WO2024141786A2 - Multitarget vaccines and therapeutics - Google Patents

Multitarget vaccines and therapeutics Download PDF

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Publication number
WO2024141786A2
WO2024141786A2 PCT/IB2023/000787 IB2023000787W WO2024141786A2 WO 2024141786 A2 WO2024141786 A2 WO 2024141786A2 IB 2023000787 W IB2023000787 W IB 2023000787W WO 2024141786 A2 WO2024141786 A2 WO 2024141786A2
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peptide
nucleic acid
sequence
protein
combination
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PCT/IB2023/000787
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French (fr)
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Tushar Ranjan MOHARANA
Soham Govindarajan SANKARAN
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Popvax Private Limited
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Publication of WO2024141786A2 publication Critical patent/WO2024141786A2/en

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  • the present disclosure relates to multitarget nucleic acid sequences, multitarget peptides, and polypeptide nanoparticle and their compositions for vaccine and therapeutic purpose.
  • pathogens such as influenza virus, HIV, human papillomavirus, SARS-CoV- 2, Streptococcus pneumoniae, Neisseria meningitidis, Neisseria gonorrhoeae, Trypanosoma brucei etc.
  • pathogens such as influenza virus, HIV, human papillomavirus, SARS-CoV- 2, Streptococcus pneumoniae, Neisseria meningitidis, Neisseria gonorrhoeae, Trypanosoma brucei etc.
  • Combination vaccines have been traditionally used to target multiple pathogens or variations within a pathogen (Skibinski, David AG et al. Journal of Global Infectious Diseases (2011) 3:63-72; Alderson, MarkR. etal. Microorganisms (2021) 9: 771).
  • mRNA based vaccines have emerged as promising alternative to classical approaches to vaccine development.
  • current mRNA based vaccines are still inadequate to address the emerging strains or variants of a pathogen.
  • the antibody titres elicited by SARS-CoV-2 vaccine against the emerging and antigenically divergent SARS-CoV-2 variants have been found to be lower, and wane over time suggesting decreased effectiveness of the vaccines targeted against a single variant/strain of the virus.
  • Bivalent vaccines encapsulating different mRNAs, each targeting a specific variant of SARS-CoV-2, are undergoing clinical trials to increase the breadth of coverage (Chalkias, Spyros et al.
  • the present disclosure relates to a nucleic acid comprising a plurality of polynucleotide sequences, wherein some or all polynucleotide sequence of the plurality comprises either a target sequence, a linker sequence, and a self-assembling sequence or a linker sequence, a target sequence, a linker sequence and a self-assembling sequence or a combination thereof.
  • each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence.
  • the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequences of the plurality.
  • 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 in 5' to 3' order.
  • a nucleic acid comprising a plurality of polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises a target sequence, a linker sequence, and a self-assembling sequence.
  • each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence.
  • the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequences of the plurality.
  • the target sequence, the linker sequence, and the self-assembling sequence are in 5' to 3' order.
  • a nucleic acid comprising a plurality of polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence.
  • each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence.
  • the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequences of the plurality.
  • the linker sequence, the target sequence, the linker sequence and the self-assembling sequence are in 5' to 3' order.
  • nucleic acid encoding a plurality of polypeptides, wherein some or all polypeptides of the plurality comprises either a target peptide, a linker peptide, and a self-assembling peptide or a linker peptide, a target peptide, a linker peptide and a self-assembling peptide or a combination thereof.
  • each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide.
  • the nucleic acid further encodes a signal peptide on the amino-terminus of one or more of the polypeptides of the plurality.
  • the target peptide, the linker peptide, and the self-assembling peptide or the linker peptide, the target peptide, the linker peptide and the self-assembling peptide are in N-terminus to C- terminus order.
  • a nucleic acid encoding a plurality of polypeptides, wherein each polypeptide of the plurality comprises a target peptide, a linker peptide, and a self-assembling peptide.
  • each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide.
  • the nucleic acid further encodes a signal peptide on the aminoterminus of one or more of the polypeptides of the plurality.
  • the target peptide, the linker peptide, and the self-assembling peptide are in N-terminus to C- terminus order.
