foods, drugs, dusts, and mites. Allergens include, but are not limited to, environmental aeroallergens; plant pollens (e.g., ragweed / hay fever); weed pollen allergens; grass pollen allergens; Johnson grass; tree pollen allergens; rye grass; spider allergens (e.g., house dust mite allergens); storage mite allergens; Japanese cedar pollen / hay fever; mold / fungal spore allergens; animal allergens (e.g., allergens from dogs, guinea pigs, hamsters, gerbils, rats, mice, etc.); food allergens (e.g., shellfish; nuts; citrus fruits; flowers; coffee); insect allergens (e.g., fleas, cockroaches); venoms: (Hymenoptera, yellow wasps) jacket, honeybee, wasp, hornet, red fire ant); bacterial allergens (e.g., streptococcal antigens); parasite allergens such as Ascaris antigens; viral antigens; drug allergens; hormones (e.g., insulin); enzymes (e.g., streptokinase); and drugs or chemicals that can act as incomplete antigens or haptens (e.g., acid anhydrides and isocyanates). When a hapten is used in the compositions of the present disclosure, it may be attached to a carrier to form a hapten-carrier adduct. The hapten-carrier adduct can initiate a humoral immune response, while the hapten itself does not elicit antibody production. Non-limiting examples of haptens are aniline, urushiol (the toxin in poison ivy), hydralazine, fluorescein, biotin, digoxigenin, and dinitrophenol.

[0116] In other embodiments, the antigen is a disease-associated antigen, such as, for example, an amyloid protein (eg, Alzheimer's disease), where it is desirable to sequester the antigen in circulation.

[0117] In some embodiments, the target sequences encoding the target peptides in the multi-target peptide may be obtained from a virus belonging to the following families, e.g., Coronaviridae, Picornaviridae, Caliciviridae, Astroviridae, Togaviridae, Flaviviridae, Arteriviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Bornaviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Retroviridae, Polyomaviridae, Herpesviridae, Poxviridae, Papillomaviridae, Hepadnaviridae, Adenoviridae, Parvoviridae, Hepeviridae, Circoviridae, or a combination thereof.

[0118] In some embodiments, target sequences encoding target peptides that may be incorporated into the multi-target peptide may be obtained from bacteria belonging to the following genera, such as 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 combinations thereof.

[0119] Coronaviridae Members of the Coronaviridae family are large, enveloped, single-stranded RNA viruses with genomes ranging from 25 to 32 kb and virions with diameters of 118 to 140 nm. The Coronaviridae family encompasses the following genera: Alphacoronavirus, Betacoronavirus, Deltacoronavirus, Gammacoronavirus, and Torovirus. Most coronaviruses cause mild respiratory infections. However, some coronaviruses, such as Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV or SARS-CoV-1), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and most recently SARS-CoV-2, have caused deadly pandemics.

[0120] In some embodiments, the target sequence encoding the target peptide is obtained or derived from a coronavirus, including SARS-CoV-1, MERS-CoV, SARS-CoV-2, OC43, HKU1, bat coronaviruses, other betacoronaviruses, or combinations thereof, including codon-optimized sequences, fragments, mutants, or variants thereof, of such target sequences.

[0121] All coronaviruses encode four major structural proteins: spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (N). These proteins or fragments thereof can serve as effective target peptides in accordance with the present disclosure.

[0122] In some embodiments, the target sequence encoding the target peptide includes, but is not limited to, a coronavirus spike protein, membrane protein, envelope protein, or nucleocapsid protein, or a codon-optimized nucleic acid sequence thereof, or a combination thereof, including a fragment, mutant, or variant thereof.

[0123] In some embodiments, the target sequence encoding the target peptide includes a spike protein or a fragment thereof from a betacoronavirus. Betacoronavirus is a genus in the subfamily Othrocoronavirinae of the family Coronaviridae. The International Committee on Taxonomy of Viruses (ICTV) has classified the genus Betacoronavirus into five subgenera: 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.

[0124] Betacoronaviruses have become increasingly clinically important as they have been found to cause pandemics (e.g., the 2002–2003 SARS pandemic caused by SARS-CoV-1 or SARS-CoV, the 2012 MERS pandemic caused by the Middle East Respiratory Syndrome coronavirus, and more recently, the 2019–2020 COVID-19 pandemic caused by SARS-CoV-2). Other betacoronaviruses known to infect humans are HKU1 and OC43.

[0125] The genome of betacoronaviruses encodes four major structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N). 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). Either or both domains may act as receptor-binding domains (RBDs) that interact with host cell receptors. The RBD contains a receptor-binding motif (RBM). The S1 subunit assembles to form a trimeric structure. The S2 subunit assists the virus in entering host cells through membrane fusion. The S2 subunit contains a 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. 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).

[0126] Receptor Binding Domain (RBD) The S1 subunit of a betacoronavirus contains a receptor-binding domain in either the N-terminal or C-terminal region. The RBD interacts with a host cell receptor. In one embodiment, the RBD comprises the full-length S1 subunit of the spike protein of a betacoronavirus or a fragment thereof, including mutants, derivatives, or variants thereof that retain the ability to interact with a host cell receptor. In some embodiments, the receptor-binding domain interacts with an angiotensin-converting enzyme 2 (ACE2) receptor, a dipeptidyl peptidase 4 (DPP4) receptor, a 9-O-acetylated sialic acid (9-O-Ac-Sia) receptor, or a combination thereof. In some embodiments, the multi-target nucleic acid sequence encodes a multi-target peptide comprising one or more receptor-binding domains obtained or derived from one or more betacoronaviruses. In some embodiments, the multi-target nucleic acid sequence encodes a multi-target peptide that includes one or more receptor-binding domains obtained or derived from SARS-CoV-1, SARS-CoV-2, MERS-CoV, OC43, HKU1, bat coronaviruses, other betacoronaviruses, or combinations thereof.

[0127] Fusion peptide The S2 subunit of the spike protein of betacoronaviruses contains a region called a fusion peptide, which promotes membrane fusion. In some embodiments, the target sequence of the present disclosure encodes a fusion peptide of a betacoronavirus. Fusion peptides include fragments, mutants, derivatives, or variants of the fusion peptide. In some embodiments, the multi-target nucleic acid sequence encodes a multi-target peptide that includes one or more fusion peptides obtained or derived from one or more betacoronaviruses.

[0128] Stem Helix The S2 subunit of the spike protein of betacoronaviruses contains a region referred to as the stem helix or S2 stem helix, which allows the heptad repeat to assume a conformation that facilitates fusion pore formation and viral entry into cells. In some embodiments, the targeting sequence of the present disclosure encodes a stem helix. The stem helix encompasses a fragment, mutant, derivative, or variant of a fusion peptide. In some embodiments, the multi-targeting nucleic acid sequence encodes a multi-targeting peptide comprising one or more stem helices obtained or derived from one or more betacoronaviruses.

[0129] Exemplary target peptides include, but are not limited to, those represented by the following amino acid sequences, or combinations thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof: RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF (SEQ ID NO: 1); RVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF (SEQ ID NO: 2); RVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF (SEQ ID NO: 338) RVVPSGDVVRFPNITNLCPFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFKCYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNTRNIDATSTGNYNYKYRYLRHGKLRPFERDISNVPFSPDGKPCTPPALNCYWPLNDYGFYTTTGIGYQPYRVVVLSFELLNAPATVCGPKLSTDLIKNQCVNF (SEQ ID NO: 339) EAKPSGSVVEQAEGVECDFSPLLSGTPPQVYNFKRLVFTNCNYNLTKLLSLFSVNDFTCSQISPAAIASNCYSSLILDYFSYPLSMKSDLSVSSAGPISQFNYKQSFSNPTCLILATVPHNLTTITKPLKYSYINKCSRLLSDDRTEVPQLVNANQYSPCVSIVPSTVWEDGDYYRKQLSPLEGGGWLVASGSTVAMTEQLQMGFGITVQYGTDTNSVCPKLEFANDTKIASQLGNCVEY (SEQ ID NO: 340) RADVYRRKPDLPNCNIEAWLNDKSVPSPLNWERKTFSNCNFNMSSLMSFIQADSFTCNNIDAAKIYGMCFSSITIDKFAIPNGRKVDLQLGNLGYLQSFNYRIDTTATSCQLYYNLPAANVSVSRFNPSTWNKRFGFIEDSVFKPQPAGVFTDHDVVYAQHCFKAPKNFCPCKSNSSLCVGSGPGKNNGIGTCPAGTNYLTCHNLCNPDPITFTGPYKCPQTKSLVGIGEHCSGLAVKSDHCGGNPCTCQPQAFLGWSADSCVQGDKCNIFANLILHDVNSGL (SEQ ID NO: 341) ADVYRRKPDLPNCNIEAWLNDKSVPSPLNWERKTFSNCNFNMSSLMSFIQADSFTCNNIDAAKIYGMCFSSITIDKFAIPNGRKVDLQLGNLGYLQSFNYRIDTTATSCQLYYNLPAANVSVSRFNPSTWNKRFGFIEDSVFKPQPAGVFTDHDVVYAQHCFKAPKNFCPCKSNSSLCVGSGPGKNNGIGTCPAGTNYLTCHNLCNPDPITFTGPYKCPQTKSLVGIGEHCSGLAVKSDHCGGNPCTCQPQAFLGWSADSCVQGDKCNIFANLILHDVNSGL (SEQ ID NO: 342) RIPDLPDCDIDKWLNNFNVPSPLNWERKIFSNCNFNLSTLLRLVHTDSFSCNNFDESKIYGSCFKSIVLDKFAIPNSRRSDLQLGSSGFLQSSNYKIDTTSSSCQLYYSLPAINVTINNYNPSSWNRRYGFNNFNLSSHSVVYSRYCFSVNNTFCPCAKPSFASSCKSHKPPSASCPIGTNYRSCESTTVLDHTDWCRCSCLPDPITAYDPRSCSQKKSLVGVGEHCAGFGVDEEKCGVLDGSYNVSCLCSTDAFLGWSYDTCVSNNRCNIFSNFILNGINSGTTCSND (SEQ ID NO: 343) RVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLLRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFN (SEQ ID NO: 344) RVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRFLRKSKLKPFERDISTEIYQVGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF (SEQ ID NO: 345) RVQPTESIVRFPNVTNLCPFHEVFNATRFASVYAWNRTRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIKGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKHSGNYDYWYRSFRKSKLKPFERDISTEIYQAGNKPCKGKGPNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFN (SEQ ID NO: 346) SFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNG (SEQ ID NO: 3); LQPELDSFKEELDKYFKNHTSPDVDLG (SEQ ID NO: 4); VNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGI (SEQ ID NO: 5); NLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTN (SEQ ID NO: 6); FSNVTWFHAIHVSGTNGTKRFDN (SEQ ID NO: 7); KSFTVEKGIYQTSNFRVQP (SEQ ID NO: 8); SKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYN (SEQ ID NO: 9); GTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGD (SEQ ID NO: 10); MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTAS (SEQ ID NO: 11); KTFPPTEPKKDKKKKADETQALPQRQKKQQ (SEQ ID NO: 12); VQERIGLFIVNFFIFT (SEQ ID NO: 13); LVQPALYLYNTGRSVY (SEQ ID NO: 14); MAAPAAPRAVSFADNNDITNTNLSRGRGRNPKPRAAPNNTVSWYTGLTQHGKVPLTFPPGQGVPLNANSTPAQNAGYWRRQDRKINTGNGIKQLAPRWYFY (SEQ ID NO: 15); KPNRTSVVSFNEW (SEQ ID NO: 16); DVANPSTPAYTFTTVKPGAAFSV (SEQ ID NO: 17); 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); LKPYGGQPVSEYHITLAL (SEQ ID NO: 23); PAFHRIESTDSIVFTYIPASGYVAALAVNVCLI (SEQ ID NO: 24); RSIIRTMVLYFLVLYNFLLAIVLVNGVHYPTGSCLIAFLVILIILWFVDRI (SEQ ID NO: 25); SMKMAPLMLLQLLGR (SEQ ID NO: 26); 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); CALPDTPSPLTPRSVRSVPGEMRLASIAF (SEQ ID NO: 32); AIPFAQSIFY(SEQ ID NO:33); AQQLVRSESAALSAQ (SEQ ID NO: 34); PVNGYFIKTNNTRIVDEWSYTGSSFYSPEPITSLNTKYVAPQVTYQNISTNLPPPLLGNSTIGIDFQDELDEFF (SEQ ID NO: 35); SFIEDLLFNKVTLADAGFIKQY (SEQ ID NO: 334); KQYGDCLGDIAARDLICAQKFN (SEQ ID NO: 335); KFNGLTVLPPLLTDEMIAQYT (SEQ ID NO: 336); GWTFGAGAALQIPFAMQMAYRFNGI (SEQ ID NO: 337);

[0130] In some embodiments, the target peptide shares at least 70% identity with a sequence described herein above, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0131] Considering the amino acid sequence of the above target peptide, those skilled in the art can deduce all possible DNA or RNA sequences that code for the above target peptide. Such DNA or RNA sequences are considered to be incorporated into the present disclosure. In some embodiments, the target peptide is encoded by a target sequence that can be either DNA, RNA or mRNA.

[0132] Herpesviridae Members of the Herpesviridae family consist of large, enveloped, double-stranded DNA viruses that are known to infect a wide variety of hosts, including mammals. Nine herpesviruses have been identified to date that have humans as their primary host: herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus types 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 (in Knipe, David M. and Howley, Peter M., eds., Fields Virology, 6th ed., 2013, pp. 1802-1822), ISBN 9781451105636). These viruses cause different clinical manifestations ranging from asymptomatic to severe disease depending on the immune status of the host.

[0133] 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 a codon-optimized sequence of such target sequence, a fragment, mutant or variant thereof.

[0134] In some embodiments, the target peptide comprises glycoprotein B, glycoprotein C, glycoprotein D, glycoprotein E, glycoprotein K, glycoprotein L, or glycoprotein M of herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0135] In some embodiments, the target peptide comprises glycoprotein B, glycoprotein H, glycoprotein L, glycoprotein M, or glycoprotein N of human cytomegalovirus (HCMV), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0136] In some embodiments, the target peptide comprises glycoprotein B, glycoprotein C, glycoprotein H, or glycoprotein L of varicella-zoster virus (VZV), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0137] In some embodiments, the target peptide includes, but is not limited to, Epstein-Barr virus (EBV) glycoprotein B, glycoprotein H, glycoprotein L, glycoprotein M, glycoprotein N, glycoprotein 42, or glycoprotein 350, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0138] Exemplary target peptides include, but are not limited to, those represented by the following amino acid sequences, or combinations thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof: TWVPKPNVEVWPVDPPPPVN (SEQ ID NO: 36); DEKEGLETTTYITSQEVQNS (SEQ ID NO: 37); GAQTPEQPAPPATTVQPTAT (SEQ ID NO: 38); 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); 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); 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); 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); TAVPTVTSTGGKANSTTGGK (SEQ ID NO: 59); ETDQMDTIY (SEQ ID NO: 60); QMDTIYQCY (SEQ ID NO: 61); 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); 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); 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); NTREYTFSY (SEQ ID NO: 77); PTNTTDITY (SEQ ID NO: 78); FLGNNSILY (SEQ ID NO: 79); HAEMQNPVY (SEQ ID NO: 80);

[0139] In some embodiments, the target peptide shares at least 70% identity with a sequence described herein above, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0140] Considering the amino acid sequence of the above target peptide, those skilled in the art can deduce all possible DNA or RNA sequences that code for the above target peptide. Such DNA or RNA sequences are considered to be incorporated into the present disclosure. In some embodiments, the target peptide is encoded by a target sequence that can be either DNA, RNA or mRNA.

[0141] Poxviridae Members of the Poxviridae family consist of large, enveloped, double-stranded DNA viruses that generally replicate in the cytoplasm of host cells. Variola (smallpox), vaccinia, cowpox, and monkeypox are the most common viruses in the Orthopoxvirus genus of the Poxviridae family.

[0142] In some embodiments, the target sequence encoding the target peptide is obtained or derived from a poxvirus, such as, but not limited to, smallpox virus, vaccinia virus, cowpox virus, or monkeypox virus, or a combination thereof, including a codon-optimized sequence of such a target sequence, a fragment, mutant, or variant thereof.

[0143] In some embodiments, the target peptide includes, but is not limited to, a poxvirus F9 membrane protein, a poxvirus H3L protein, a poxvirus A4 protein, a poxvirus A27 protein, a poxvirus A33 protein, a poxvirus A56 protein, a poxvirus B5 protein, or a poxvirus L1 protein, or a combination thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof.

[0144] Exemplary target peptides include, but are not limited to, those represented by the following amino acid sequences, or combinations thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof. SNEEFDPVDDGPVSDYVSELY (SEQ ID NO: 81); LPAVVYSTCTVPTMNNAKLT (SEQ ID NO: 82); 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); SSSNHEGKPHYITENYRNPY (SEQ ID NO: 88); VRINFKGGYISGGFLPNEYV (SEQ ID NO: 89); RSNEEFDPV (SEQ ID NO: 90); AVVYSTCTV (SEQ ID NO: 91); 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); SNHEGKPHY (SEQ ID NO: 97); GFLPNEYVL (SEQ ID NO: 98); GKWNPILPTCVRSNE (SEQ ID NO: 99); 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); GEINLVHWNKKKYSS (SEQ ID NO: 105); KFRTLLSSSNHEGKP (SEQ ID NO: 106); GGFLPNEYVLSTIHI (SEQ ID NO: 107);

[0145] In some embodiments, the target peptide shares at least 70% identity with a sequence described herein above, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0146] Considering the amino acid sequence of the above target peptide, those skilled in the art can deduce all possible DNA or RNA sequences that code for the above target peptide. Such DNA or RNA sequences are considered to be incorporated into the present disclosure. In some embodiments, the target peptide is encoded by a target sequence that can be either DNA, RNA or mRNA.

[0147] Flaviviridae Members of the Flaviviridae family consist of enveloped single-stranded RNA viruses. Dengue virus, Japanese encephalitis virus, Zika virus, yellow fever virus, West Nile virus, and hepatitis C virus are the most common viruses in the genera Flavivirus and Hepacivirus, respectively.

[0148] 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, West Nile virus, or Hepatitis C virus, or a combination thereof, including a codon-optimized sequence of such target sequence, a fragment, mutant, or variant thereof.

[0149] In some embodiments, the target peptide includes, but is not limited to, a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of the Flavivirus or Hepacivirus genus, or a combination thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof.

[0150] In some embodiments, the target peptide comprises a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Japanese encephalitis virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0151] In some embodiments, the target peptide includes a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Zika virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0152] In some embodiments, the target peptide comprises a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Yellow Fever Virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0153] In some embodiments, the target peptide comprises a West Nile virus capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0154] In some embodiments, the target peptide comprises a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Hepatitis C virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0155] In some embodiments, the target peptide comprises a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a dengue virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0156] Exemplary target peptides include, but are not limited to, those represented by the following amino acid sequences, or combinations thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof. 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); FRCKKNMEGKVVQPENLEYT (SEQ ID NO: 113); LTGYGTVTMECSPRTGLDFN (SEQ ID NO: 114); IDGPETAECPNTNRAWNSLE (SEQ ID NO: 115); 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); 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); 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); FTMRYKKATYEPDVDLGSGT (SEQ ID NO: 131); QGRGPLKLYMALVAFLRFLTI (SEQ ID NO: 132); NSTSTWVTY (SEQ ID NO: 133); TTEAELTGY (SEQ ID NO: 134); 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); 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); LINRFTMRY (SEQ ID NO: 145); RSLIGNEEY (SEQ ID NO: 146); KTWAYHGSY (SEQ ID NO: 147); MTDTTPFGQ (SEQ ID NO: 148); ASSMVNGVF (SEQ ID NO: 149); MSTYGWNLV (SEQ ID NO: 150); QEHETSWHY (SEQ ID NO: 151); WGTIKKSKAINVLRGFR (SEQ ID NO: 152); 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); IPMTGPLVAGGPLTVCYV (SEQ ID NO: 158); IFRKRRLTIMDLHPGAG (SEQ ID NO: 159); GVLWDVPSPPPMGKA (SEQ ID NO: 160); FLLVAHYAIIGPALQAKASREAQKRAAA (SEQ ID NO: 161);

[0157] In some embodiments, the target peptide shares at least 70% identity with a sequence described herein above, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0158] Considering the amino acid sequence of the above target peptide, those skilled in the art can deduce all possible DNA or RNA sequences that code for the above target peptide. Such DNA or RNA sequences are considered to be incorporated into the present disclosure. In some embodiments, the target peptide is encoded by a target sequence that can be either DNA, RNA or mRNA.

[0159] Togaviridae Members of the Togaviridae family consist of enveloped, single-stranded RNA viruses. The Togaviridae family consists of two genera: Alphavirus and Rubivirus. Alphaviruses cause severe human illness, including persistent arthritis and fatal encephalitis, and are an emerging cause of human disease. Some alphaviruses (e.g., Chikungunya (CHIKV), Ross River (RRV), Mayaro (MAYV), Semliki Forest (SFV), Sindbis (SINV), and Onyong-Nyong (ONNV)) cause acute inflammatory musculoskeletal joint-related syndromes that can become chronic, while others (Eastern (EEEV), Western (WEEV), and Venezuelan (VEEV) equine encephalitis viruses) cause infection in the brain and neurological disease (Holmes, Autumn C. et al. 2020 PLoS Pathogens 16(10): e1008876).

[0160] 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) virus, Eastern (EEEV), Western (WEEV), or Venezuelan (VEEV) equine encephalitis virus, or a combination thereof, including a codon-optimized sequence of such target sequence, a fragment, mutant, or variant thereof.

[0161] In some embodiments, the target peptide includes, but is not limited to, an alphavirus capsid protein or envelope protein such as E1, E2, and E3, or a combination thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof.

[0162] In some embodiments, the target peptide includes, but is not limited to, domain A, domain B, or domain C of the E2 protein of an alphavirus, or a combination thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof.

[0163] In some embodiments, the target peptide includes, but is not limited to, a capsid protein or envelope protein such as E1, E2, and E3 of the Chikungunya virus, or a combination thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof.

[0164] In some embodiments, the target peptide includes, but is not limited to, Domain A, Domain B, or Domain C of the E2 protein of Chikungunya virus, or a combination thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof.

[0165] Exemplary target peptides include, but are not limited to, those represented by the following amino acid sequences, or combinations thereof, including codon-optimized nucleic acid sequences thereof, 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); YEKEPEETL (SEQ ID NO: 166); LLANTTFPC (SEQ ID NO: 167); GTLKIQVSL (SEQ ID NO: 168); 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); 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); 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); 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); 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); LTTTDKVINNCKVDQCHAAVTNHKKW (SEQ ID NO: 194); HAAVTNHKKWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYOKNQV (SEQ ID NO: 195); PTVTYGKNQVIMLLYPDHPTLLSYRN (SEQ ID NO: 196); RNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNWEPYKYWPQLSTHGT (SEQ ID NO: 197); LLSMVGMAAGMCHCARRRCITPYELTPGATVPFL (SEQ ID NO: 198); LAHCPDCGEGHSCHS (SEQ ID NO: 199); 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); 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); 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); 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); VPTEGLEVTWGNNEPY (SEQ ID NO: 220); LSTNGTAHGHPHE (SEQ ID NO: 221); HEIILYYYELYP (SEQ ID NO: 222);

[0166] In some embodiments, the target peptide shares at least 70% identity with a sequence described herein above, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0167] Considering the amino acid sequence of the above target peptide, those skilled in the art can deduce all possible DNA or RNA sequences that code for the above target peptide. Such DNA or RNA sequences are considered to be incorporated into the present disclosure. In some embodiments, the target peptide is encoded by a target sequence that can be either DNA, RNA or mRNA.

[0168] Retroviridae Members of the Retroviridae family consist of enveloped, single-stranded RNA viruses. They constitute a large family of viruses that primarily infect both human and vertebrate hosts and cause a wide range of diseases, from malignant lesions to immunodeficiencies and neurological disorders. The Retroviridae family is organized into several genera: alpharetroviruses, betaretroviruses, gammaretroviruses, deltaretroviruses, epsilonretroviruses, lentiviruses, and spumaviruses. Of these genera, lentiviruses have become the subject of considerable interest, the most prominent of which is the human immunodeficiency virus (HIV).

[0169] In some embodiments, the target sequence encoding the target peptide is obtained or derived from an alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus, epsilonretrovirus, lentivirus, spumavirus, or a combination thereof, including a codon-optimized sequence of such a target sequence, a fragment, mutant or variant thereof.

[0170] In some embodiments, the target peptide includes, but is not limited to, a retroviral gag, pol, or env protein, or a codon-optimized nucleic acid sequence thereof, or a combination thereof, including a fragment, mutant, or variant thereof.

[0171] In some embodiments, the target peptide is a lentiviral-derived p17 Gag , p24 Gag , p7 Gag , p6 Gag , gp12 Env , gp41 Env or pol protein, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0172] In some embodiments, the target peptide is p17 from human immunodeficiency virus (HIV). Gag , p24 Gag , p7 Gag , p6 Gag , gp12 Env , gp41 Env or pol protein, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0173] Exemplary target peptides include, but are not limited to, those represented by the following amino acid sequences, or combinations thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof. PEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPAQAGPIAPGQIREPRGSDIAGTTSSLQEQITWMTXNPPIPVGEIYKRWIILGLNKIVRMYSPVGILDIRQGPKEPFRDYVDRFYKTLRAEQASQDVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVG (SEQ ID NO: 223); MGARASVLSGGKLDTWERIRLRPGGKKKYALKHLIWASRELERFKLNPGLLETSEGCKQIIGQLQPSIQTGSEEIRSLYNTVATLYCVHERIEVKDTKEAVEKMEEEQNKSKKKAQQAADSSQVSQNY (SEQ ID NO: 224); GSLAEEEVVIRSENFTDNAKTIIVQLNESVEINCTRPNNNTSKSIPIGPGRAFYATDRIIGDIRQAHCNLSRTKWDSTLEKIVIKLREQVGNKTIVFNQS (SEQ ID NO: 225); MGAASLTLTVQARQLLSGIVQQQNDLLRAIEAQQHLLQLTVWGIKQLQARVLAVERYLKDQQLLGIWGCSGKLICTTAVPWNSSWSNKSQDQIWHNMTWMEWEREIENYTDLIYTLIEKSQNQQEKNEQELLELDKWASLWNW (SEQ ID NO: 226); KIRLRPGGK (SEQ ID NO: 227); KYKLKHIVW (SEQ ID NO: 228); GHQAAMQMLKETI (SEQ ID NO: 229); 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); QMAVFIHHFKRK (SEQ ID NO: 235); NTSVITQACPKVSFEPEPIHYCAPA (SEQ ID NO: 236); GFAILKCNNKT (SEQ ID NO: 237); SLYNTVATL (SEQ ID NO: 238);

[0174] In some embodiments, the target peptide shares at least 70% identity with a sequence described herein above, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0175] Considering the amino acid sequence of the above target peptide, those skilled in the art can deduce all possible DNA or RNA sequences that code for the above target peptide. Such DNA or RNA sequences are considered to be incorporated into the present disclosure. In some embodiments, the target peptide is encoded by a target sequence that can be either DNA, RNA or mRNA.

[0176] Paramyxoviridae Members of the Paramyxoviridae family consist of enveloped, single-stranded RNA viruses. Diseases caused by these viruses continue to result in high mortality and morbidity worldwide. The Paramyxovirinae family is divided into two subfamilies: Paramyxovirinae and Pneumovirinae. The former contains seven genera: Respirovirus, Rubulavirus, Morbillivirus, Henipavirus, Aquaparamyxovirus, Avulavirus, and Ferravirus, while the latter contains two genera: Pneumovirus and Metapneumovirus.

[0177] Some of the important members of the Paramyxoviridae family known to cause disease outbreaks are: mumps virus (MuV), parainfluenza virus type 5 (PIV5), human parainfluenza virus types 2, 4a, and 4b (HPIV2 / 4a / 4b), Newcastle disease virus (NDV), human parainfluenza virus types 1 and 3 (HPIV1 / 3), Nipah virus (NiV), measles virus (MeV), human respiratory syncytial virus (HRSV) A2, B1, S2, and human metapneumovirus (HMPV).

[0178] In some embodiments, the target sequence encoding the target peptide is obtained or derived from mumps virus (MuV), parainfluenza virus type 5 (PIV5), human parainfluenza virus types 2, 4a and 4b (HPIV2 / 4a / 4b), Newcastle disease virus (NDV), human parainfluenza virus types 1 and 3 (HPIV1 / 3), Nipah virus (NiV), measles virus (MeV), human respiratory syncytial virus (HRSV) A2, B1, S2, or human metapneumovirus (HMPV), or a combination thereof, including a codon-optimized sequence of such target sequence, a fragment, mutant or variant thereof.

[0179] In some embodiments, target peptides include, but are not limited to, the 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 codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof of mumps virus (MuV), parainfluenza virus type 5 (PIV5), human parainfluenza virus types 2, 4a, and 4b (HPIV2 / 4a / 4b), Newcastle disease virus (NDV), human parainfluenza virus types 1 and 3 (HPIV1 / 3), Nipah virus (NiV), measles virus (MeV), human respiratory syncytial virus (HRSV) A2, B1, S2, human metapneumovirus (HMPV), or combinations thereof.

[0180] In some embodiments, the target peptide includes, but is not limited to, the 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 of human respiratory syncytial virus (HRSV) A2, B1, S2, or a combination thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof.

[0181] Exemplary target peptides include, but are not limited to, those represented by the following amino acid sequences, or combinations thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof. 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); 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); 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);

[0182] In some embodiments, the target peptide shares at least 70% identity with a sequence described herein above, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0183] Considering the amino acid sequence of the above target peptide, those skilled in the art can deduce all possible DNA or RNA sequences that code for the above target peptide. Such DNA or RNA sequences are considered to be incorporated into the present disclosure. In some embodiments, the target peptide is encoded by a target sequence that can be either DNA, RNA or mRNA.

[0184] Papillomaviridae Members of the Papillomaviridae family are small, non-enveloped, double-stranded DNA viruses. The most common genus in the Papillomaviridae family is the papillomavirus. Human papillomaviruses are the most prominent papillomaviruses known to cause papillomas or warts, and are responsible for cervical, vaginal, vulvar, penile, anal, and oropharyngeal cancers.

[0185] In some embodiments, the target sequence encoding the target peptide is obtained or derived from a papillomavirus, preferably a human papillomavirus, including codon-optimized sequences of such target sequences, fragments, mutants, or variants thereof.

[0186] In some embodiments, the target sequence encoding the target peptide is obtained or derived from a human papillomavirus selected from the group including: HPV1, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV10, HPV11, HPV12, HPV13, HPV14, HPV15, HPV16, HPV17, HPV18, HPV19, HPV20, HPV21, HPV22, HPV23, HPV24, HPV 25, HPV26, HPV27, HPV28, HPV29, HPV29, HPV30, HPV31, HPV32, HPV33, HPV34, HPV35, HPV36, H PV37, HPV38, HPV39, HPV40, HPV41, HPV42, HPV43, HPV44, HPV45, HPV46, HPV47, HPV48, HPV49, HPV50, HPV51, HPV52, HPV53, HPV54, HPV55, HPV56, HPV57, HPV58, HPV59, HPV60, HPV61, HPV6 2, HPV63, HPV64, HPV65, HPV66, HPV67, HPV68, HPV69, HPV70, HPV71, HPV72, HPV73, HPV74, HPV 75, HPV76, HPV77, HPV78, HPV79, HPV80, HPV81, HPV82, HPV83, HPV84, HPV85, HPV86, HPV87, H PV88, 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.

[0187] In some embodiments, the target peptide includes, but is not limited to, an early (E) protein or a late (L) protein of a papillomavirus, preferably a human papillomavirus, or a combination thereof, including a codon-optimized sequence, fragment, mutant, or variant thereof.

[0188] In some embodiments, the target peptide includes, but is not limited to, the E1, E2, E4, E5, E6, or E7 protein of a papillomavirus, preferably a human papillomavirus, or a combination thereof, including a codon-optimized sequence, fragment, mutant, or variant thereof.

[0189] In some embodiments, the target peptide includes, but is not limited to, the L1 (major capsid) protein, L2 (minor capsid) protein, or combinations thereof, including codon-optimized sequences, fragments, mutants, or variants thereof, of a papillomavirus, preferably a human papillomavirus.

[0190] In some embodiments, the target peptide comprises the L1 (major capsid) protein of a papillomavirus, preferably a human papillomavirus, that has lost its ability to self-assemble into virus-like particles. In some embodiments, the L1 protein is modified or mutated such that it loses its ability to self-assemble into virus-like particles.

[0191] In some embodiments, the target peptide comprises the L2 (minor capsid) protein of a papillomavirus, preferably a human papillomavirus, including codon-optimized sequences, fragments, mutants, or variants thereof.