  • nucleic acid encoding a plurality of polypeptides, wherein each polypeptide of the plurality comprises a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide.
  • each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide.
  • the nucleic acid further encodes a signal peptide on the amino-terminus of one or more of the polypeptides of the plurality.
  • the linker peptide, the target peptide, the linker peptide and the selfassembling peptide are in N-terminus to C- terminus order.
  • total number of the polynucleotide sequences is not more than 100. In some embodiments, total number of the polynucleotide sequences is between 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-99.
  • the nucleic acid is a DNA or an RNA. In some embodiments, the RNA is an mRNA.
  • the linker sequence encodes a linker peptide. In some embodiments, the linker peptide is an amino acid linker, foldon, scaffold or a combination thereof. In some embodiments, the amino acid linker comprises 2 to 49 amino acids. In some embodiments, the amino acid linker is glycine serine linker, glycine proline linker, glycine threonine linker, alanine serine linker, any combination of two amino acids, or a combination thereof.
  • the linker peptide has an amino acid sequence of any one of SEQ ID NOs: 262-299, 330, and 350.
  • the self-assembling sequence encodes a self-assembling peptide.
  • the self-assembling peptide is lumazine synthase from Aquifex species, hepatitis B surface antigen (HBsAg) from Hepatitis B Virus, hepatitis B core antigen (HBcAg) from Hepatitis B virus, human papillomavirus LI (HPV LI) protein, matrix protein Ml from influenza A virus, ferritin, riboflavin synthase, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • HBsAg hepatitis B surface antigen
  • HBcAg hepatitis B core antigen
  • HPV LI human papillomavirus LI
  • the ferritin comprises of ferritin subunit or ferritin peptide.
  • the ferritin peptide is derived from Helicobacter pylori ferritin.
  • the self-assembling peptide has an amino acid sequence of any one of SEQ ID NOs: 254-261, 331 and 333.
  • the cleavage sequence encodes one or more cleavage peptide.
  • the one or more cleavage peptides are optionally connected to each other by a linker.
  • the cleavage peptide is a golgi specific cleavage peptide or self-cleaving peptide.
  • the cleavage peptide has an amino acid sequence of any one of SEQ ID NOs: 300-311 and 347-349.
  • the signal sequence encodes a signal peptide. In some embodiments, the signal peptide is present on the amino-terminus of the first polypeptide. In some embodiments, the nucleic acid further encodes a second signal peptide on the amino-terminus of all or some polypeptides. In some embodiments, the signal peptide has an amino acid sequence of any one of SEQ ID NOs: 312-329.
  • the target sequence encodes a target peptide.
  • the target peptide is encoded by a codon optimized nucleic acid sequence, or fragments, mutants, or variants thereof.
  • 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.
  • the virus is selected from the family comprising picomaviride, calciviridae, astroviridae, togaviridae, flaviviridae, coronaviridae, arteriviridae, rhabndoviridae, filoviridae, paramyxoviridae, bomaviridae, orthomyxoviridae, bunyaviridae, arenaviridae, reoviridae, retroviridae, polyomaviridae, herpesviridae, poxviridae, papillomaviridae, hepadnaviridae, adenoviridae, parvoviridae, hepeviridae, circoviridae or a combination thereof.
  • the bacterium is selected from the genus comprising Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio, Yersinia, or a combination thereof
  • the virus is selected from the family consisting of coronaviridae, herpesviridae, poxviridae, flaviviridae, togaviridae, retroviridae, paramyxoviridae, or a combination thereof.
  • the virus is alphacoronavirus, betacoronavirus
  • the betacoronavirus is SARS-CoV-1, SARS-CoV-2, MERS- CoV, OC43, HKU1, bat coronavirus, other betacoronavirus or a combination thereof.
  • the target peptide is a spike protein, a membrane protein, an envelope protein or a nucleocapsid protein of coronaviruses.
  • the target peptide is a receptor binding domain, fusion peptide, or stem helix of the spike protein, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants or variants thereof.
  • the target peptide is a receptor binding domain obtained or derived from a betacoronavirus comprising SARS-CoV-1, SARS- CoV-2, MERS-CoV, OC43, HKU1, bat coronavirus, other betacoronavirus, or a combination thereof.