[0192] Exemplary target peptides include, but are not limited to, those represented by the following amino acid sequences, or combinations thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof. QLYKTCKQAGTCPPDIIPKV (SEQ ID NO: 353) KTCKQAGTCPPDIIPKVEG (SEQ ID NO: 354) GGLGIGTGSGTGGRTGYIPL (SEQ ID NO: 355) RTGYIPLGTRPPT (SEQ ID NO: 356) LVEETSFIDAGAP (SEQ ID NO: 357) MAHSRARRRKRASATQLYQTCKLTGTCPPDVIPKVEHNTIADQILKWGSLGVFFGGLGIGTGSGTGGRTGYVPLGTSAKPSITSGMARPPPVVVEPVAPSDPSIVSLIEESAIINAG APEIVPPAHGGFTITSSETTTPAILDVSVTSHTTTSIFRNPVFTEPSVTQPQPPVEANGHILISAPTITSHPIEEIPLDTFVISSSDSGPTSSTPVPGTAPRPRVGLYSRALHQVQV TDPAFLSTPQRLITYDNPVYEGEDVSVQFSHDSIHNAPDEAFMDIIRLHRPAIASRRGLVRYSRIGQRGSMHTRSGKHIGARIHYFYDISPIAQAAEEIEMHPLVAAQEDTFDIYAESFEPDINPTQHPVTNISDTYLTSTPNTVTQPWGNTTVPLSIPNDLFLQSGPDITFPTAPMGTPFSPVTPALPTGPVFITGSGFYLHPAWYFARKRRKRIPLFFSDVAA (SEQ ID NO: 358) MKPRARRRKRASATQLYQTCKATGTCPPDVIPKVEHTTIADQILKWGSLGVFFGGLGIGTGAGSGGRAGYIPLGSSPKPAITGGPAARPPVLVEPVAPSDPSIVSLIEESAIINAG APEVVPPTQGGFTITSSESTTPAILDVSVTNHTTTSVFQNPLFTEPSVIQPQPPVEANGHILISAPTITSQHVEDIPLDTFVVSSSDSGPTSSTPLPRAFPRPRVGLYSRALQQVQ VRDPAFLSTPQRLVTYDNPVYEGEDVSLQFTHESIHNAPDEAFMDIIRLHRPAITSRRGLVRFSRIGQRGSMYTRSGQHIGARIHYFQDISPVTQAAEEIELHPLVAAENDTFDIYAEPFDPIPDPVQHSVTQSYLTSTPNTLSQSWGNTTVPLSIPSDWFVQSGPDITFPTASMGTPFSPVTPALPTGPVFITGSDFYLHPTWYFARRRRKRIPLFFTDVAA (SEQ ID NO: 359) MRHKRSAKRTKRASATQLYKTCKQAGTCPPDIIPKVEGKTIADQILQYGSMGVFFGGLGIGTGSGTGGRTGYIPLGTRPPTATDTLAPVRPPLTVDPVGPSDPSIVSLVEETSFIDAGAP TPVPSIPPDVSGFSITTSTDTTPAILDINNTVTTVTTHNNPTFTDPSVLQPPTPAETGGHFTLSSSTISTHNYEEIPMDTFIVSTNPNTVTSSTPIPGSRPVARLGLYSRTTQQVKVVDPA FVTAPTKLITYDNPAYEGIDVDNTFYFPSNDNSINIAPDPDFLDIVALHRPALTSRRTGIRYSRIGNKQTLRTRSGKSIGAKVHYYYDLSTINPAEEIELQTITPSTYTTTSHAASPTSINNGLYDIYADDFITDTVTTPVPAIPSTSLSGYIPANTTIPFGGAYNIPLVSGPDIPINTTDQTPSLIPIVPGSPQYTIIADGGDFYLHPSYYMLRKRRKRLPYFFSDVSLAA (SEQ ID NO: 360) MVSHRAARRKRASVTDLYKTCKQSGTCPSDVVNKVEGTTLADKILQWSSLGIFLGGLGIGTGSGTGGRTGYIPLGGRSNTVVDVGPTRPPVVIEPVGPTDPSIVTLIEDSSVVTSGA PRPTFTGTSGFDITSAGTTTPAVLDITPSSTSVSISTTNFTNPAFSDPSIIEVPQTGEVSGNVFVGTPTSGTHGYEEIPLQTFASSGTGEEPISSTPLPTVRRVTGPRLYSRAYQQVS VANPEFLTRPSSLITYDNPAFEPMDTTLTFEPRSNVPDSDFMDIIRLHRPASTSRRGTVRFSRLGQRATMFTRSGTQIGARVHFYHDISPIAPSPEYIELQPLVSATEDNGLFDIYADDIDPALPVPSRPTTSSAVSTYSPTISSASSYSNVTVPLTSSWDVPVYTGPDITLPSTTSVWPIVSPTDPASTQYIGIHGTHYYLWPLYYFIPKKRKRVPYFFADGFVAA (SEQ ID NO: 361) MRSKRSTKRTKRASATQLYQTCKAAGTCPSDVIPKIEHTTIADQILRYGSMGVFFGGLGIGSGSGTGGRTGYVPLSTRPSTVSEASIPIRPPVSIDPVGPLDPSIVSLVEESGIVDVG APAPIHPPTTSGFDIATTADTTPAILDVTSVSTHENPTFTDPSVLQPPTPAETSGHLLLSSSSISTHNYEEIPMDTFIVSTNNENITSSTPIPGVRRRPARLGLYSKATQQVKVIDPTF LSAPKQLITYENPAYETVNAEESLYFSNTSHNIAPDPDFLDIIALHRPALTSRRNTVRYSRLGNKQTLRTRSGATIGARVHYYYDISSINPAGESIEMQPLGASATTTSTLNDGLYDIYADTDFTVDTPATHNVSPSTAVQSTSAVSAYVPTNTTVPLSTGFDIPIFSGPDVPIEHAPTQVFPFPLAPTTPQVSIFVDGGDFYLHPSYYMLKRRRKRVSYFFTDVSVAA (SEQ ID NO: 362) MRHKRSTRRKRASATQLYQTCKATGTCPPDVIPKVEGSTIADQILKYGSLGVFFGGLGIGTGSGSGGRTGYVPIGTDPPTAAIPLQPIRPPVTVDTVGPLDSSIVSLIEETSFIEAGAP APSIPTPSGFDVTTSADTTPAIINVSSVGESSIQTISTHLNPFTEPSVLHPPAPAEASGHFIFSSPTVSTQSYENIPMDTFVVSTDSSNVTSSTPIPGSRPVARLGLYSRNTQQVKVV DPAFLTSPHKLITYDNPAFESFDPEDTLQFQHSDISPAPDPDFLDIIALHRPAITSRRHTVRFSRVGQKATLKTRSGKQIGARIHYYQDLSPIVPLDHTVPNEQYELQPLHDTSTSSYSINDGLYDVYADDVDNVHTPMQHSYSTFATTRTSNVSIPLNTGFDTPVMSGPDIPSPLFPTSSPFVPISPFFPFDTIVVDGADFVLHPSYFILRRRRKRFPYFFTDVRVAA (SEQ ID NO: 363) MVSHRAARRKRASATDLYKTCKQSGTCPPDVINKVEGTTLADRILQWSSLGIFLGGLGIGTGSGSGGRTGYVPLGGRSNTVVDVGPTRPPVVIDPVGPTDPSIVTLVEESSVVSSGAP VPTFTGTSGFEITSSGTTTPAVLDITPTVDSVSISSTSFTNPAFSDPSIIEVPQTGEVSGNIFVGTPTSGSHGYEEIPLQTFASSGSGTEPISSTPLPTVRRVAGPRLYSRANQQVRV STSQFLTRPSSLVTFDNPAYEPLDTTLSFEPTSNVPDSDFMDIIRLHRPALSSRRGTVRFSRLGQRATMFTRSGKQIGGRVHFYHDISPIAATEEIELQPLLSATDDSDLFDVYADFPPPASTTPSTINKSFTYPKYSLTMPSTAASSYSNVTVPLTSAWDVPIYTGPDIILPSHTPMWPSTSPTNAATSTYIGIHGTQYYLWPWYYYFPKKRKRIPYFFADGFVAA (SEQ ID NO: 364) MRYRRSTRHKRASATQLYQTCKASGTCPPDVIPKVEGTTIADQLLKYGSLGVFFGGLGIGTGAGSGGRAGYVPLSTRPPTSSITTSTIRPPVTVEPIGPLEPSIVSMIEETTFIES GAPAPSIPSATGFDVTTSANNTPAIINVTSIGESSVQSVSTHLNPTFTEPSIIQPPAPAEASGHVLFSSPTISTHTYEEIPMDTFVTSTDSSSVTSSTPIPGSRPTTRLGLYSRATQ QVKVVDPAFMSSPQKLVTYNNPVFEGVDTDETIIFDRSQLLPAPDPDFLDIIALHRPALTSRRGTVRFSRLGNKATLRTRSGKQIGARVHYYHDISPIQPAEVQEDIELQPLLPQS VSPYTINDGLYDVYADSLQQPTFHLPSTLSTHNNTFTVPINSGIDFVYQPTMSIESGPDIPLPSLPTHTPFVPIAPTAPSTSIIVDGTDILHPSYFLLRRRRKRFPYFFTDVRVAA (SEQ ID NO: 365) MRHKRSTRRKRASATQLYQTCKASGTCPPDVIPKVEGTTIADQILRYGSLGVFFGGLGIGTGSGTGGRTGYVPLGSTPPSEAIPLQPIRPPVTVDTVGPLDSSIVSLIEESSFIDAGAPA PSIPTPSGFDITTSADTTPAILNVSSIGESSIQTVSTHLNPSFTEPSVLRPPAPAEASGHLIFSSPTVSTHSYENIPMDTFVISTDSGNVTSSTPIPGSRPVARLGLYSRNTQQVKVVDP AFLTSPHRLVTYDNPAFEGFNPEDTLQFQHSDISPAPDPDFLDIVALHRPALTSRRGTVRYSRVGQKATLRTRSGKQIGAKVHYYQDLSPIQPVQEQVQQQQQFELQSLNTSVSPYSINDGLYDIYADDADTIHDFQSPLHSHTSFATTRTSNVSIPLNTGFDTPLVSLEPGPDIASSVTSMSSPFIPISPLTPFNTIIVDGADFMLHPSYFILRRRRKRFPYFFADVRVAA (SEQ ID NO: 366) KRAAPKDIYPSCKISNTCPPDIQNKIEHTTIADKILQYGSLGVFLGGLGIGTARGSGGRIGYTPLGEGGGVRVATRPT (SEQ ID NO: 367) KRASATQLYQTCKLTGTCPPDVIPKVEHNTIADQILKWGSLGVFFGGLGIGTGSGTGGRTGYVPLGTSAKPSITSGPM (SEQ ID NO: 368) KRDSVTHIYQTCKQAGTCPPDVINKVEQTTVADNILKYGSAGVFFGGLGISTGRGTGGATGYVPLGEGPGVRVGGTPT (SEQ ID NO: 369) SATQLYQTCKAAGTCPSDVIPKIEHTTIADQILRYGSMGVFFGGLGIGSGSGTGGRTGYVPLSTRPSTVSEASIPRA (SEQ ID NO: 370) MAHSRARRRKRASATQLYQTCKLTGTCPPDVIPKVEHNTIADQILKWGSLGVFFGGLGIGTGSGTGGRTGYVPLGTSAKPSITSGPMARPPVVVEPVAPS (SEQ ID NO: 371) MKPRARRRKRASATQLYQTCKATGTCPPDVIPKVEHTTIADQILKWGSLGVFFGGLGIGTGAGSGGRAGYIPLGSSPKPAITGGPAARPPVLVEPVAPSD (SEQ ID NO: 372) MRHKRSAKRTKRASATQLYKTCKQAGTCPPDIIPKVEGKTIADQILQYGSMGVFFGGLGIGTGSGTGGRTGYIPLGTRPPTATDTLAPVRPPLTVDPVGP (SEQ ID NO: 373) MVSHRAARRKRASVTDLYKTCKQSGTCPSDVVNKVEGTTLADKILQWSSLGIFLGGLGIGTGSGTGGRTGYIPLGGRSNTVVDVGPTRPPVVIEPVGPTD (SEQ ID NO: 374) MRSKRSTKRTKRASATQLYQTCKAAGTCPSDVIPKIEHTTIADQILRYGSMGVFFGGLGIGSGSGTGGRTGYVPLSTRPSTVSEASIPIRPPVSIDPVGP (SEQ ID NO: 375) MRHKRSTRRKRASATQLYQTCKATGTCPPDVIPKVEGSTIADQILKYGSLGVFFGGLGIGTGSGSGGRTGYVPIGTDPPTAAIPLQPIRPPVTVDTVGPL (SEQ ID NO: 376) MVSHRAARRKRASATDLYKTCKQSGTCPPDVINKVEGTTLADRILQWSSLGIFLGGLGIGTGSGSGGRTGYVPLGGRSNTVVDVGPTRPPVVIDPVGPTD (SEQ ID NO: 377) MRYRRSTRHKRASATQLYQTCKASGTCPPDVIPKVEGTTIADQLLKYGSLGVFFGGLGIGTGAGSGGRAGYVPLSTRPPTSSITTSTIRPPVTVEPIGPL (SEQ ID NO: 378) MRHKRSTRRKRASATQLYQTCKASGTCPPDVIPKVEGTTIADQILRYGSLGVFFGGLGIGTGSGTGGRTGYVPLGSTPPSEAIPLQPIRPPVTVDTVGPL (SEQ ID NO: 379) MAHSRARRRKRASATQLYQTCKLTGTCPPDVIPKVEHNTIADQILKWGSLGVFFGGLGIGTGSGTGGRTGYVPLGTSAKPSITSGPMARPPVVVEPVAPSDPSIVSLIEESAIINAGAPEIVPPAHGGFTITSSETTTPAILDVSVTSHTTTSIFRNPVFTEPSVTQPQPPVEANGHILISAPTITSHPIEEIPLDTFVI (SEQ ID NO: 380) MKPRARRRKRASATQLYQTCKATGTCPPDVIPKVEHTTIADQILKWGSLGVFFGGLGIGTGAGSGGRAGYIPLGSSPKPAITGGPAARPPVLVEPVAPSDPSIVSLIEESAIINAGAPEVVPPTQGGFTITSSESTTPAILDVSVTNHTTTSVFQNPLFTEPSVIQPQPPVEANGHILISAPTITSQHVEDIPLDTFVVS (SEQ ID NO: 381) MRHKRSAKRTKRASATQLYKTCKQAGTCPPDIIPKVEGKTIADQILQYGSMGVFFGGLGIGTGSGTGGRTGYIPLGTRPPTATDTLAPVRPPLTVDPVGPSDPSIVSLVEETSFIDAGAPTPVPSIPPDVSGFSITTSTDTTPAILDINNTVTTVTTHNNPTFTDPSVLQPPTPAETGGHFTLSSSTISTHNYEEIPMDT (SEQ ID NO: 382) MVSHRAARRKRASVTDLYKTCKQSGTCPSDVVNKVEGTTLADKILQWSSLGIFLGGLGIGTGSGTGGRTGYIPLGGRSNTVVDVGPTRPPVVIEPVGPTDPSIVTLIEDSSVVTSGAPRPTFTGTSGFDITSAGTTTPAVLDITPSSTSVSISTTNFTNPAFSDPSIIEVPQTGEVSGNVFVGTPTSGTHGYEEIPLQTF (SEQ ID NO: 383) MRSKRSTKRTKRASATQLYQTCKAAGTCPSDVIPKIEHTTIADQILRYGSMGVFFGGLGIGSGSGTGGRTGYVPLSTRPSTVSEASIPIRPPVSIDPVGPLDPSIVSLVEESGIVDVGAPAPIPHPPTTSGFDIATTADTTPAILDVTSVSTHENPTFTDPSVLQPPTPAETSGHLLLSSSSISTHNYEEIPMDTFIVST (SEQ ID NO: 384) MRHKRSTRRKRASATQLYQTCKATGTCPPDVIPKVEGSTIADQILKYGSLGVFFGGLGIGTGSGSGGRTGYVPIGTDPPTAAIPLQPIRPPVTVDTVGPLDSSIVSLIEETSFIEAGAPAPSIPTPSGFDVTTSADTTPAIINVSSVGESSIQTISTHLNPTFTEPSVLHPPAPAEASGHFIFSSPTVSTQSYENIPMDT (SEQ ID NO: 385) MVSHRAARRKRASATDLYKTCKQSGTCPPDVINKVEGTTLADRILQWSSLGIFLGGLGIGTGSGSGGRTGYVPLGGRSNTVVDVGPTRPPVVIDPVGPTDPSIVTLVEESSVVSSGAPVPTFTGTSGFEITSSGTTTPAVLDITPTVDSVSISSTSFTNPAFSDPSIIEVPQTGEVSGNIFVGTPTSGSHGYEEIPLQTF (SEQ ID NO: 386) MRYRRSTRHKRASATQLYQTCKASGTCPPDVIPKVEGTTIADQLLKYGSLGVFFGGLGIGTGAGSGGRAGYVPLSTRPPTSSITTSTIRPPVTVEPIGPLEPSIVSMIEETTFIESGAPAPSIPSATGFDVTTSANNTPAIINVTSIGESSVQSVSTHLNPTFTEPSIIQPPAPAEASGHVLFSSPTISTHTYEEIPMDT (SEQ ID NO: 387) MRHKRSTRRKRASATQLYQTCKASGTCPPDVIPKVEGTTIADQILRYGSLGVFFGGLGIGTGSGTGGRTGYVPLGSTPPSEAIPLQPIRPPVTVDTVGPLDSSIVSLIEESSFIDAGAPAPSIPTPSGFDITTSADTTPAILNVSSIGESSIQTVSTHLNPSFTEPSVLRPPAPAEASGHLIFSSPTVSTHSYENIPMDT (SEQ ID NO: 388)

[0193] In some embodiments, the target peptide shares at least 70% identity with a sequence described herein above, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0194] Considering the amino acid sequence of the above target peptide, those skilled in the art can deduce all possible DNA or RNA sequences that code for the above target peptide. Such DNA or RNA sequences are considered to be incorporated into the present disclosure. In some embodiments, the target peptide is encoded by a target sequence that can be either DNA, RNA or mRNA.

[0195] Self-assembling sequences and self-assembling peptides The multi-target nucleic acid sequence and the multi-target peptide respectively comprise a self-assembling sequence and a self-assembling peptide.The self-assembling sequence is composed of a sequence of nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes a self-assembling peptide.The self-assembling sequence comprises a codon-optimized sequence, a fragment, a mutant, a variant, or a combination thereof.In some embodiments, the self-assembling sequence is DNA, RNA, or mRNA.

[0196] Any self-assembling peptide capable of self-assembling into polypeptide nanoparticles can be employed in accordance with the present disclosure. In some embodiments, the self-assembling peptide may be a full-length protein or a fragment thereof, a mutant, or a variant thereof.

[0197] In some embodiments, the self-assembling peptides include, but are 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 combinations thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof.

[0198] 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 generally consists of 24 subunits or peptides that self-assemble into ferritin nanoparticles.

[0199] In some aspects, the multi-target nucleic acid sequence and the multi-target peptide comprise a ferritin sequence and a ferritin peptide, respectively.The ferritin sequence is composed of a sequence of nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes the ferritin peptide.In some embodiments, the ferritin sequence is DNA, RNA, or mRNA.

[0200] Any ferritin peptide capable of self-assembling into nanoparticles can be employed in accordance with the present disclosure. In some embodiments, the ferritin peptide is obtained or derived from Helicobacter pylori ferritin, including its codon-optimized nucleic acid sequence, fragments, mutants, or variants.

[0201] Exemplary self-assembling peptides include, but are not limited to, those represented by the following amino acid sequences, or combinations thereof, including codon-optimized sequences, fragments, mutants, or variants thereof: LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGI (SEQ ID NO: 331); LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIRRKR (SEQ ID NO: 333); LSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO: 254); MQIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGREEDITLVRVPGSWEIPVAAGELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLANLSLELRKPITFGVITADTLEQAIERAGTKHGNKGWEAALSAIEMANLFKSLR (SEQ ID NO: 255); MTKKVGIVDTTFARVDMASIAIKKLKELSPNIKIIRKTVPGIKDLPVACKKLLEEEGCDIVMALGMPGKAEKDKVCAHEASLGLMLAQLMTNKHIIEVFVHEDEAKDDKELDWLAKRRAEEHAENVYYLLFKPEYLTRMAGKGLRQGFEDAGPARE (SEQ ID NO: 256); MTEKEKMLAEKWYDANFDQYLINERARAKDICFELNHTRPSATNKRKELIDQLFQTTTDNVSISIPFDTDYGWNVKLGKNVYVNTNCYFMDGGQITIGDNVFIGPNCGFYTATHPLNFHHRNEGFEKAGPIHIGSNTWFGGHVAVLPGVTIGEGSVIGAGSVVTKDIPPHSLAVGNPCKVVRKIDNDLPSETLNDETIK (SEQ ID NO: 257); MENTTSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFQGGAPTCPGQNSQSPTSNHSPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLLPGTSTTGTGPCRTCTIPAQGTSMFPSCCCTKPSDGNCTCIPIPSSWAFARFLWEWASVRFSWLSLLVPFVQWFAGLSPTVWLSVIWMMWYRGPSLYNTLSPFLPLLPISFCLWVYI (SEQ ID NO: 258); MIFVLGGCRHKLVCSPAPCNFFHLCLIISCSCPTVHASKLCLGWLWGMHIDPYKEFGASVELLSFLPSDFFPSIRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC (SEQ ID NO: 259); MSLWLPSEATVYLPPVPVSKVVSTDEYVARTNIYYHAGTSRLLAVGHPYFPIKKPNNNKILVPKVSGLQYRVFRIHLPDPNKFGFPDTSFYNPDTQRLVWACVGVEVGRGQPLGVGISGHPLLNKLDD TENASAYAANAGVDNRECISMDYKQTQLCLIGCKPPIGEHWGKGSPCTNVAVNPGDCPPLELINTVIQDGDMVDTGFGAMDFTTLQANKSEVPLDICTSICKYPDYIKMVSEPYGDSLFFYLRREQMFV RHLFNRAGAVGENVPDDLYIKGSGSTANLASSNYFPTPSGSMVTSDAQIFNKPYWLQRAQGHNNGICWGNQLFVTVVDTTRSTNMSLCAAISTSETTYKNTNFKEYLRHGEEYDLQFIFQLCKITLTADVMTYIHSMNSTILEDWNFGLQPPPGGTLEDTYRFVTSQAIACQKHTPPAPKEDPLKKYTFWEVNLKEKFSADLDQFPLGRKFLLQAGLKAKPKFTLGKRKATPTTSSTSTTAKRKKRKL (SEQ ID NO: 260); MSLLTEVETYVLSIVPSGPLKAEIAQRLEDVFAGKNTDLEALMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDRAVKLYRKLKREITFHGAKEIALSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQMVTTTNPLIRHENRMVLASTTAKAMEQMAGSSEQAAEAMEVASQARQMVQAMRAIGTHPRSSAGLKDDLLENLQAYQKRMGVQMQRFK (SEQ ID NO: 261);

[0202] In some embodiments, the self-assembling peptides share at least 70% identity with a sequence described herein above, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0203] Considering the amino acid sequence of the above self-assembling peptide, those skilled in the art can deduce all possible DNA or RNA sequences that code for the above self-assembling peptide. Such DNA or RNA sequences are considered to be incorporated into the present disclosure. In some embodiments, the self-assembling peptide is coded by a self-assembling sequence, which can be either DNA, RNA or mRNA.

[0204] Linker Sequences and Linker Peptides The multi-target nucleic acid sequence and the multi-target peptide each comprise a linker sequence and a linker peptide.The linker sequence is composed of a nucleotide sequence, either deoxyribonucleotide or ribonucleotide, that encodes the linker peptide.The linker sequence can comprise a codon-optimized sequence, a fragment, a mutant, a variant, or a combination thereof.In some embodiments, the linker sequence is DNA, RNA, or mRNA.

[0205] In some embodiments, a linker peptide connects a target peptide to a self-assembling peptide in a polypeptide. In some embodiments, a linker peptide connects a signal peptide to a polypeptide. In some other embodiments, a linker peptide connects a signal sequence to a first polypeptide. In some embodiments, one linker peptide connects a cleavage peptide to a target peptide, and another linker peptide connects the target peptide to a self-assembling peptide in a polypeptide. In some embodiments, a linker peptide connects two cleavage peptides. Any suitable linker peptide can be employed in accordance with the present disclosure.

[0206] In some embodiments, the linker peptide is an amino acid linker, a foldon, a scaffold, or a combination thereof. In some embodiments, the linker peptide consists of a combination of an amino acid linker and a foldon. In some embodiments, the linker peptide consists of a combination of an amino acid linker and a scaffold. In some embodiments, the linker peptide consists of a combination of a foldon and a scaffold. In some embodiments, the linker peptide consists of a combination of an amino acid linker, a foldon, and a scaffold.

[0207] In some embodiments, the amino acid linker is composed of about 2 to 49 amino acids, 2 to 40 amino acids, 2 to 40 amino acids, 2 to 20 amino acids, 2 to 15 amino acids, or 2 to 10 amino acids. In some embodiments, the amino acid linker may include a glycine-serine linker, a glycine-proline linker, a glycine-threonine linker, an alanine-serine linker, or the like.

[0208] A glycine proline linker is composed of consecutive glycine (G) and proline (P) amino acids, with no preference for the order in which either the glycines or prolines appear. In some embodiments, the glycine proline linker is 2 to 49 amino acids in length.

[0209] A glycine-threonine linker is composed of consecutive glycine (G) and threonine (T) amino acids, with no preference for the order in which either the glycines or threonines appear. In some embodiments, the glycine-threonine linker is 2 to 49 amino acids in length.

[0210] An alanine-serine linker is composed of alanine (A) and serine (S) amino acids consecutively, with no preference for the order in which either alanine or serine appears. In some embodiments, the alanine-serine linker is 2 to 49 amino acids in length.

[0211] A glycine-serine linker is composed of consecutive glycine (G) and serine amino acids, with no preference for the order in which either the glycines or serines appear. In some embodiments, the glycine-serine linker is 2 to 49 amino acids in length.

[0212] Exemplary glycine serine linkers include, but are not limited to, those represented by the following amino acid sequences, or combinations thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof. GSG (SEQ ID NO: 350) GSGG (SEQ ID NO: 330); 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); GGGGSGGGGSGGGGS (SEQ ID NO: 267);

[0213] In some embodiments, the glycine serine linker shares at least 70% identity with a sequence described herein above, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0214] Considering the amino acid sequence of the above glycine serine linker, a person skilled in the art can deduce all possible DNA or RNA sequences that encode the above glycine serine linker. Such DNA or RNA sequences are considered to be incorporated into the present disclosure. In some embodiments, the glycine serine linker is encoded by a glycine serine linker sequence, which may be either DNA, RNA or mRNA.

[0215] In some embodiments, the linker peptide is a foldon. A foldon is composed of the amino acid sequence encoded by a foldon sequence. A foldon sequence includes a codon-optimized sequence, a fragment, a mutant, a variant, or a combination thereof. A foldon allows two or more homologous polypeptides to assemble to form an oligomeric complex. In some embodiments, a foldon also assists in the orientation of a polypeptide so that a domain or epitope on a target peptide is exposed or presented for interaction or communication with a cell, a biomolecule, or the immune system.

[0216] Exemplary foldons include, but are not limited to, those represented by the following amino acid sequences, or combinations thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof: YIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 268); HENEISHHAKEIERLQKEIERHKQSIKKLKQSE (SEQ ID NO: 269);

[0217] In some embodiments, the foldon shares at least 70% identity with a sequence described herein above, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0218] Considering the amino acid sequence of above-mentioned foldon, those skilled in the art can deduce all possible DNA or RNA sequences that code above-mentioned foldon.This DNA or RNA sequence is considered to be incorporated into the present disclosure.In some embodiments, foldon is coded by foldon sequence, which can be either DNA, RNA or mRNA.

[0219] In some embodiments, the linker peptide is a scaffold. The scaffold is composed of an amino acid sequence encoded by the scaffold sequence. The scaffold sequence includes a codon-optimized sequence, a fragment, a mutant, a variant, or a combination thereof. The scaffold provides structural and / or functional integrity or support to the target peptide, and may also assist in orienting the target peptide so that the domain or epitope of the target peptide is exposed or presented for interaction or communication with cells, biomolecules, or the immune system.

[0220] Exemplary scaffolds include, but are not limited to, those represented by the following amino acid sequences, or combinations thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof: VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 270); QDSTSDLIPAPPLSKVPLQQNFQDNQFHGKWYVVGKAGNHDLREDKDPRKMQATIYELKEDKSYNVTNVRFVHKKCNYRIWTFVPGSQPGEFTLGNIKSWPGLTSWLVRVVSTNYNQHAMVFFKRVYQNRELFEITLYGRTKELTNELKENFIRFSKSLGLPENHIVFPVPIDQCIDGSAWSHPQFEK (SEQ ID NO: 271); VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 272); GCPRILMRCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 273); MRGSHHHHHHGSDLGKKLLEAARAGQDDEVRILMANGADVNATDNDGYTPLHLAASNGHLEIVEVLLKNGADVNASDLTGITPLHLAAATGHLEIVEVLLKHGADVNAYDNDGHTPLHLAAKYGHLEIVEVLLKHGADVNAQDKFGKTAFDISIDNGNEDLAEILQ (SEQ ID NO: 274); MRGSHHHHHHGSVKVKFFWNGEEKEVDTSKIVWVKRAGKSVLFIYDDNGKNGYGDVTEKDAPKELLDMLARAEREKKL (SEQ ID NO: 275); MLPAPKNLVVSEVTEDSARLSWDDPAAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGHHHHHH (SEQ ID NO: 276); ETDICKLPKDEGTCRDFILKWYYDPNTKSCARFWYGGCGGNENKFGSQKECEKVCAPV (SEQ ID NO: 277); MIPGGLSEAKPATPEIQEIVDKVKPQLEEKTNETYGKLEAVQYKTQVVAGTNYYIKVRAGDNKYMHLKVFKSLPGQNEDLVLTGYQVDKNKDDELTGF (SEQ ID NO: 278); PCSAFEFHCLSGECIHSSWRCDGGPDCKDKSDEENCA (SEQ ID NO: 279); MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (SEQ ID NO: 280); MGSIIFLEDRAFQGRIYGCTTDCPNLQPYFSRCNSIVVQSGCWMIYERPNYQGHQYFLRRGEYPDYQQWMGLSDSIRSCCLIPPHSGAYRMKIYDRDELRGQMSELTDDCLSVQDRFHLTEIHSLNVLEGSWILYEMPNYRGRQYLLRPGEYRRFLDWGAPNAKVGSLRRVMDLYLEHHHHHH (SEQ ID NO: 281); AGHRIAWLLMMGHPRQQLAIIFGIGVSTLYRYFPA (SEQ ID NO: 282); AFSKSEEARHSSLERECIEEICDHAEAWDIMM(SEQ ID NO:283); RECDYCGTDIEPGTGGMAVHGDGATTHFCSHRCAWDAMMGAEARNLEWTDTAR (SEQ ID NO: 284); CSQNEYFDSLLHACIPCQLRCSGAPHRCAWDCMM (SEQ ID NO: 285); EHIPGTLAARLSHRAAWDLMMHSLDASQGTATGPRGIFTAEDALKLVQLKQTGK; TFPTYKCGHRFAWDCMMGSGLNGAACFAVKIADLPVYSCECAIGFMGQRCEYKE (SEQ ID NO: 286); ACYGHRCAWDCMMLGFSSGKCINSKCKCYK (SEQ ID NO: 287); GEYVVEKVLDKRVVKGKVEYLLKWKGFSDEDNTWEPDENLDGHRLAWDFMM; ADVYEVEAILADRVNKNGINEYYIKWAGYDWYDNTWEPEQNLFGAGHRLAWWMMR (SEQ ID NO: 288); AGTIKITQTRSAIGRLPAHKATLLGLGLRRIGHTVEREDGHRIAWDIMMVSFMVKVEG (SEQ ID NO: 289); GIPCGESCGSPCISSAIGCSCKLINTNGSWHIVCYRN (SEQ ID NO: 290); GKCPETFDAWYCLNDAHCFAVLINTNGSWHIVYSCECAIGFMGQRCEYKE (SEQ ID NO: 291); QEEADRTVFVGNLEARVREEILYELFLQAGPLTKVTICKDREGKPKSFGFVCFKHPESVSYAIALAGLINLNGSWIIVSGPSSG (SEQ ID NO: 292); NEEDAGKMFVGGLSWDTSKKDLKDYFTKFGEVVDCTIKMDPNTGRSRGFGFILFKDAASVEKVLDAGLHNLNGSWIIPKKA (SEQ ID NO: 293); SGNIFIKNLDKSIDNKALYDTFSAFGNILSCKVVCDEQGSKGYGFVHFETQEAAERAIAKMGLMNLNGSWVIVGRFKSRKE (SEQ ID NO: 294); PSRVVYLGSIPYDQTEEQILDLCSNVGPVINLKMMFDPQTGRSKGYAFIEFRDLESSASAVGALGLYNLNGSWLICGYSSNSDISGVSLEHHHH (SEQ ID NO: 295); LAILVFGYPETMANQVIAYFQEFGTILEDFEVLRKPQAMTVGLQDRQFVPIFSGNSWTKITYDNPASAVDALAEGLANFNGSWLLVIPYTKDAVERLQ (SEQ ID NO: 296); RLVNCNGSWLIGLDRPPYPGAKGEDIYNNVSRKAWDEWQKHQTMLINERRLNMMNAEDRKFLQQEMDKFLSGEDY (SEQ ID NO: 297); FAVESIEKLRNRNGSWEILVKWRGWSPKYNTWEPEENIG (SEQ ID NO: 298); MRDFFVITNSLYNFNGSWYIKGAVLHVSPTQKRAFWVIADQENFIKQVNKNIEYVEKQASPAFLQRIVEIYQVKFEGKNVG (SEQ ID NO: 299);

[0221] In some embodiments, the scaffold peptide shares at least 70% identity with a sequence described herein above, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0222] Considering the amino acid sequence of the above scaffold peptide, a person skilled in the art can deduce all possible DNA or RNA sequences that encode the above scaffold peptide. Such DNA or RNA sequences are considered to be incorporated into the present disclosure. In some embodiments, the target peptide is encoded by a target sequence, which may be DNA, RNA, or mRNA.

[0223] In some embodiments, the linker peptide consists of a glycine serine linker followed by a foldon. In other embodiments, 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.

[0224] Cleavage Sequences and Cleavage Peptides The multi-target nucleic acid sequence and the multi-target peptide comprise a cleavage sequence and a cleavage peptide, respectively. The cleavage sequence is composed of a sequence of nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes the cleavage peptide. The cleavage sequence may comprise a codon-optimized sequence, a fragment, a mutant, a variant, or a combination thereof. In some embodiments, the cleavage sequence is DNA, RNA, or mRNA.

[0225] A cleavage peptide connects one polypeptide to another polypeptide, e.g., an adjacent polypeptide. The cleavage peptide carries one or more cleavage sites. In some embodiments, the cleavage peptide comprises one or more cleavage sequences, e.g., cleavage peptide-1, cleavage peptide-2, ..., etc. In some embodiments, the cleavage peptide optionally comprises a linker peptide between two cleavage peptides.

[0226] In some embodiments, the cleavage peptide facilitates the cleavage of the multi-target polypeptide into individual polypeptides by cellular proteases, or the self-cleavage of the multi-target polypeptide into individual polypeptides. In some embodiments, the resulting polypeptide may 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. In some embodiments, the polypeptide may also comprise some residues (amino acids) of the cleavage peptide in addition to these peptides. Any cleavage peptide that is susceptible to the action of cellular proteases or that has the ability to undergo self-cleavage can be employed in accordance with the present disclosure.

[0227] In some embodiments, the cleavage peptide is a Golgi-specific cleavage peptide, ie, is susceptible to the action of a Golgi-specific protease.

[0228] Exemplary truncation peptides include, but are not limited to, those represented by the following amino acid sequences, or combinations thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof: RRKRSVS (SEQ ID NO: 300); GIRRKRSVSH (SEQ ID NO: 301); VQREKRAVGI (SEQ ID NO: 302); 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); THRTRRSTSD (SEQ ID NO: 308); SRRKRRSAST (SEQ ID NO: 309); 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)

[0229] In some embodiments, the truncated peptides share at least 70% identity with the sequences described herein above, or combinations thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof.

[0230] Considering the amino acid sequences of the above cleavage peptides, those skilled in the art can deduce all possible DNA or RNA sequences that encode the above cleavage peptides. Such DNA or RNA sequences are considered to be incorporated into the present disclosure. In some embodiments, the cleavage peptides are encoded by cleavage sequences, which may be DNA, RNA, or mRNA.

[0231] Signal Sequences and Signal Peptides The multi-target nucleic acid sequence and the multi-target peptide each include a signal sequence and a signal peptide. The signal sequence is composed of a sequence of nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes the signal peptide. The signal sequence may be a codon-optimized sequence, a fragment, a mutant, a variant, or a combination thereof. In some embodiments, the signal sequence is DNA, RNA, or mRNA.

[0232] The signal peptide is present upstream (N-terminal or amino-terminal side) of one or more polypeptides. In some embodiments, the signal peptide may be present at the N-terminal side of all or some polypeptides. In some embodiments, the signal peptide is present at the N-terminal side of the first polypeptide. In some embodiments, the signal peptide is present upstream (N-terminal side) of some polypeptides. In some embodiments, the signal peptide is present upstream (N-terminal side) of each of the polypeptides.

[0233] In some embodiments, the signal peptide transports the multi-target peptide to an organelle. In some embodiments, the signal peptide transports the multi-target peptide to the Golgi apparatus or Golgi apparatus. Any signal peptide that transports the multi-target peptide to the Golgi apparatus 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 multi-target peptide to the Golgi complex.

[0234] Exemplary signal peptides include, but are not limited to, those represented by the following amino acid sequences, or combinations thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof: MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAA (SEQ ID NO: 312); MDMRAPAGIFGFLLVLFPGYRS (SEQ ID NO: 313); 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); 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); 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); MGVKVLFALICIAVAEA (SEQ ID NO: 329);

[0235] In some embodiments, the signal peptide shares at least 70% identity with a sequence described herein above, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0236] Taking into consideration the amino acid sequences of the above signal peptides, one skilled in the art could deduce all possible DNA or RNA sequences encoding the above signal peptides. Such DNA or RNA sequences are considered to be incorporated into the present disclosure. In some embodiments, the signal peptide is encoded by a signal sequence, which may be either DNA, RNA, or mRNA.

[0237] Synthesis of multi-target nucleic acid sequences The multi-target nucleic acid sequence according to the present disclosure can be either DNA or RNA or mRNA.The multi-target nucleic acid sequence as described herein can be synthesized by molecular biology or genetic engineering techniques well known in the art, for example, by using recombinant expression systems, chemical synthesis, or in vitro transcription (IVT).

[0238] In some embodiments, the multiple target nucleic acid sequences are obtained through a single IVT process. In some embodiments, the multiple target nucleic acid sequences obtained through a single IVT process are mRNA.

[0239] In some embodiments, the multi-target nucleic acid sequence is messenger RNA (mRNA), which encodes a multi-target peptide as described herein.

[0240] Typically, mRNA includes at least a coding region (which encodes a multi-target peptide), a 5'UTR, a 3'UTR, a 5'cap, and a 3'poly(A) tail. The UTR (untranslated region) flanks the coding region or open reading frame (ORF). The 5'UTR and 3'UTR are sections of mRNA before the start codon and after the stop codon, respectively. The 5'UTR has a cap (5'cap) made of modified nucleotides. The mRNA also contains a polyadenylation region at its 3' end with adenine nucleotides, called a poly(A) tail.

[0241] In some embodiments, mRNA can be unmodified, modified, or a combination of both. Modification can be in the nucleobase of nucleotide, or in the sugar moiety of nucleotide, or in the phosphate of nucleotide. In some embodiments, unmodified mRNA can include naturally occurring nucleosides, such as adenosine, guanosine, cytidine, and uridine. mRNA can include one or more modified nucleosides, such as adenosine analogs, guanosine analogs, cytidine analogs, or uridine analogs.

[0242] In some embodiments, the one or more modified nucleosides are nucleoside analogs selected from 2-aminoadenosine, 3-methyladenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, or 8-oxoguanosine, or combinations thereof.

[0243] In some embodiments, the one or more modified nucleosides are uridine analogs selected from propynyl-uridine, pseudouridine, C5-bromouridine, C5-fluorouridine, C5-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-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-pseudouridine, 1-methyl-1 deaza-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 combinations thereof.

[0244] In some embodiments, the one or more modified nucleosides are cytidine analogs 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-1-1 deaza-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine or a combination thereof.

[0245] Methods for making modified nucleosides are well known in the art (WO 2020168466, US8278036; US8691966; US8748089; US8835108; US9750824; US10232055; WO2007024708; WO2012135805; WO2013052523; WO2011012316).

[0246] In some embodiments, the modified nucleoside is a pseudouridine, e.g., 1-methyl-pseudouridine, 1-propynyl-pseudouridine, 1-carboxymethyl-pseudouridine, 1-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.

[0247] In some embodiments, mRNA is produced using a recombinant expression system, chemically synthesized, or obtained through in vitro transcription. In some embodiments, multiple target nucleic acid sequences are obtained through a single IVT process.

[0248] The mRNA according to the present disclosure may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically carried out using a DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system (which 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 depending on the specific application. Methods for producing mRNA via IVT reactions are well known in the art (see, e.g., Beckert, Bertrand and Masquida, Benoit Methods 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 and Biochemistry (2018) 48:1915-1927; WO2018157153; WO2020185811; WO2022082001).

[0249] In some embodiments, in vitro transcription occurs in a single batch. In some embodiments, the IVT reaction includes capping and tailing reactions, either simultaneously with transcription or separately. A cap analog is added to the in vitro transcription reaction and incorporated into the 5' end of the mRNA during the reaction. An alternative method of capping involves adding a cap through an enzymatic reaction after transcription. A poly(A) tail can be incorporated into the DNA template sequence, and thus the poly(A) tail will be incorporated into the mRNA during in vitro transcription by T7 RNA polymerase. An alternative method of tailing involves adding a poly(A) tail through an enzymatic reaction after transcription. In some embodiments, the capping and tailing reactions are performed simultaneously with transcription, i.e., during the IVT reaction. In some embodiments, the capping and tailing reactions are performed separately from the IVT reaction, i.e., after transcription.

[0250] The mRNA produced as a result of the IVT reaction may be purified using techniques well known in the art, such as centrifugation, filtration, and / or chromatography. Purification of mRNA may be achieved before the capping and tailing step is performed or after the capping and tailing step. The synthesized mRNA may be purified by ethanol precipitation, filtration, or chromatography. In some embodiments, tangential flow filtration is used to purify the mRNA. In some embodiments, the mRNA is purified by a chromatography step. In other embodiments, the mRNA is purified by a combination of filtration and chromatography steps.

[0251] In some embodiments, suitable mRNA sequences are mRNA sequences that encode proteins, peptides, or polypeptides.In some embodiments, suitable mRNA sequences are codon-optimized for efficient expression in host cells or organisms.Codon optimization typically involves modifying naturally occurring or wild-type nucleic acid sequences that encode peptides, polypeptides, or proteins to achieve the highest possible expression of peptides, polypeptides, proteins, or antibodies without changing amino acid sequences.

[0252] 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. A multi-target nucleic acid sequence as described herein expresses a multi-target peptide.

[0253] Polypeptide Nanoparticles A multi-target peptide comprises multiple repeats of a polypeptide comprising a target peptide, a linker peptide, and a self-assembling peptide, or a combination of a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide, with a cleavage peptide interposed therebetween (see legend in the figures). In some embodiments, the total number of polypeptides present in a multi-target peptide can be up to 100 polypeptides. In some embodiments, one or more polypeptides in a multi-target peptide can have the same target peptide (homologous polypeptides). In some embodiments, one or more polypeptides in a multi-target peptide can have different target peptides (heterologous polypeptides).

[0254] The multi-target peptides as described herein are encoded by the multi-target nucleic acid sequences as described herein. Each multi-target peptide comprises two or more polypeptides, wherein some or all of the polypeptides 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. The polypeptides are connected to each other through a cleavage peptide. The multi-target peptide comprises a signal peptide upstream (N-terminal side) of one or more polypeptides. In some embodiments, the multi-target peptide comprises a signal peptide upstream (N-terminal side) of each of all or some of the polypeptides. In some embodiments, the multi-target peptide optionally comprises a signal peptide upstream (N-terminal side) of each polypeptide. In some embodiments, the multi-target peptide comprises a signal peptide upstream (N-terminal side) of some of the polypeptides. In some embodiments, the multi-target peptide comprises a signal peptide upstream (N-terminal side) of all of the polypeptides.

[0255] The signal peptide transports the multi-target peptide to the Golgi apparatus. Cellular proteases act on the cleavage site present in the cleavage peptide, or the cleavage peptide undergoes self-cleavage to cleave the multi-target peptide into individual polypeptides (including any one or combination of the target peptide, linker peptide, and self-assembling peptide, or linker peptide, target peptide, linker peptide, and self-assembling peptide, or signal peptide, target peptide, linker peptide, target peptide, linker peptide, and self-assembling peptide). The polypeptide may additionally also have some residues (amino acids) of the cleavage peptide.

[0256] In some embodiments, the linker peptide may be an amino acid linker, a foldon, a scaffold, or a combination thereof. In some embodiments, the polypeptide may be a homologous polypeptide. In some embodiments, two or more homologous polypeptides may assemble to form an oligomeric complex, hi 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, etc.

[0257] In some embodiments, the polypeptide may be a heterologous polypeptide. Polypeptide nanoparticles are formed by the self-assembly of two or more homologous polypeptides, or two or more heterologous polypeptides, or one or more oligomeric complexes, or combinations thereof.