  • the target peptide is glycoprotein B, glycoprotein C, glycoprotein D, glycoprotein E, glycoprotein K, glycoprotein L, and glycoprotein M, of herpes simplex virus 1 (HSV-1) or herpes simplex virus 2 (HSV-2), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants or variants thereof.
  • the target peptide is glycoprotein B, glycoprotein H, glycoprotein L, glycoprotein M, or glycoprotein N of human cytomegalovirus (HCMV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the target peptide is glycoprotein B, glycoprotein C, glycoprotein H, or glycoprotein L of varicella-zoster virus (VZV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of flaviviruses or hepaciviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • a non-structural proteins such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5
  • the target peptide is nucleocapsid protein, P protein, V protein, W protein, D protein, I protein, C protein, L protein, M protein, H (hemagglutinin) protein, HN (hemagglutinin-neuraminidase) protein, G protein or F protein of human respiratory syncytial virus A2, Bl, S2 (HRSV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • nucleic acid sequence disclosed herein in the manufacture of a medicament for the treatment or prevention of a disease in a subject.
  • a multitarget peptide encoded by the nucleic acid disclosed herein is provided herein.
  • nucleic acid sequence comprising a multitarget nucleic acid sequence comprising two or more polynucleotide sequences, wherein some or all polynucleotide sequences comprises either a target sequence, a linker sequence, and a self-assembling sequence, or a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence, or a combination thereof, wherein one polynucleotide sequence is connected to another polynucleotide sequence by a cleavage sequence, wherein the multitarget nucleic acid sequence includes a signal sequence upstream of one or more of the polynucleotide sequences.
  • the multitarget nucleic acid sequence is a DNA or an RNA. In some embodiments, the multitarget nucleic acid sequence is an mRNA. In some embodiments, the multitarget nucleic acid sequence encodes a multitarget peptide. In some embodiments, the multitarget nucleic acid is encapsulated in a lipid nanoparticle composition. In some embodiments, the multitarget nucleic acid sequence is synthesized through a single in vitro transcription (IVT) process. In some embodiments, the disclosure relates to a nucleic acid sequence encoding a multitarget peptide described herein.
  • the disclosure also relates to a multitarget peptide comprising two or more polypeptides, wherein some or all polypeptides comprises either a target peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide, or a combination thereof, wherein one polypeptide is connected to another polypeptide by a cleavage peptide, wherein the multitarget peptide includes a signal peptide upstream (amino-terminus) of one or more of the polypeptides.
  • signal peptide may be present upstream (aminoterminus) of all or some of the polypeptides. In some embodiments, signal peptide may be present upstream (amino-terminus) of the first polypeptide. In some embodiments, signal peptide may be present upstream (amino-terminus) of all the polypeptides.
  • the linker peptide connects signal peptide with the first polypeptide in a multitarget peptide. In some embodiments, the linker peptide connects target peptide with the self-assembling peptide in a polypeptide. In some embodiments, one linker peptide connects cleavage peptide with the target peptide and another linker peptide connects the target peptide with the self-assembling peptide in a polypeptide. In some embodiments, the multitarget peptide comprise homologous polypeptides. In some other embodiments, the multitarget peptide comprise heterologous polypeptides. In some embodiments, multitarget peptide comprise homologous polypeptides, or heterologous polypeptides.
  • the disclosure relates to a polypeptide nanoparticle formed from the self-assembly of two or more polypeptides, wherein some or all polypeptides comprises either a target peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide or a combination thereof.
  • the polypeptides in the polypeptide nanoparticle may also have some residues of cleavage peptide.
  • the polypeptide nanoparticle comprises of homologous polypeptides, heterologous polypeptides, oligomeric complexes, or a combination thereof.
  • FIG. 1 - shows representative schematic illustration of multitarget nucleic acid sequence wherein each polynucleotide sequence (PS) comprises a target sequence (TS), a linker sequence (LS), and a self-assembling sequence (SAS) or a linker sequence (LS), a target sequence (TS), a linker sequence (LS), and a self-assembling sequence (SAS) .