[0258] In some embodiments, the polypeptide nanoparticles comprise homologous polypeptides, heterologous polypeptides, oligomeric complexes, or combinations 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-shaped, or a combination thereof.

[0259] In some embodiments, the polypeptide nanoparticles may be enveloped or non-enveloped, or a combination thereof. In some embodiments, the polypeptide nanoparticles may be unilamellar or multilamellar, or a combination thereof. In some embodiments, the polypeptide nanoparticles may comprise at least 2 or up to 100 polypeptides.

[0260] In some embodiments, polypeptide nanoparticles may comprise 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-99 polypeptides. In one embodiment, the polypeptide nanoparticles may comprise at least two or up to 100 homologous polypeptides. In some embodiments, the polypeptide nanoparticles may comprise at least two or up to 100 heterologous polypeptides.

[0261] In some embodiments, a polypeptide nanoparticle may comprise two or more oligomeric complexes, such that the total number of polypeptides in the polypeptide nanoparticle is 100 or less. In some embodiments, a polypeptide nanoparticle may comprise some homologous polypeptides and some heterologous polypeptides, such that the total number of polypeptides in the polypeptide nanoparticle is 100 or less.

[0262] In some embodiments, a polypeptide nanoparticle may comprise several homologous polypeptides and several oligomeric complexes, such that the total number of polypeptides in the polypeptide nanoparticle is 100 or less.

[0263] In some embodiments, a polypeptide nanoparticle may comprise several heterologous polypeptides and several oligomeric complexes, such that the total number of polypeptides in the polypeptide nanoparticle is 100 or less. In some embodiments, a polypeptide nanoparticle may comprise several homologous polypeptides, several heterologous polypeptides, several oligomeric complexes, or a combination thereof, such that the total number of polypeptides in the polypeptide nanoparticle is 100 or less.

[0264] Lipid Nanoparticle (LNP) Composition Multi-target nucleic acid sequences as described herein may be encapsulated in lipid nanoparticle compositions. In some embodiments, the lipid nanoparticle composition comprises a lipid component, an ionizable polymer, or a combination thereof, and a multi-target nucleic acid sequence as described herein.

[0265] In some embodiments, the lipid nanoparticle composition comprises lipid components such as cationic lipids, phospholipids, sterols, PEG-lipids, and multi-targeting nucleic acid sequences as described herein. In another embodiment, the lipid nanoparticle composition comprises an ionizable polymer, a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and a multi-targeting nucleic acid sequence as described herein.

[0266] Lipid components The lipid component of the lipid nanoparticle composition may include one or more lipids, such as cationic lipids, phospholipids, sterols, and PEG-lipids.

[0267] cationic lipids Cationic lipids refer to lipids that have a net positive charge at a selected pH. Cationic lipids generally consist of a hydrophilic head group and a hydrophobic tail that carry the charge.

[0268] Exemplary cationic lipids for use in 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-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)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-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), N-(1,2-dimyristyl (oxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienooxy)propane (Clin-DMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienooxy)propane Dienoxy)propane (CpLin-DMA), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLin-DAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarb-DAP), 1,2-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-(dimethyl amino)butanoate (DLin-MC3-DMA), heptadecan-9-yl-8-[2-hydroxyethyl-(6-oxo-6-undecaoxyhexyl)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 combinations thereof;

[0269] Cationic lipids with amine head groups are preferred cationic lipids. The amine group can be in the primary, secondary, or tertiary position. The cationic lipid may contain one (monoamine) or more (polyamine) such amine groups.

[0270] In some embodiments, the cationic lipid is positively charged at acidic pH, i.e., pH 1.0 to pH 6.9. In some embodiments, the cationic lipid is neutral at a specific pH, i.e., near physiological pH (pH 7.0 to pH 7.5). Cationic lipids that can exist in either a positively charged or neutral form depending on the pH are referred to as ionizable lipids. Preferred cationic lipids are ionizable so that they can exist in either a positively charged or neutral form depending on the pH. For example, ionizable lipids may be neutral near physiological pH (pH 7.0 to pH 7.5) and cationic near acidic pH (pH 1.0 to pH 6.9).

[0271] In some embodiments, the cationic lipid present in the lipid nanoparticle composition is an ionizable lipid.

[0272] Methods for making cationic and / or ionizable lipids or imparting the ability to behave as ionizable lipids to cationic lipids are well known in the art (WO2005121348; 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).

[0273] The percentage of cationic lipid present in the lipid nanoparticle composition is from about 25 mol % to about 70 mol %, or any range therein.

[0274] In some embodiments, the percentage of cationic lipid present in the lipid nanoparticle composition is about 25 mol% to about 70 mol%, about 25 mol% to about 65 mol%, about 25 mol% to about 60 mol%, about 25 mol% to about 55 mol%, about 25 mol% to about 50 mol%, about 25 mol% to about 48 mol%, about 25 mol% to about 46 mol%, about 25 mol% to about 45 mol%, about 25 mol% to about 44 mol%, about 25 mol% to about 43 mol%, about 25 mol% to about 42 mol%, about 25 mol% to about 41 mol%, about 25 mol% to about 40 mol%, or any range therein.

[0275] In some embodiments, the percentage of cationic lipid present in the lipid nanoparticle composition 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 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%, about 66 mol%, about 67 mol%, about 68 mol%, about 69 mol%, about 70 mol%, or any portion or fraction thereof.

[0276] phospholipids Phospholipids include lipids that contain a hydrophilic head bearing a phosphate group and a hydrophobic tail composed of fatty acid chains attached to a glycerol or sphingosine backbone.

[0277] Exemplary phospholipids for use in lipid nanoparticle compositions include, but are not limited to, the following: 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-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-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-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-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC), sphingomyelin, or a combination thereof.

[0278] The percentage of phospholipids present in the lipid nanoparticle composition is from about 2 mol % to about 30 mol %, or any range therein.

[0279] In some embodiments, the percentage of phospholipids present in the lipid nanoparticle composition is between about 2 mol% and about 30 mol%, between about 2 mol% and about 28 mol%, between about 2 mol% and about 26 mol%, between about 2 mol% and about 24 mol%, between about 2 mol% and about 22 mol%, between about 2 mol% and about 20 mol%, between about 3 mol% and about 19 mol%, between about 3 mol% and about 18 mol%, between about 3 mol% and about 17 mol%, between about 3 mol% and about 16 mol%, between about 3 mol% and about 15 mol%, between about 3 mol% and about 14 mol%, between about 3 mol% and about 13 mol%, between about 3 mol% and about 12 mol%, or any range therein.

[0280] In some embodiments, the percentage of phospholipids present in the lipid nanoparticle composition 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 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 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, or any portion or fraction thereof.

[0281] sterols The lipid nanoparticle composition disclosed herein may include sterol and / or sterol derivative.The term "sterol" as used herein includes, but is not limited to, cholesterol, sitosterol, fecosterol, ergosterol, campesterol, stigmasterol, or derivatives thereof.In some embodiments, the lipid nanoparticle composition includes cholesterol and / or cholesterol derivative.Non-limiting examples of cholesterol and cholesterol derivatives include 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, 6-ketocholestanol, 5α-cholestan, cholestenone, 5α-cholestanone, 5β-cholestanone, cholesteryl decanoate, or mixtures thereof.Methods for producing cholesterol and cholesterol derivatives are well known in the art (WO2009127060; WO2019152557).

[0282] The percentage of sterol present in the lipid nanoparticle composition can be from about 30 mol % to about 65 mol %, or any range therein. In some embodiments, the percentage of sterol present in the lipid nanoparticle composition is between about 30 mol% and about 65 mol%, between about 31 mol% and about 60 mol%, between about 32 mol% and about 60 mol%, between about 33 mol% and about 60 mol%, between about 34 mol% and about 60 mol%, between about 35 mol% and about 60 mol%, or any range therein.

[0283] In some embodiments, the percentage of sterols present in the lipid nanoparticle composition 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%, about 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.

[0284] PEG-lipid The terms PEG-lipid, PEGylated lipid, PEG-linked lipid, PEG-conjugated lipid, PEG-lipid conjugate, and PEG-modified lipid are used interchangeably and refer to polyethylene glycol linked to lipid moiety.Lipid moiety can also be directly linked to PEG molecule through linker.In some embodiments, PEG-lipid comprises PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and / or PEG-modified cholesterol, and / or their mixture.The method of making PEG-lipid is well known to those skilled in the art (see, for example, US20030077829;US2005008689;US5885613;US7404969;WO2005026372;WO2009086558).

[0285] In some embodiments, the PEG-lipid is selected from mPEG-dimyristoylglycerol (mPEG-DMG), mPEG-N,N-ditetradecylacetamide (mPEG-DTA or ALC0159), mPEG-cholesterol (mPEG-CLS), mPEG-DSPE, mPEG-DMPE, mPEG-DPPE, mPEG-DLPE, 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 a mixture thereof.

[0286] The PEG portion of the PEG-lipid may comprise an average molecular weight 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, or 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.

[0287] The percentage of PEG-lipid present in the lipid nanoparticle composition can be from about 0.2 mol % to about 2.0 mol %, or any range therein. In some embodiments, the percentage of PEG-lipid present in the lipid nanoparticle composition 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.

[0288] In some embodiments, the percentage of PEG-lipid present in the lipid nanoparticle composition 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.

[0289] In some embodiments, the lipid nanoparticle composition may additionally contain an ionizable polymer.

[0290] ionizable polymer As used herein, the term "polymer" refers to a compound formed from multiple repeating units called monomers. Polymers are produced through a process called polymerization, in which two or more monomers are linked through chemical bonds to form a polymer. In some embodiments, polymers are branched or unbranched. In some embodiments, polymers may be homopolymers, i.e., composed of the same type of repeating unit or monomer, or heteropolymers, i.e., composed of more than one type of repeating unit or monomer. The terms heteropolymer and copolymer are used interchangeably herein.

[0291] 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; for example, an ionizable polymer will be cationic (positively charged) near acidic pH (pH 1.0 to pH 6.9) and neutral (uncharged) near physiological pH (pH 7.0 to pH 7.5).

[0292] In some embodiments, the ionizable polymer is a biocompatible polymer or a biodegradable polymer. The terms "biocompatible polymer" and "biodegradable polymer" are used interchangeably and refer to a polymer that is substantially free of any adverse effects when introduced into a biological system. Such polymers are capable of undergoing degradation when introduced into a biological system and are not expected to produce significant toxicity or immunological responses.

[0293] In some embodiments, the lipid nanoparticle composition comprises an ionizable polymer, which may be selected from chitosan, chitosan derivatives, cellulose derivatives, poly-L-lysine (PLL), poly-L-glutamic acid, protamine, polyethyleneimine, derivatives thereof, or combinations thereof.

[0294] In some embodiments, the ionizable polymer is positively charged at acidic pH, ie, pH 1.0-6.9, and neutral at about physiological pH (pH 7.0-7.5). The percentage of ionizable polymer present in the lipid nanoparticle composition may be from about 1 mol % to about 25 mol %.

[0295] In some embodiments, the percentage of ionizable polymer present in the lipid nanoparticle composition 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 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 range therein.

[0296] In some embodiments, the percentage of ionizable polymer present in the lipid nanoparticle composition is between about 1 mol% and about 25 mol%, between about 1 mol% and about 24 mol%, between about 1 mol% and about 23 mol%, between about 1 mol% and about 22 mol%, between about 1 mol% and about 21 mol%, between about 1 mol% and about 20 mol%, between about 1 mol% and about 19 mol%, between about 1 mol% and about 18 mol%, between about 1 mol% and about 17 mol%, between about 1 mol% and about 16 mol%, between about 1 mol% and about 15 mol%, or any range therein.

[0297] In some embodiments, the percentage of ionizable polymer present in the lipid nanoparticle composition is about 1 mol%, 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 mol%, 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.

[0298] In some embodiments, preferred ionizable polymers include chitosan, chitosan derivatives, cellulose derivatives, or combinations thereof.

[0299] Chitosan Chitosan is a neutral polymer composed of glucosamine units. Chemically, chitosan is poly-β-(1-4)-2-amino-2-deoxy-D-glucose. Chitosan is prepared by partial deacetylation of chitin, typically by alkaline hydrolysis. Thus, chitosan may contain not only acetylated units (N-acetyl-D-glucosamine) but also deacetylated units (β-(1->4)-linked D-glucosamine). Typically, chitosan molecules have a degree of deacetylation greater than 60% compared to chitin. The molecular weight of chitosan typically varies between 10 kDa and 1000 kDa. Chitosan nanoparticles have been used for drug delivery, including delivery of nucleic acids. However, chitosan nanoparticles alone are insufficient for effective delivery of nucleic acids (Ragelle, Heloise et al. Journal of Controlled Release (2013) 172: 207-218).

[0300] In some embodiments, the lipid nanoparticle composition comprises an ionizable polymer, such as chitosan, along with the lipid components and nucleic acid. In some embodiments, the ionizable polymer is chitosan or a derivative thereof, hi some embodiments, the ionizable polymer includes a chitosan derivative or a dialdehyde chitosan derivative or a combination thereof.

[0301] The chitosan or chitosan derivative employed in the present disclosure may have a molecular weight of about 25 kDa to about 400 kDa. In some embodiments, the molecular weight of chitosan or its derivative is about 25 kDa to about 375 kDa, about 30 kDa to about 350 kDa, about 35 kDa to about 325 kDa, about 40 kDa to about 300 kDa, about 40 kDa to about 250 kDa, about 40 kDa to about 225 kDa, about 40 kDa to about 220 kDa, about 40 kDa to about 210 kDa, or about 40 kDa to about 200 kDa, or any range therein.

[0302] The proportion of chitosan or its derivatives or combinations thereof present in the lipid nanoparticle composition may be from about 1 mol % to about 25 mol %. In some embodiments, the percentage of chitosan or a derivative thereof or a combination thereof present in the lipid nanoparticle composition 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 16 mol%, from about 1 mol% to about 15 mol%, or any range therein.

[0303] In some embodiments, the proportion of chitosan or its derivatives or combinations thereof present in the lipid nanoparticle composition is from about 1 mol% to about 25 mol%, preferably from about 1 mol% to 20 mol%, and most preferably from about 1 mol% to about 15 mol%.

[0304] In some embodiments, the percentage of chitosan or its derivatives or combinations thereof present in the lipid nanoparticle composition 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%, 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.

[0305] cellulose Cellulose is a polymer composed of a linear chain of d-glucose units linked via β-1,4 glycosidic bonds. It typically contains hundreds to thousands of repeating glucose units. Native cellulose is not ideal for mRNA delivery, and therefore requires modification according to the present disclosure. In some 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 small molecule drug delivery (Amalin Kavitha, K. Thomas Paul, Parambath Anilkumar, Chapter 18 - Cellulose-derived materials for drug delivery applications (Faruq Mohammad, Hamad A. Al-Lohedan, Mohammad Jawaid, eds., Micro and Nano Technologies, Sustainable Nanocellulose and Nanohydrogels from Natural Sources, Elsevier, 2020, pp. 367-390), ISBN 9780128167892).

[0306] In some embodiments, the lipid nanoparticle composition comprises a cellulose derivative, which may be a dialdehyde cellulose derivative. The proportion of the cellulose derivative or dialdehyde cellulose derivative or combination thereof present in the lipid nanoparticle composition may be from about 1 mol % to about 25 mol %.

[0307] In some embodiments, the percentage of cellulose derivative or dialdehyde cellulose derivative or combination thereof present in the lipid nanoparticle composition 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 16 mol%, from about 1 mol% to about 15 mol%, or any range therein.

[0308] In some embodiments, the percentage of cellulose derivative or dialdehyde cellulose derivative or combination thereof present in the lipid nanoparticle composition is from about 1 mol% to about 25 mol%, preferably from about 1 mol% to about 20 mol%, most preferably from about 1 mol% to about 15 mol%, or any range therein.

[0309] In some embodiments, the percentage of cellulose or its derivatives or combinations thereof present in the lipid nanoparticle composition 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%, 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.

[0310] Treatment method In some aspects, provided herein are methods of treating or preventing a disease, the methods comprising administering a multi-target nucleic acid sequence as described herein to a subject in need thereof. In some aspects, provided herein are methods of treating or preventing a disease, the methods comprising administering to a subject in need thereof a lipid nanoparticle composition comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and a multi-targeting nucleic acid sequence as described herein.

[0311] In some aspects, provided herein are methods of treating or preventing disease, the methods comprising administering to a subject in need thereof a lipid nanoparticle composition comprising an ionizable polymer, a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and a multi-targeting nucleic acid sequence as described herein.

[0312] In some aspects, the present disclosure relates to the use of a lipid nanoparticle composition comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and a multi-targeting nucleic acid sequence as described herein in the manufacture of a medicament for the treatment or prevention of a disease in a subject.

[0313] In some embodiments, the multi-target nucleic acid sequence is present in a biologically effective or therapeutically effective amount. In some embodiments, the biologically effective amount of the multi-target nucleic acid sequence is between 0.1 μg and 2000 μg, between 0.1 μg and 1800 μg, between 0.1 μg and 1600 μg, between 0.1 μg and 1600 μg, between 0.1 μg and 1400 μg, between 0.1 μg and 1200 μg, between 0.1 μg and 1000 μg, between 0.1 μg and 950 μg, between 0.1 μg and 900 μg, between 0.1 μg and 10 ... 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 the multi-target nucleic acid sequence is about 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, or any range therein.

[0314] In some embodiments, a biologically effective amount of a multi-target 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, 40 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, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg, 300 μg, 310 μg, 320 μg, 330 μg, 340 μg, 350 μg, 360 μg, 370 μg, 380 μg, 390 μg, 400 μg, 410 μg, 420 μg, 430 μg, 440 μg, 450 μg, 460 μg, 470 μg, 480 μg, 490 μg, 510 μg, 52 30 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, 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 part or fraction thereof.

[0315] In one aspect, provided herein is a nucleic acid comprising a plurality of polynucleotide sequences, wherein some or all of the plurality of polynucleotide sequences 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, wherein each of the plurality of polynucleotide sequences is connected to adjacent ones of the plurality of polynucleotide sequences by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the plurality of polynucleotide sequences. In another aspect, provided herein is a nucleic acid comprising a plurality of polynucleotide sequences, wherein each of the plurality of polynucleotide sequences 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 of the plurality of polynucleotide sequences is connected to adjacent ones of the plurality of polynucleotide sequences by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the plurality of polynucleotide sequences. In another aspect, provided herein is a nucleic acid comprising a plurality of polynucleotide sequences, wherein each of the plurality of polynucleotide sequences comprises a target sequence, a linker sequence, and a self-assembling sequence, wherein each of the plurality of polynucleotide sequences is connected to adjacent ones of the plurality of polynucleotide sequences by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the plurality of polynucleotide sequences. In yet another aspect, provided herein is a nucleic acid comprising a plurality of polynucleotide sequences, wherein each of the plurality of polynucleotide sequences comprises a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence, wherein each of the plurality of polynucleotide sequences is connected to adjacent ones of the plurality of polynucleotide sequences by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the plurality of polynucleotide sequences.In one aspect, provided herein is a nucleic acid comprising a first plurality of polynucleotide sequences and a second plurality of polynucleotide sequences, each of the first plurality of polynucleotide sequences comprising a first target sequence, a first linker sequence, and a first self-assembling sequence, wherein each of the second plurality of polynucleotide sequences comprises a second linker sequence, a second target sequence, a third linker sequence, and a second self-assembling sequence, wherein each of the first plurality of polynucleotide sequences and each of the second plurality of polynucleotide sequences comprises a cleavage sequence that cleaves the first plurality of polynucleotide sequences. a first polynucleotide sequence in the nucleic acid is a polynucleotide sequence in the first plurality of polynucleotide sequences or a polynucleotide sequence in the second plurality of polynucleotide sequences, and the nucleic acid further comprises a signal sequence upstream of the first polynucleotide sequence in the first plurality of polynucleotide sequences or the second polynucleotide sequence in the second plurality of polynucleotide sequences, or a combination thereof. In another aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein some or all of the plurality of polypeptides comprise a target peptide, a linker peptide, and a self-assembling peptide, or either a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide, or a combination thereof, wherein each of the plurality of polypeptides is connected to an adjacent one of the plurality of polypeptides by a cleavage peptide, and wherein the nucleic acid further comprises a signal peptide amino-terminal to one or more of the plurality of polypeptides.In another aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein some or all of the plurality of polypeptides comprise 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 a combination thereof, wherein each of the plurality of polypeptides is connected to an adjacent one of the plurality of polypeptides by a cleavage peptide, and wherein the nucleic acid further comprises a signal peptide amino-terminal to a first polypeptide of the plurality of polypeptides, a second polypeptide of the plurality of polypeptides, or a combination thereof. In one aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein each of the plurality of polypeptides comprises a target peptide, a linker peptide, and a self-assembling peptide, wherein each of the plurality of polypeptides is connected to an adjacent one of the plurality of polypeptides by a cleavage peptide, and wherein the nucleic acid further encodes a signal peptide amino-terminal to one or more of the plurality of polypeptides. In another aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein each of the plurality of polypeptides comprises a linker peptide, a targeting peptide, a linker peptide, and a self-assembling peptide, wherein each of the plurality of polypeptides is connected to an adjacent one of the plurality of polypeptides by a cleavage peptide, and wherein the nucleic acid further encodes a signal peptide at the amino terminus of one or more of the plurality of polypeptides.In yet another aspect, provided herein is a nucleic acid encoding a first plurality of polypeptides and a second plurality of polypeptides, wherein each of the first plurality of polypeptides comprises a target peptide, a linker peptide, and a self-assembling peptide; wherein each of the second plurality of polypeptides comprises a second linker peptide, a second target peptide, a third linker peptide, and a second self-assembling peptide; wherein each of the first plurality of polypeptides and each of the second plurality of polypeptides is connected to an adjacent polypeptide of the first plurality of polypeptides or an adjacent polypeptide of the second plurality of polypeptides by a cleavage peptide; and wherein the first polypeptide encoded by the nucleic acid is a polypeptide of the first plurality of polypeptides or a polypeptide of the second plurality of polypeptides, and wherein the nucleic acid further encodes a signal peptide amino-terminal to a first polypeptide of the first plurality of polypeptides or a second polypeptide of the second plurality of polypeptides, or a combination thereof.

[0316] Aspects Some of the aspects of the disclosure are described in the following numbered paragraphs: 1. A nucleic acid comprising a plurality of polynucleotide sequences, wherein some or all of the plurality of polynucleotide sequences comprise 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 of the plurality of polynucleotide sequences is connected to an adjacent one of the plurality of polynucleotide sequences by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the plurality of polynucleotide sequences.

[0317] 2. A nucleic acid comprising a plurality of polynucleotide sequences, wherein each of the plurality of polynucleotide sequences comprises a target sequence, a linker sequence, and a self-assembling sequence, wherein each of the plurality of polynucleotide sequences is connected to adjacent ones of the plurality of polynucleotide sequences by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the plurality of polynucleotide sequences.

[0318] 3. A nucleic acid comprising a plurality of polynucleotide sequences, wherein each of the plurality of polynucleotide sequences comprises a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence, wherein each of the plurality of polynucleotide sequences is connected to adjacent ones of the plurality of polynucleotide sequences by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the plurality of polynucleotide sequences.

[0319] 4. A nucleic acid encoding a plurality of polypeptides, wherein some or all of the plurality of polypeptides 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, wherein each of the plurality of polypeptides is connected to an adjacent one of the plurality of polypeptides by a cleavage peptide, and wherein the nucleic acid further comprises a signal peptide amino-terminal to one or more of the plurality of polypeptides.

[0320] 5. A nucleic acid encoding a plurality of polypeptides, wherein each of the plurality of polypeptides comprises a target peptide, a linker peptide, and a self-assembling peptide, wherein each of the plurality of polypeptides is connected to an adjacent one of the plurality of polypeptides by a cleavage peptide, and wherein the nucleic acid sequence further encodes a signal peptide amino-terminal to one or more of the plurality of polypeptides.

[0321] 6. A nucleic acid encoding a plurality of polypeptides, wherein each of the plurality of polypeptides comprises a linker peptide, a targeting peptide, a linker peptide, and a self-assembling peptide, wherein each of the plurality of polypeptides is connected to an adjacent one of the plurality of polypeptides by a cleavage peptide, and wherein the nucleic acid sequence further encodes a signal peptide amino terminal to one or more of the plurality of polypeptides.

[0322] 7. The nucleic acid of any of the preceding paragraphs, wherein the total number of polynucleotide sequences is 100 or less. 8. The nucleic acid of any of the preceding paragraphs, wherein the total number of polynucleotide sequences is between 2 and 10, 10 and 20, 20 and 30, 30 and 40, 40 and 50, 50 and 60, 60 and 70, 70 and 80, 80 and 90, or 90 and 99. 9. The nucleic acid of any of the preceding paragraphs, wherein the nucleic acid is DNA or RNA.

[0323] 10. The nucleic acid of paragraph 9, wherein the RNA is mRNA. 11. The nucleic acid of any of the preceding paragraphs, wherein the linker sequence encodes a linker peptide. 12. The nucleic acid of paragraph 11, wherein the linker peptide is an amino acid linker, a foldon, a scaffold, or a combination thereof.

[0324] 13. The nucleic acid of paragraph 12, wherein the amino acid linker comprises 2 to 49 amino acids. 14. The nucleic acid of paragraph 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 of any of the preceding paragraphs, wherein the linker peptide has the amino acid sequence of any one of SEQ ID NOs: 262-299, 330, and 350.

[0325] 16. The nucleic acid of any of the preceding paragraphs, wherein the self-assembling sequence encodes a self-assembling peptide. 17. The nucleic acid of paragraph 16, wherein the self-assembling peptide is lumazine synthase from Aquifyx 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 a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0326] 18. The nucleic acid of paragraph 17, wherein the ferritin is composed of ferritin subunits or ferritin peptides. 19. The nucleic acid according to paragraph 18, wherein the ferritin peptide is derived from ferritin of Helicobacter pylori. 20. The nucleic acid of any of the preceding paragraphs, wherein the self-assembling peptide has the amino acid sequence of any one of SEQ ID NOs: 254-261, 331 and 333.

[0327] 21. The nucleic acid of any of the preceding paragraphs, wherein the cleavage sequence encodes one or more cleavage sequences. 22. The nucleic acid of paragraph 21, wherein the one or more cleavage sequences are optionally connected to each other by a linker peptide. 23. The nucleic acid of paragraph 22, wherein the cleavage peptide is a Golgi-specific cleavage peptide or a self-cleavage peptide.

[0328] 24. The nucleic acid of any of the preceding paragraphs, wherein the truncated peptide has the amino acid sequence of any one of SEQ ID NOs: 300-311 and 347-349. 25. The nucleic acid of any of the preceding paragraphs, wherein the signal sequence encodes a signal peptide. 26. The nucleic acid of paragraph 25, wherein the signal peptide is present amino-terminally to one or more of the multiple polypeptides.

[0329] 27. The nucleic acid of paragraph 26, wherein the nucleic acid further encodes a second signal peptide amino-terminal to all or some of the plurality of polypeptides. 28. The nucleic acid of any of the preceding paragraphs, wherein the signal peptide has the amino acid sequence of any one of SEQ ID NOs: 312-329. 29. The nucleic acid of any of the preceding paragraphs, wherein the target sequence encodes a target peptide.

[0330] 30. The nucleic acid sequence of paragraph 29, wherein the target peptide is encoded by a codon-optimized nucleic acid sequence, or a fragment, mutant, or variant thereof. 31. The nucleic acid of 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 parasite, a mycoplasma, an animal, a human, or a combination thereof.

[0331] 32. The nucleic acid of paragraph 31, wherein the virus is selected from a family comprising Picornaviridae, Caliciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Arteriviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Bornaviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Retroviridae, Polyomaviridae, Herpesviridae, Poxviridae, Papillomaviridae, Hepadnaviridae, Adenoviridae, Parvoviridae, Hepeviridae, Circoviridae, or a combination thereof.

[0332] 33. The nucleic acid of paragraph 31, wherein the bacterium is selected from a 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.

[0333] 34. The nucleic acid of paragraph 32, wherein the virus is selected from a family comprising Coronaviridae, Herpesviridae, Poxviridae, Flaviviridae, Togaviridae, Retroviridae, Paramyxoviridae, or a combination thereof.

[0334] 35. The nucleic acid according to paragraph 31, wherein the virus is an alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus, torovirus or a combination thereof. 36. The nucleic acid according to paragraph 35, wherein the betacoronavirus is SARS-CoV-1, SARS-CoV-2, MERS-CoV, OC43, HKU1, a bat coronavirus, another betacoronavirus, or a combination thereof.

[0335] 37. The nucleic acid of paragraph 30, wherein the target peptide is a spike protein, a membrane protein, an envelope protein or a nucleocapsid protein of a coronavirus, or a combination thereof. 38. The nucleic acid of paragraph 37, wherein the target peptide is spike protein, or a fragment thereof.

[0336] 39. The nucleic acid of paragraph 38, wherein the target peptide is the receptor binding domain of the spike protein, a fusion peptide, a stem helix, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof. 40. The nucleic acid of paragraph 39, wherein the target peptide is a receptor binding domain obtained or derived from a betacoronavirus, including SARS-CoV-1, SARS-CoV-2, MERS-CoV, OC43, HKU1, bat coronaviruses, other betacoronaviruses, or combinations thereof.

[0337] 41. The nucleic acid of paragraph 30, wherein the target peptide is glycoprotein B, glycoprotein C, glycoprotein D, glycoprotein E, glycoprotein K, glycoprotein L, or glycoprotein M of herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0338] 42. The nucleic acid of paragraph 30, wherein the target peptide is glycoprotein B, glycoprotein H, glycoprotein L, glycoprotein M, or glycoprotein N of human cytomegalovirus (HCMV), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0339] 43. The nucleic acid of paragraph 30, wherein the target peptide is glycoprotein B, glycoprotein C, glycoprotein H, or glycoprotein L of varicella-zoster virus (VZV), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0340] 44. The nucleic acid of paragraph 30, wherein 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 a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0341] 45. The nucleic acid of paragraph 30, wherein the target peptide is a poxvirus F9 membrane protein, a poxvirus H3L protein, a poxvirus A4 protein, a poxvirus A27 protein, a poxvirus A33 protein, a poxvirus A56 protein, a poxvirus B5 protein, or a poxvirus L1 protein, or a codon-optimized nucleic acid sequence thereof, or a combination thereof, including a fragment, mutant, or variant thereof.

[0342] 46. ​​The nucleic acid of paragraph 30, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a Flavivirus or Hepacivirus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0343] 47. The nucleic acid of paragraph 30, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Japanese encephalitis virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0344] 48. The nucleic acid of paragraph 30, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Zika virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0345] 49. The nucleic acid of paragraph 30, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Yellow Fever Virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0346] 50. The nucleic acid of paragraph 30, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of West Nile virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0347] 51. The nucleic acid of paragraph 30, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Hepatitis C virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0348] 52. The nucleic acid of paragraph 30, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a dengue virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0349] 53. The nucleic acid of paragraph 30, wherein the target peptide is a capsid protein or envelope protein such as E1, E2 and E3 proteins of an alphavirus, or a combination thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof. 54. The nucleic acid of paragraph 30, wherein the target peptide is domain A, domain B, or domain C of the E2 protein of an alphavirus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0350] 55. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid protein or an envelope protein such as the E1, E2 and E3 proteins of Chikungunya virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof. 56. The nucleic acid of paragraph 30, wherein the target peptide is a retroviral gag, pol or env protein, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0351] 57. The target peptide is p17 derived from lentivirus. Gag , p24 Gag , p7 Gag , p6 Gag , gp12 Env , gp41 Env 31. The nucleic acid of paragraph 30, which is a pol protein or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof. 58. The target peptide is p17 derived from human immunodeficiency virus (HIV). Gag , p24 Gag , p7 Gag , p6 Gag , gp12 Env , gp41 Env 31. The nucleic acid of paragraph 30, which is a pol protein or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0352] 59. The nucleic acid of paragraph 30, wherein the target peptide is a 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 virus (MuV), parainfluenza virus type 5 (PIV5), human parainfluenza virus types 2, 4a, and 4b (HPIV2 / 4a / 4b), Newcastle disease virus (NDV), human parainfluenza virus types 1 and 3 (HPIV1 / 3), Nipah virus (NiV), measles virus (MeV), human respiratory syncytial virus (HRSV) A2, B1, S2, or human metapneumovirus (HMPV), or a combination thereof, or a combination thereof comprising a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0353] 60. The nucleic acid of paragraph 30, wherein the target peptide is the 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 (HRSV) A2, B1, S2, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0354] 61. The nucleic acid according to paragraph 30, wherein the target peptide is the E1, E2, E4, E5, E6, E7, L1 or L2 protein of a papillomavirus, preferably a human papillomavirus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0355] 62. The nucleic acid of any of the preceding paragraphs, wherein the target peptide has the amino acid sequence of any one of SEQ ID NOs: 1-253, 334-337, 338-346 and 353-388. 63. A lipid nanoparticle composition comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and a nucleic acid according to any of the preceding paragraphs. 64. The lipid nanoparticle composition of paragraph 63, wherein the cationic lipid comprises an ionizable lipid.

[0356] 65. The lipid nanoparticle composition of paragraph 64, wherein the ionizable lipid is present in an amount of 25 mol percent to 70 mol percent. 66. The lipid nanoparticle composition of paragraph 63, wherein the phospholipid is present in an amount of 2 mol percent to about 30 mol percent. 67. The lipid nanoparticle composition of paragraph 63, wherein the sterol is present in an amount of 30 mol percent to about 65 mol percent.

[0357] 68. The lipid nanoparticle composition of paragraph 63, wherein the PEG-lipid is present in an amount of 0.2 mol percent to about 2.0 mol percent. 69. The lipid nanoparticle composition of paragraph 63, additionally comprising an ionizable polymer. 70. The lipid nanoparticle composition of paragraph 69, wherein the ionizable polymer is present in an amount between 1 mol percent and 25 mol percent.

[0358] 71. The lipid nanoparticle composition according to paragraph 70, wherein the ionizable polymer is selected from the group comprising chitosan, cellulose derivatives, poly-L-lysine, poly-L-glutamic acid, and / or derivatives thereof or combinations thereof. 72. A method for treating or preventing a disease, the method comprising administering to a subject in need thereof a nucleic acid according to any one of claims 1 to 62.

[0359] 73. A method for treating or preventing a disease, comprising administering to a subject in need thereof a lipid nanoparticle composition described in any one of paragraphs 63 to 71. 74. Use of a nucleic acid sequence according to any one of paragraphs 1 to 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 described in any one of paragraphs 63 to 71 in the manufacture of a medicament for the treatment or prevention of a disease in a subject.

[0360] 76. A multi-target peptide encoded by a nucleic acid according to any one of paragraphs 1 to 62. 77. A multi-target peptide comprising two or more polypeptides, wherein some or all of the polypeptides 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, wherein one polypeptide is connected to another polypeptide by a cleavage peptide, and wherein the multi-target peptide includes a signal peptide amino-terminal to one or more of the polypeptides.

[0361] 78. The multi-target peptide according to paragraph 77, wherein the linker peptide is an amino acid linker, a foldon, a scaffold or a combination thereof. 79. The multi-target peptide of paragraph 78, wherein the amino acid linker comprises 2 to 49 amino acids. 80. The multi-target peptide according to paragraph 78, wherein the amino acid linker is selected from 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.

[0362] 81. The multi-target peptide of paragraph 77, wherein the self-assembling peptide is selected from the group comprising lumazine synthase from Acwifex 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 codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof.

[0363] 82. The multi-target peptide of paragraph 81, wherein the ferritin comprises a ferritin subunit or a ferritin peptide. 83. The multi-target peptide according to paragraph 82, wherein the ferritin peptide is derived from ferritin of Helicobacter pylori. 84. The multi-target 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.

[0364] 85. The multi-target peptide according to paragraph 77, wherein a signal peptide may be present at the amino terminus of all or some of the polypeptides. 86. The multi-target peptide of 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 parasite, a mycoplasma, an animal, a human, or a combination thereof.

[0365] 87. The multi-target peptide of paragraph 86, wherein the virus is selected from a family comprising Picornaviridae, Caliciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Arteriviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Bornaviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Retroviridae, Polyomaviridae, Herpesviridae, Poxviridae, Papillomaviridae, Hepadnaviridae, Adenoviridae, Parvoviridae, Hepeviridae, Circoviridae, or a combination thereof.

[0366] 88. The multi-target peptide of paragraph 86, wherein the bacteria is selected from a 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.

[0367] 89. The multi-target peptide of paragraph 87, wherein the virus is selected from a family including Coronaviridae, Herpesviridae, Poxviridae, Flaviviridae, Togaviridae, Retroviridae, Paramyxoviridae, or a combination thereof. 90. The multi-target peptide according to paragraph 89, wherein the virus is an alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus, torovirus, or a combination thereof.

[0368] 91. The multi-target peptide according to paragraph 90, wherein the betacoronavirus is selected from the group comprising SARS-CoV-1, SARS-CoV-2, MERS-CoV, OC43, HKU1, bat coronavirus, other betacoronavirus, or a combination thereof. 92. The multi-target peptide according to paragraph 77, wherein the target peptide is a coronavirus spike protein, membrane protein, envelope protein or nucleocapsid protein, or a combination thereof, including a codon-optimized nucleic acid sequence fragment thereof, a mutant or variant thereof.

[0369] 93. The multi-target peptide according to paragraph 92, wherein the target peptide is a spike protein or a fragment thereof. 94. The multi-target peptide according to paragraph 93, wherein the target peptide is the receptor binding domain of the spike protein, a fusion peptide, a stem helix, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0370] 95. The multi-target peptide of paragraph 77, wherein the target peptide is glycoprotein B, glycoprotein C, glycoprotein D, glycoprotein E, glycoprotein K, glycoprotein L, or glycoprotein M of herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0371] 96. The multi-target peptide of paragraph 77, wherein the target peptide is glycoprotein B, glycoprotein H, glycoprotein L, glycoprotein M, or glycoprotein N of human cytomegalovirus (HCMV), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0372] 97. The multi-target peptide according to paragraph 77, wherein the target peptide is glycoprotein B, glycoprotein C, glycoprotein H, or glycoprotein L of varicella-zoster virus (VZV), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0373] 98. The multi-target peptide according to paragraph 77, wherein the target peptide is Epstein-Barr virus (EBV) glycoprotein B, glycoprotein H, glycoprotein L, glycoprotein M, glycoprotein N, glycoprotein 42, glycoprotein 350, or a combination thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof.

[0374] 99. The multi-target peptide of paragraph 77, wherein the target peptide is a poxvirus F9 membrane protein, a poxvirus H3L protein, a poxvirus A4 protein, a poxvirus A27 protein, a poxvirus A33 protein, a poxvirus A56 protein, a poxvirus B5 protein, or a poxvirus L1 protein, or a codon-optimized nucleic acid sequence thereof, or a combination thereof, including a fragment, mutant, or variant thereof.

[0375] 100. The multi-target peptide of paragraph 77, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or non-structural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of the Flavivirus or Hepacivirus genus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0376] 101. The multi-target peptide described in paragraph 77, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or non-structural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Japanese encephalitis virus, or a combination thereof including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0377] 102. The multi-target peptide of paragraph 77, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Zika virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0378] 103. The multi-target peptide according to paragraph 77, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or non-structural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Yellow Fever Virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0379] 104. The multi-target peptide of paragraph 77, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or non-structural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of West Nile virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0380] 105. The multi-target peptide according to paragraph 77, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or non-structural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Hepatitis C virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0381] 106. The multi-target peptide according to paragraph 77, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or non-structural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of the dengue virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0382] 107. The multi-target peptide according to paragraph 77, wherein the target peptide is a capsid protein or envelope protein such as E1, E2 and E3 proteins of an alphavirus, or a combination thereof including codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof. 108. The multi-target peptide of paragraph 77, wherein the target peptide is domain A, domain B, domain C of the E2 protein of an alphavirus, or a combination thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof.

[0383] 109. The multi-target 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 Chikungunya virus, or a combination thereof including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0384] 110. The multi-target peptide according to paragraph 77, wherein the target peptide is a retroviral gag, pol and env protein, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0385] 111. The target peptide is p17 derived from lentivirus. Gag , p24 Gag , p7 Gag , p6 Gag , gp12 Env , agp41 Env 78. The multi-target peptide of paragraph 77, which is a pol protein or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0386] 112. The target peptide is p17 derived from human immunodeficiency virus (HIV). Gag , p24 Gag , p7 Gag , p6 Gag , gp12 Env , gp41 Env 78. The multi-target peptide of paragraph 77, which is a pol protein or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0387] 113. The multi-target peptide of 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, an L protein, an M protein, an H (hemagglutinin) protein, an HN (hemagglutinin-neuraminidase) protein, a G protein, an F protein, or a combination thereof comprising a codon-optimized nucleic acid sequence thereof, a fragment, a mutant or a variant thereof, of mumps virus (MuV), parainfluenza virus type 5 (PIV5), human parainfluenza virus types 2, 4a and 4b (HPIV2 / 4a / 4b), Newcastle disease virus (NDV), human parainfluenza virus types 1 and 3 (HPIV1 / 3), Nipah virus (NiV), measles virus (MeV), human respiratory syncytial virus (HRSV) A2, B1, S2, or human metapneumovirus (HMPV), or a combination thereof.

[0388] 114. The multi-target peptide according to paragraph 77, wherein the target peptide is the 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 (HRSV) A2, B1, S2, or a combination thereof including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

[0389] 115. The multi-target peptide according to paragraph 77, wherein the target peptide is a papillomavirus, preferably a human papillomavirus, E1, E2, E4, E5, E6, E7, L1, or L2 protein, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

[0390] 116. The multi-target peptide according to any one of paragraphs 77 to 115, wherein the total number of polypeptides is 100 or less. 117. The multi-target peptide of paragraph 116, wherein the total number of polypeptides is 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-99.

[0391] 118. A polypeptide nanoparticle comprising at least two or up to 100 polypeptides according to any one of paragraphs 1-62 or 77-115. 119. The polypeptide nanoparticle according to paragraph 118, wherein the polypeptide is a homologous polypeptide, a heterologous polypeptide, an oligomeric complex, or a combination thereof. 120. The polypeptide nanoparticle of paragraph 118, wherein the polypeptide nanoparticle is icosahedral, helical, spherical, rod-shaped, or a combination thereof.

[0392] example Example 1: Synthesis of multi-target nucleic acid sequences (mRNA) Plasmid DNA construction A multi-target nucleic acid sequence containing a signal sequence and three repeats of polynucleotide sequences, each separated by a cleavage sequence, was codon-optimized for human expression. The multi-target 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 sequence (encoding the receptor binding domain (RBD) of SARS-CoV-2), linker sequences (a glycine-serine linker sequence encoding a glycine-serine linker, a foldon sequence encoding a foldon, and a glycine-serine linker sequence encoding a glycine-serine linker), and a self-assembling sequence (a ferritin sequence encoding ferritin). The second polynucleotide sequence consisted of a linker sequence (a scaffold sequence encoding a scaffold and a glycine serine linker sequence encoding a glycine serine linker), a target sequence (encoding a fusion peptide of SARS-CoV-2), a linker sequence (a glycine serine linker sequence encoding a glycine serine linker, a scaffold sequence encoding a scaffold and a glycine serine linker sequence encoding a glycine serine linker), and a self-assembling sequence (a ferritin sequence encoding ferritin). The third polynucleotide sequence consisted of a linker sequence (a scaffold sequence encoding a scaffold and a glycine serine linker sequence encoding a glycine serine linker), a target sequence (encoding the stem helix of SARS-CoV-2), a linker sequence (a glycine serine linker sequence encoding a glycine serine linker, a scaffold sequence encoding a scaffold and a glycine serine linker sequence encoding a glycine serine linker), and a self-assembling sequence (a ferritin sequence encoding ferritin). A signal sequence (Golgi targeting signal sequence) was included upstream of the first polynucleotide sequence. 5'UTR, cap, and 3'UTR sequences were also included in the multi-target nucleic acid sequence. The multi-target nucleic acid sequence construct was inserted between the HindIII and BamHI restriction sites of PTwist Kan High Copy (Twist BioScience, USA).The sequences of the multi-target peptides encoded by the above multi-target nucleic acid sequences are provided in Table 1.

[0393] Table 1 shows the sequences of the multi-target peptides encoded by the above multi-target nucleic acid sequences. [Table 1] The amino acid sequences of the various peptides present in the multi-target peptides encoded by the multi-target nucleic acid sequences of Example 1 are individually identified below:

[0394] The complete sequence of the multi-target peptide of the multi-target nucleic acid of Example 1

number

[0395] Plasmid DNA isolation and linearization Plasmid DNA was obtained using standard techniques. Briefly, plasmid DNA was introduced into E. coli DH5 alpha cells (Stellar™ Competent Cells, Catalog No. 636763, Takara Bio) by heat shock at 42°C for 60–90 seconds, followed by cold shock on ice for 5 minutes. The cells were transferred to LB (Lauria-Bertani) medium in a tube and incubated for 1 hour at 37°C at 600–900 rpm on a thermal shaker. The cells were harvested and centrifuged at 4000 g for 2 minutes, and the pellet was collected and resuspended in the same medium. The resuspended cells were transferred to an agar plate containing LB medium supplemented with kanamycin, evenly spread with sterile glass beads, and incubated for 14–16 hours at 37°C. Well-grown colonies were picked and resuspended in LB medium supplemented with kanamycin. Colony PCR was performed to check for the presence of pDNA. Colonies were further cultured overnight, and pDNA was isolated using a NucleoSpin® Plasmid Miniprep Kit (catalog number 740588, Takara Bio Inc.). pDNA was linearized by digestion with BbsI-HF restriction enzyme (catalog number R3539L, New England Biolabs Inc.). pDNA was quantified using a NanoDrop™ One / OneC Microvolume UV-Vis Spectrophotometer (catalog number ND-ONE-W, Thermo Scientific).

[0396] In vitro transcription (IVT) IVT was performed according to the standard procedure described in the HiScribe™ T7 High Yield RNA Synthesis Kit (Cat. No. E2040S, New England BioLabs Inc.). Briefly, an IVT mix containing four ribonucleotide triphosphates (ATP, UTP, GTP, CTP), NEB T7 buffer, NEB T7 enzyme mix, CleanCap® Reagent AG (3'OMe) (Cat. No. N-7413, TriLink BioTechnologies), and linearized plasmid DNA was placed in a PCR tube containing nuclease-free sterile water. The mixture was incubated at 37°C for approximately 2 hours. This was followed by DNase I (Cat. No. M0303S, New England BioLabs Inc.) treatment to degrade any plasmid DNA. mRNA was purified according to the instructions provided in the Monarch® RNA Cleanup Kit (Cat. No. T2050L, New England BioLabs Inc.). A poly(A) tail was added to the purified mRNA, followed by a standard post-tailing procedure using E. coli poly(A) polymerase (catalog number M0276L, New England BioLabs). The poly(A)-tailed mRNA was purified using the Monarch® RNA Cleanup Kit (catalog number T2050L, New England BioLabs Inc.). RNA was quantified using the Qubit™ RNA BR Assay Kit (catalog number Q10210, Thermo Scientific) on a Qubit 4 fluorometer (Thermo Scientific).

[0397] Transfection HEK293T cells (catalog no. CRL3216, ATCC) were cultured at 0.25–1 × 10 in 6-well plates containing Gibco DMEM medium, high glucose, pyruvate (catalog no. 11995065, ThermoFisher Scientific) supplemented with 10% fetal bovine serum and 100 units / mL penicillin-streptomycin. 6 Cells were seeded and cultured until 70-80% confluency was achieved. A transfection mix containing multi-target nucleic acid (mRNA, 3 μg of Example 1) and Lipofectamine™ 2000 (9 μL) in 100 μL of serum-free medium was incubated at room temperature for 15 minutes, added to the cells, and incubated for 24 and 48 hours. The spent medium was collected by aspiration, and the cells were recovered by vigorous pipetting with ice-cold 1x PBS (1 mL). The cells were centrifuged at 4000 g for 5 minutes at 4°C. Excess PBS was aspirated. The cells were gently suspended in 300 μL of 1x NETN lysis buffer with inhibitor and kept on ice for 20 minutes. The lysate was centrifuged at 16,000 g for 20 minutes at 4°C. The supernatant was aspirated into a microtube.

[0398] Western blot 6x Laemmli buffer was added to each sample (supernatant or cell lysate) to a final concentration of 1x. Samples were heated at 95°C for 5 minutes and centrifuged at 10,000g for 5 minutes. Approximately 21 µL of cell lysate and 42 µL of supernatant, along with molecular weight standards, were added to a gel (NuPAGE™ 4-12% Bis-Tris gel), and the gel was run using mops-SDS running buffer. Proteins were transferred onto a PVDF membrane using the iBlot 2 blotting system. Blots were blocked with 5% skim milk in 1x TBST for 1 hour and incubated with primary antibodies (SARS-CoV-2 (2019-nCoV) spike antibody, rabbit PAb (catalog number 40589-T62, Sino Biological Inc.) or SARS-CoV-2 spike RBD polyclonal antibody (catalog number E-AB-V-1006, Elab Sciences) for 1 hour at room temperature. The membranes were washed three times and incubated with secondary antibodies (anti-rabbit IgG HRP (catalog number 7074S, manufacturer)) for 1 hour. The membranes were washed three times and signal development was performed with 50 μL of luminol and 50 μL of peroxide solution. Images were captured using a ChemiDoc™ imaging system (Bio-Rad). The results are shown in Figures 3a and 3b.

[0399] ELISA ELISA was performed on the cell lysates and supernatants according to the instructions provided in the SARS-CoV-2 (2019-nCoV) Spike Detection ELISA Kit (Cat. No. KIT40591, Sino Biological, Inc.). The results are shown in Figures 4a and 4b.

[0400] Transmission electron microscopy (TEM) After 48 hours of transfection, 1 mL of spent medium was concentrated to 0.5 mL by ultracentrifugation at 3000 g for 3 minutes using a 100 kDa concentrator (Cat. No. UFC210024, Millipore Amicon Ultra-2 mL). The sample was buffer-exchanged 10 times with 1x PBS using a Centricon™ filter. Finally, the sample was further concentrated to 200-250 μL by ultracentrifugation. 10 μL of the sample was placed on a glow-discharged 400-mesh carbon-coated copper grid and incubated for 1 minute. Excess sample was removed by careful blotting with absorbent paper. 10 μL of UranyLess staining reagent (Cat. No. 22409, Electron Microscopy Sciences) was added and incubated for 1 minute. Excess sample was removed as above. The sample was allowed to air dry by incubating at room temperature for 1 hour. TEM images were captured on a Talos™ L120C TEM (ThermoFisher Scientific), and the results are shown in Figure 5.

[0401] Analytical or immunogenicity assays Testing of multi-target nucleic acid sequences involves immunizing animals (typically mice) with the multi-target nucleic acid sequences in an appropriate formulation according to a prime-boost immunization strategy at predetermined doses. Serum is collected at appropriate intervals, and antibody responses to the target peptides are measured by ELISA.

[0402] The effectiveness of the multi-target nucleic acid sequence is evaluated by a pseudovirus neutralization assay well known to those skilled in the art. The method typically involves incubating the pseudovirus in the presence of various concentrations of immune serum containing the antibody of interest (i.e., antibody raised against the target peptide), adding this mixture to cells, further incubating it, and measuring luminescence to determine the titer of inhibition or neutralization.

[0403] Example 2: Synthesis of multi-target nucleic acid sequence (mRNA)-trivalent RBD constructs Plasmid DNA construction The multi-target nucleic acid sequence of Example 2, which contains three repeats of a polynucleotide sequence, each separated by a cleavage sequence, was codon-optimized for human expression. The multi-target 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 sequence (encoding the receptor binding domain (RBD) of the SARS-CoV-2 SBB.1.5 variant), a linker sequence (a glycine-serine linker sequence encoding a glycine-serine linker), and a self-assembling sequence (a ferritin sequence encoding ferritin). The second polynucleotide sequence consisted of a target sequence (encoding the receptor binding domain (RBD) of SARS-CoV-1), a linker sequence (a glycine-serine linker sequence encoding a glycine-serine linker), and a self-assembling sequence (a ferritin sequence encoding ferritin). The third polynucleotide sequence consisted of a target sequence (encoding the receptor binding domain (RBD) of MERS-CoV), a linker sequence (a glycine-serine linker sequence encoding a glycine-serine linker), and a self-assembly sequence (a 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 (a glycine-serine linker sequence encoding a 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 multi-target nucleic acid sequence. The multi-target nucleic acid sequence construct was inserted between the HindIII and BamHI restriction sites of PTwist Kan High Copy (Twist BioScience, USA). The sequence of the multi-target peptide encoded by the multi-target nucleic acid sequence of Example 2 above is provided in Table 2.

[0404] Table 2: Multi-target nucleic acid sequences of Example 2 - Shows the sequences of the multi-target peptides encoded by the trivalent RBD constructs. [Table 2] The amino acid sequences of the various peptides present in the multi-target peptides encoded by the multi-target nucleic acid sequences of Example 1 are individually identified below:

[0405] The complete sequence of the multi-target peptide of the multi-target nucleic acid of Example 2

number

[0406] Transfection HEK293T cells (Cat. No. CRL3216, ATCC) were cultured at 0.05 × 10 6Cells were seeded into 24-well plates containing Gibco DMEM medium, high glucose, and pyruvate (catalog number 11995065, ThermoFisher Scientific) supplemented with 10% fetal bovine serum and 100 units / mL penicillin-streptomycin and cultured until 70-80% confluency was achieved. A transfection mix containing the multi-target nucleic acid sequence (mRNA, 0.5-1 μg of Example 2) and Lipofectamine™ 2000 (2.5 μL) in 100 μL of serum-free medium was incubated at room temperature for 15 minutes, added to the cells, and allowed to stand for 24 and 48 hours of incubation. The spent medium was removed by aspiration, and the cells were harvested by vigorous pipetting in ice-cold 1x PBS (1 mL). The cells were centrifuged at 4000 g for 5 minutes at 4°C. Excess PBS was aspirated. The cells were gently suspended in 100 μL of 1×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 minutes. The lysate was centrifuged at 16,000 g for 20 minutes at 4°C. The supernatant was aspirated into a microtube.

[0407] Western blot Western blotting was performed on the cell lysate obtained in the previous step using a Jess instrument (Automated Western Blot System, Catalog No. 004-650, ProteinSimple, Bio-Techne). The cell lysate was diluted and combined with one part 5x fluorescence master mix (a component of the Jess-compatible Separation Module, Catalog No. SM-W001, ProteinSimple, Bio-Techne) and heated at 95°C for 5 minutes. After protein denaturation, the sample was mixed with luminol-S and peroxide according to the manufacturer's protocol (Separation Module, Catalog No. SM-W001, ProteinSimple, Bio-Techne). The primary antibody (SARS-CoV-2 spike RBD polyclonal antibody (catalog number E-AB-V-1006, Elab Sciences) was diluted 1:200 with a ready-to-use secondary HRP anti-rabbit antibody and chemiluminescent substrate (both components of the Jess-compatible Detection Module, catalog number DM-001, ProteinSimple, Bio-Techne) and dispensed into assigned wells in a microplate provided by the manufacturer. The plate was then inserted into the Jess instrument, where samples were drawn into individual capillaries arranged on a 25-capillary cassette (12-230 kDa Separation Module, catalog number SM-W001, ProteinSimple, Bio-Techne). Electrophoresis and immunodetection were performed by the Jess instrument in an automated manner. Data were obtained using Compass software accompanying the Jess system. The results are shown in Figure 6.

[0408] Immunogenicity assays The immunogenicity of the multi-target nucleic acid encoding the multi-target peptide of Example 2 was evaluated by ELISA. Mice were immunized with the multi-target nucleic acid sequence (mRNA)-trivalent RBD construct encapsulated in lipid nanoparticles, and ELISA was performed using serum collected on day 28 to find titers against each of the target peptides encoded by the construct of Example 2.

[0409] Plate preparation: Three ELISA plates were each coated with the S1 subunit of spike protein from either SARS-CoV-2 (XBB 1.5 variant), SARS-CoV-1, or MERS-CoV at a concentration of 100 ng per well in 100 μL of coating buffer (1x PBS, pH 7.4). The ELISA plates were gently tapped to ensure uniform coating of the S1 subunit of spike protein on the bottom of each well of the three ELISA plates. The ELISA plates were covered with plate sealers and incubated overnight at 4°C. After overnight incubation, the coating buffer was discarded, and the ELISA plates were washed three times with 300 μL of wash buffer (1x PBS supplemented with 0.05% (v / v) Tween 20). The ELISA plates were blotted on paper towels to remove residual liquid from the wells. 200 μL of blocking buffer (1×PBS containing 3% NFDM) was added to each well and the plate was incubated at room temperature for 2 hours.

[0410] Sample preparation: Mouse serum samples were serially diluted 3-fold in a dilution plate from 1:100 up to 1:218700 in 1x PBS containing 1% NFDM.

[0411] Test: The blocking solution was discarded from the ELISA plate, and residual liquid was removed from the wells using paper towels. The ELISA plate was washed three times with wash buffer (composition as described above). 100 μL of the diluted serum samples from the dilution plate were transferred (in duplicate) to the ELISA plate according to the specified plate layout. The ELISA plate was covered with a plate sealer and incubated at room temperature for 1 hour. After incubation, the serum samples were discarded from the ELISA plate, and the plate was washed three times with wash buffer (composition as described above). 100 μL of diluted secondary antibody solution (1:5000 in 1×PBS) was added to each well, and the ELISA plate was covered with a plate sealer and incubated at room temperature for 1 hour. The ELISA plate was then washed three times with wash buffer (composition as described above). 100 μL of TMB substrate was added to each well and incubated for 15 minutes. The enzyme-substrate reaction was stopped by adding 50 μL of 1 M HCl to each well. After 5 minutes, absorbance was recorded at dual wavelengths of 450 nm and 630 nm using a Spark multimode microplate reader (Tecan Trading AG). The endpoint titer was calculated in the following manner: Cutoff = (average of negative controls (NC) across all plates) x 4 NC = wells without any sample, ie containing PBS (phosphate buffered saline). Endpoint titer = (respective dilution / cutoff value) x respective mean absorbance value The endpoint titer was taken as the highest dilution sample above the cutoff. The results are shown in Figure 7.

[0412] Pseudovirus production and pseudovirus neutralization assay Lentiviral HIV-1-based pseudoviruses carrying the SARS-CoV-2 XBB.1.5, SARS-CoV-1, and MERS-CoV spike glycoproteins were generated by transfecting Lenti-X™ 293T cells (catalog no. 632180, Takara Bio) with the second-generation packaging plasmid psPAX2 vector (catalog no. V010353, Novopro Labs), a spike-expressing plasmid (Twist Biosciences), and a lentiviral firefly luciferase reporter plasmid (catalog no. LR151, Alstem Inc.) using Lipofectamine™ 3000 transfection reagent (catalog no. L3000150, Invitrogen). After 12–16 h of incubation at 37°C and 5% CO, the transfection medium was replaced with DMEM + 10% FBS. The supernatant containing the pseudovirions was collected 72 hours after transfection, centrifuged, filtered, aliquoted, and stored at -80°C.

[0413] A pseudovirus neutralization assay was performed to assess neutralizing antibodies in pooled sera from groups of six mice immunized with the multi-target nucleic acid sequence-trivalent RBD construct of Example 2. Briefly, 1 x 10 IgG antibodies were immunized per well in tissue culture-treated, opaque 96-well plates. 4 HEK293 cells (catalog no. 79951, BPS Biosciences) stably expressing ACE2 were seeded in growth medium (DMEM + 10% FBS + 1% Pen-Strep) and incubated overnight at 37°C and 5% CO2. Next, three-fold serial dilutions of pooled mouse sera were prepared in growth medium, starting from an initial dilution of 1:10, and diluted 1:1 with 0.5 x 10 5 RLU ~ 5 × 10 5The mixtures were mixed with the respective pseudoviruses to produce relative luminescence units (RLU) values ​​in the RLU range. The mixtures were incubated at 37°C for 1 hour, added to HEK293-ACE cells, and incubated at 37°C and 5% CO2. After 72 hours, luminescence activity was measured using a Bright-Glo™ Luciferase Assay System (Cat. No. E2650, Promega) on a Tecan Spark multimode Reader via Spark control Magellan 3.1 software (Tecan Trading AG). Percentage neutralization was calculated by normalizing the test RLU to the RLU of the virus control and cell-only control. The reciprocal IC50 (half-maximal inhibitory concentration) titer of pseudovirus neutralization was calculated using a nonlinear regression curve fit of "log(inhibitor) vs. response - variable slope (4 parameters)" in GraphPad Prism 10. The results are shown in Figures 8a, 8b, and 8c.

[0414] Example 3: Synthesis of multi-target nucleic acid sequence (mRNA)-pentavalent RBD constructs Plasmid DNA construction The multi-target nucleic acid sequence of Example 3, which contains five signal sequences, each consisting of five repeats of a polynucleotide sequence separated by a cleavage sequence, was codon-optimized for human expression. The multi-target 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 sequence (encoding the receptor binding domain (RBD) of the SARS-CoV-2 SBB.1.5 variant), a linker sequence (a glycine-serine linker sequence encoding a glycine-serine linker), and a self-assembling sequence (a ferritin sequence encoding ferritin). The second polynucleotide sequence consisted of a target sequence (encoding the receptor binding domain (RBD) of SARS-CoV-1), a linker sequence (a glycine-serine linker sequence encoding a glycine-serine linker), and a self-assembling sequence (a ferritin sequence encoding ferritin). The third polynucleotide sequence consisted of a target sequence (encoding the receptor binding domain (RBD) of MERS-CoV), a linker sequence (a glycine-serine linker sequence encoding a glycine-serine linker), and a self-assembly sequence (a ferritin sequence encoding ferritin). The fourth polynucleotide sequence consisted of a target sequence (encoding the receptor binding domain (RBD) of OC43), a linker sequence (a glycine-serine linker sequence encoding a glycine-serine linker), and a self-assembly sequence (a ferritin sequence encoding ferritin). The fifth polynucleotide sequence consisted of a target sequence (encoding the receptor binding domain (RBD) of HKU1), a linker sequence (a glycine-serine linker sequence encoding a glycine-serine linker), and a self-assembly sequence (a 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 (a glycine-serine linker sequence encoding a 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 included in the multi-target nucleic acid sequence.The multi-target nucleic acid sequence construct was inserted between the HindIII and BamHI restriction sites of PTwist Kan High Copy (Twist BioScience, USA). Example 3 Multi-target nucleic acid sequence - The sequence of the multi-target peptide encoded by the pentavalent RBD construct is provided in Table 3.

[0415] Table 3: Example 3 Multi-target nucleic acid sequences - Shows the sequences of multi-target peptides encoded by pentavalent RBD constructs. [Table 3-1] [Table 3-2] The amino acid sequences of the various peptides present in the multi-target peptides encoded by the multi-target nucleic acid sequence-pentavalent RBD construct sequences of Example 3 are individually identified below:

[0416] The complete sequence of the multi-target peptide encoded by the multi-target nucleic acid of Example 3

number

[0417] Transfection HEK293T cells (Cat. No. CRL3216, ATCC) were cultured at 0.05 × 10 6Cells were seeded into 24-well plates containing Gibco DMEM medium, high glucose, and pyruvate (catalog number 11995065, ThermoFisher Scientific) supplemented with 10% fetal bovine serum and 100 units / mL penicillin-streptomycin and cultured until 70-80% confluency was achieved. A transfection mix containing the multi-target nucleic acid (mRNA, 0.5-1 μg of Example 3) and Lipofectamine™ 2000 (2.5 μL) in 100 μL of serum-free medium was incubated at room temperature for 15 minutes, added to the cells, and incubated for 24 and 48 hours. The spent medium was removed by aspiration, and the cells were harvested by vigorous pipetting in ice-cold 1x PBS (1 mL). The cells were centrifuged at 4000 g for 5 minutes at 4°C. Excess PBS was aspirated. The cells were gently suspended in 100 μL of 1×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 minutes. The lysate was centrifuged at 16,000 g for 20 minutes at 4°C. The supernatant was aspirated into a microtube.

[0418] Western blot Western blotting was performed on the cell lysate obtained in the previous step using a Jess instrument (Automated Western Blot System, Catalog No. 004-650, ProteinSimple, Bio-Techne). The cell lysate was diluted and combined with one part 5x fluorescence master mix (a component of the Jess-compatible Separation Module, Catalog No. SM-W001, ProteinSimple, Bio-Techne) and heated at 95°C for 5 minutes. After protein denaturation, the sample was mixed with luminol-S and peroxide according to the manufacturer's protocol (Separation Module, Catalog No. SM-W001, ProteinSimple, Bio-Techne). The primary antibody (SARS-CoV-2 spike RBD polyclonal antibody (catalog number E-AB-V-1006, Elab Sciences) was diluted 1:200 with a ready-to-use secondary HRP anti-rabbit antibody and chemiluminescent substrate (all components of the Jess-compatible Detection Module, catalog number DM-001, ProteinSimple, Bio-Techne) and dispensed into assigned wells in a microplate provided by the manufacturer. The plate was then inserted into the Jess instrument, where samples were drawn into individual capillaries arranged on a 25-capillary cassette (12-230 kDa Separation Module, catalog number SM-W001, ProteinSimple, Bio-Techne). Electrophoresis and immunodetection were performed by the Jess instrument in an automated manner. Data were obtained using Compass software accompanying the Jess system. The results are shown in Figure 9.

[0419] Incorporation by Reference Each of the patents, published patent applications, and non-patent references cited herein is hereby incorporated by reference in its entirety.

[0420] equivalent 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 invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A nucleic acid comprising a plurality of polynucleotide sequences, wherein some or all of the plurality of polynucleotide sequences comprise 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 of the plurality of polynucleotide sequences is connected to an adjacent one of the plurality of polynucleotide sequences by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the plurality of polynucleotide sequences.

2. A nucleic acid comprising a plurality of polynucleotide sequences, wherein each of the plurality of polynucleotide sequences comprises a target sequence, a linker sequence, and a self-assembling sequence, wherein each of the plurality of polynucleotide sequences is connected to adjacent ones of the plurality of polynucleotide sequences by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the plurality of polynucleotide sequences.

3. A nucleic acid comprising a plurality of polynucleotide sequences, wherein each of the plurality of polynucleotide sequences comprises a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence, wherein each of the plurality of polynucleotide sequences is connected to adjacent ones of the plurality of polynucleotide sequences by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the plurality of polynucleotide sequences.

4. A nucleic acid encoding a plurality of polypeptides, wherein some or all of the plurality of polypeptides 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, wherein each of the plurality of polypeptides is connected to an adjacent one of the plurality of polypeptides by a cleavage peptide, and wherein the nucleic acid further comprises a signal peptide amino-terminal to one or more of the plurality of polypeptides.

5. A nucleic acid encoding a plurality of polypeptides, wherein each of the plurality of polypeptides comprises a target peptide, a linker peptide, and a self-assembling peptide, wherein each of the plurality of polypeptides is connected to an adjacent one of the plurality of polypeptides by a cleavage peptide, and wherein the nucleic acid sequence further encodes a signal peptide amino-terminal to one or more of the plurality of polypeptides.

6. A nucleic acid encoding a plurality of polypeptides, wherein each of the plurality of polypeptides comprises a linker peptide, a targeting peptide, a linker peptide, and a self-assembling peptide, wherein each of the plurality of polypeptides is connected to an adjacent one of the plurality of polypeptides by a cleavage peptide, and wherein the nucleic acid sequence further encodes a signal peptide amino-terminal to one or more of the plurality of polypeptides.

7. The nucleic acid according to any one of claims 1 to 6, wherein the total number of polynucleotide sequences is 100 or less.

8. 8. The nucleic acid of any one of claims 1 to 7, wherein the total number of polynucleotide sequences is 2 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 99.

9. The nucleic acid according to any one of claims 1 to 8, wherein the nucleic acid is DNA or RNA.

10. The nucleic acid of claim 9 , wherein the RNA is mRNA.

11. The nucleic acid according to any one of claims 1 to 10, wherein the linker sequence encodes a linker peptide.

12. The nucleic acid of claim 11 , wherein the linker peptide is an amino acid linker, a foldon, a scaffold, or a combination thereof.

13. The nucleic acid of claim 12, wherein the amino acid linker comprises 2 to 49 amino acids.

14. 14. The nucleic acid of 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 one of claims 1 to 14, wherein the linker peptide has an amino acid sequence of any one of SEQ ID NOs: 262 to 299, 330, and 350.

16. The nucleic acid of any one of claims 1 to 15, wherein the self-assembling sequence encodes a self-assembling peptide.

17. 17. The nucleic acid of claim 16, wherein the self-assembling peptide is lumazine synthase from Acwifex 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 codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof.

18. The nucleic acid of claim 17, wherein the ferritin is composed of ferritin subunits or ferritin peptides.

19. 19. The nucleic acid of claim 18, wherein the ferritin peptide is derived from ferritin of Helicobacter pylori.

20. The nucleic acid of any one of claims 1 to 19, wherein the self-assembling peptide has an amino acid sequence of any one of SEQ ID NOs: 254 to 261, 331 and 333.

21. The nucleic acid of any one of claims 1 to 20, wherein the cleavage sequence encodes one or more cleavage sequences.

22. 22. The nucleic acid of claim 21, wherein the one or more cleavage sequences are optionally connected to each other by a linker peptide.

23. 23. The nucleic acid of claim 22, wherein the cleavage peptide is a Golgi-specific cleavage peptide or a self-cleavage peptide.

24. 24. The nucleic acid of any one of claims 1 to 23, wherein the truncated peptide has the amino acid sequence of any one of SEQ ID NOs: 300 to 311 and 347 to 349.

25. The nucleic acid according to any one of claims 1 to 24, wherein the signal sequence encodes a signal peptide.

26. 26. The nucleic acid of claim 25, wherein the signal peptide is present at the amino terminal end of one or more of the multiple polypeptides.

27. 27. The nucleic acid of claim 26, further encoding a second signal peptide amino-terminal to all or some of the multiple polypeptides.

28. 28. The nucleic acid of any one of claims 1 to 27, wherein the signal peptide has the amino acid sequence of any one of SEQ ID NOs: 312 to 329.

29. The nucleic acid of any one of claims 1 to 28, wherein the target sequence encodes a target peptide.

30. 30. The nucleic acid of claim 29, wherein the target peptide is encoded by a codon-optimized nucleic acid sequence, or a fragment, mutant, or variant thereof.

31. 31. The nucleic acid of 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 parasite, a mycoplasma, an animal, a human, or a combination thereof.

32. 32. The nucleic acid of claim 31 , wherein the virus is selected from a family comprising: Picornaviridae, Caliciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Arteriviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, Bornaviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Retroviridae, Polyomaviridae, Herpesviridae, Poxviridae, Papillomaviridae, Hepadnaviridae, Adenoviridae, Parvoviridae, Hepeviridae, Circoviridae, or a combination thereof.

33. 32. The nucleic acid of claim 31 , wherein the bacterium is selected from a 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. 33. The nucleic acid of claim 32, wherein the virus is selected from the family consisting of Coronaviridae, Herpesviridae, Poxviridae, Flaviviridae, Togaviridae, Retroviridae, Paramyxoviridae, and combinations thereof.

35. 32. The nucleic acid of claim 31 , wherein the virus is an alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus, torovirus, or a combination thereof.

36. 36. The nucleic acid of claim 35, wherein the betacoronavirus is SARS-CoV-1, SARS-CoV-2, MERS-CoV, OC43, HKU1, a bat coronavirus, another betacoronavirus, or a combination thereof.

37. 31. The nucleic acid of claim 30, wherein the target peptide is a spike protein, a membrane protein, an envelope protein, or a nucleocapsid protein of a coronavirus.

38. 31. The nucleic acid of claim 30, wherein the target peptide is glycoprotein B, glycoprotein C, glycoprotein D, glycoprotein E, glycoprotein K, glycoprotein L, or glycoprotein M of herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

39. 31. The nucleic acid of claim 30, wherein the target peptide is glycoprotein B, glycoprotein H, glycoprotein L, glycoprotein M, or glycoprotein N of human cytomegalovirus (HCMV), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

40. 31. The nucleic acid of claim 30, wherein the target peptide is glycoprotein B, glycoprotein C, glycoprotein H, or glycoprotein L of varicella-zoster virus (VZV), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

41. 31. The nucleic acid of claim 30, wherein the target peptide is Epstein-Barr virus (EBV) glycoprotein B, glycoprotein H, glycoprotein L, glycoprotein M, glycoprotein N, glycoprotein 42, glycoprotein 350, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

42. 31. The nucleic acid of claim 30, wherein the target peptide is a poxvirus F9 membrane protein, a poxvirus H3L protein, a poxvirus A4 protein, a poxvirus A27 protein, a poxvirus A33 protein, a poxvirus A56 protein, a poxvirus B5 protein, or a poxvirus L1 protein, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, a mutant or a variant thereof.

43. 31. The nucleic acid of claim 30, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a flavivirus or hepacivirus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

44. The nucleic acid of claim 30, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Japanese encephalitis virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

45. 31. The nucleic acid of claim 30, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Zika virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

46. 31. The nucleic acid of claim 30, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of yellow fever virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

47. 31. The nucleic acid of claim 30, wherein the target peptide is a West Nile virus capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

48. 31. The nucleic acid of claim 30, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Hepatitis C virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

49. The nucleic acid of claim 30, wherein the target peptide is a capsid protein, membrane protein, envelope protein, or nonstructural protein (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a dengue virus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

50. The nucleic acid of claim 30, wherein the target peptide is an alphavirus capsid protein or envelope protein such as E1, E2 and E3 proteins, or a combination thereof including their codon-optimized nucleic acid sequences, fragments, mutants or variants thereof.

51. The nucleic acid of claim 30, wherein the target peptide is domain A, domain B, or domain C of the E2 protein of an alphavirus, or a combination thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants, or variants thereof.

52. 31. The nucleic acid of claim 30, wherein the target peptide is a capsid protein or an envelope protein such as E1, E2 and E3 protein of Chikungunya virus, or a combination thereof including a codon-optimized nucleic acid sequence thereof, a fragment, a mutant or a variant thereof.

53. 31. The nucleic acid of claim 30, wherein the target peptide is a retroviral gag, pol and env protein, or a combination thereof, including codon-optimized nucleic acid sequences thereof, fragments, mutants or variants thereof.

54. The target peptide is p17 derived from lentivirus. Gag , p24 Gag , p7 Gag , p6 Gag , gp12 Env , gp41 Env 31. The nucleic acid of claim 30, which is a pol protein or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

55. The target peptide is p17 derived from human immunodeficiency virus (HIV). Gag , p24 Gag , p7 Gag , p6 Gag , gp12 Env , gp41 Env or pol protein, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant or variant thereof.

56. 31. The nucleic acid of 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, an L protein, an M protein, an H (hemagglutinin) protein, an HN (hemagglutinin-neuraminidase) protein, a G protein, or an F protein of mumps virus (MuV), parainfluenza virus type 5 (PIV5), human parainfluenza virus types 2, 4a, and 4b (HPIV2 / 4a / 4b), Newcastle disease virus (NDV), human parainfluenza virus types 1 and 3 (HPIV1 / 3), Nipah virus (NiV), measles virus (MeV), human respiratory syncytial virus (HRSV) A2, B1, S2, or human metapneumovirus (HMPV), or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, a mutant, or a variant thereof.

57. 31. The nucleic acid of claim 30, wherein the target peptide is a 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 (HRSV) A2, B1, or S2, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

58. 31. The nucleic acid of claim 30, wherein the target peptide is aE1, E2, E4, aE5, E6, E7, L1, or L2 protein of a papillomavirus, preferably a human papillomavirus, or a combination thereof, including a codon-optimized nucleic acid sequence thereof, a fragment, mutant, or variant thereof.

59. 59. The nucleic acid of any one of claims 1 to 58, wherein the target peptide has an amino acid sequence of any one of SEQ ID NOs: 1 to 253, 334 to 337, 338 to 346 and 353 to 388.

60. A lipid nanoparticle composition comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and a nucleic acid according to any one of claims 1 to 59.

61. 61. The lipid nanoparticle composition of claim 60, wherein the cationic lipid comprises an ionizable lipid.

62. 62. The lipid nanoparticle composition of claim 61, wherein the ionizable lipid is present in an amount of 25 mol percent to 70 mol percent.

63. 61. The lipid nanoparticle composition of claim 60, wherein the phospholipid is present in an amount of from 2 mol percent to about 30 mol percent.

64. 61. The lipid nanoparticle composition of claim 60, wherein the sterol is present in an amount of from 30 mol percent to about 65 mol percent.

65. 61. The lipid nanoparticle composition of claim 60, wherein the PEG-lipid is present in an amount of 0.2 mol percent to about 2.0 mol percent.

66. 61. The lipid nanoparticle composition of claim 60, additionally comprising an ionizable polymer.

67. 67. The lipid nanoparticle composition of claim 66, wherein the ionizable polymer is present in an amount of 1 mol percent to 25 mol percent.

68. 67. The lipid nanoparticle composition of claim 66, wherein the ionizable polymer is selected from the group comprising chitosan, cellulose derivatives, poly-L-lysine, poly-L-glutamic acid, and / or derivatives thereof or combinations thereof.

69. A method for treating or preventing a disease, comprising administering to a subject in need thereof a nucleic acid according to any one of claims 1 to 59.

70. A method for treating or preventing a disease, comprising administering to a subject in need thereof the lipid nanoparticle composition of any one of claims 60 to 68.

71. 60. Use of a nucleic acid sequence according to any one of claims 1 to 59 in the manufacture of a medicament for the treatment or prevention of a disease in a subject.

72. Use of a lipid nanoparticle composition according to any one of claims 60 to 68 in the manufacture of a medicament for the treatment or prevention of a disease in a subject.

73. A multi-target peptide encoded by a nucleic acid according to any one of claims 1 to 59.

74. A multi-target peptide comprising two or more polypeptides, wherein some or all of the polypeptides 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, wherein one polypeptide is connected to another polypeptide by a cleavage peptide, and wherein the multi-target peptide includes a signal peptide upstream of one or more of the polypeptides.

75. A polypeptide nanoparticle comprising at least two or up to 100 polypeptides according to any one of claims 1 to 59.

76. The polypeptide nanoparticle of claim 75, wherein the polypeptide is a homologous polypeptide, a heterologous polypeptide, an oligomeric complex, or a combination thereof.

77. 77. The polypeptide nanoparticle of claim 76, wherein the polypeptide nanoparticle is icosahedral, helical, spherical, rod-shaped, or a combination thereof.

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