  • PS polynucleotide sequence
  • LS linker sequence
  • SAS self-assembling sequence
  • Multiple polynucleotide sequences are connected through a cleavage sequence (CS) such that between any two polynucleotide sequences there is present a cleavage sequence.
  • the multitarget nucleic acid sequence has a signal sequence (SS) upstream of the first polynucleotide sequence.
  • the letter ‘n’ in figure 1 represents any number between 1 to 98.
  • the multitarget nucleic acid sequence may additionally have 5’ cap and 3’ poly(A) tail. This multitarget nucleic acid sequence
  • FIG. 2 - shows representative schematic illustration of multitarget peptide wherein 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).
  • PP polypeptide
  • LP linker peptide
  • SAP self-assembling peptide
  • Multiple polypeptides are connected through a cleavage peptide (CP) such that between any two polypeptides there is present a cleavage peptide.
  • the multitarget peptide has a signal peptide (SP) on the N-terminus of the first polypeptide.
  • SP signal peptide
  • Figures 7a, 7b, and 7c - show ELISA data showing generation of antibodies (IgG) against the respective target peptides encoded by the multitarget nucleic acid sequence - trivalent RBD construct of example 2.
  • Figures 8a, 8b, and 8c - show pseudovirus neutralization of the antibodies generated against the respective target peptides encoded by the multitarget nucleic acid sequence - trivalent RBD construct of example 2.
  • Figure 9 - shows a western blot of cell lysate (lane 3) showing expression of target peptides encoded by multitarget nucleic acid sequence - pentavalent RBD construct of example 3 using polyclonal anti-spike antibody against SARS-CoV-2. Lanes 1 and 2 are protein ladder and negative control respectively.
  • an individual “at risk” of developing a particular disease, disorder, or condition may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment methods described herein.
  • “At risk” denotes that an individual has one or more risk factors, which are measurable parameters that correlate with development of a particular disease, disorder, or condition, as known in the art. An individual having one or more of these risk factors has a higher probability of developing a particular disease, disorder, or condition than an individual without one or more of these risk factors.
  • multitarget nucleic acid sequence may comprise multiple repeats of polynucleotide sequences wherein each polynucleotide sequence may comprise either a target sequence, a linker sequence, and a self-assembling sequence, or a linker sequence, a target sequence, a linker sequence and a self-assembling sequence, or a combination thereof, such that total number of polynucleotide sequences in a multitarget nucleic acid sequence are not more than 100.
  • the linker sequence connects signal sequence with the first polynucleotide sequence.
  • the signal sequence is present upstream of each of some or all of the polynucleotide sequences.
  • the multitarget nucleic acid sequence encodes multitarget peptide.
  • polynucleotide sequence as used herein means a sequence of nucleotides that encodes a polypeptide.
  • polypeptide as used herein means a sequence of amino acids that comprise either a target peptide, a linker peptide, and a self-assembling peptide or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide.
  • the polypeptide comprises a target peptide, a linker peptide, and a selfassembling peptide.
  • the polypeptide comprises a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide.
  • the polypeptide may have some residues (amino acids) of cleavage peptide.
  • the polypeptide may have signal peptide.
  • target sequence means a sequence of nucleotides that encodes a target peptide.
  • target peptide as used herein means a sequence of amino acids that has immunostimulatory or immunomodulatory effect.
  • Target peptide also means a peptide of interest.
  • target peptide is an antigen.
  • the target peptide in two or more polypeptides may be identical i.e., homologous polypeptides. In some other embodiments, the target peptides in two or more polypeptides may be different i.e., heterologous polypeptides.
  • signal sequence means a sequence of nucleotides that encodes a signal peptide.
  • signal peptide as used herein means a sequence of amino acids that transports the multitarget peptide to specific cell organelles. In some embodiments the signal peptide transports the multitarget peptide to golgi apparatus or golgi body.
  • the signal peptide is present on the N-terminus (amino-terminus) of one or more polypeptides. In some embodiments, the signal peptide is present on the N-terminus of some or all polypeptides. In some embodiments, the signal peptide is present on the N-terminus of the first polypeptide. In some embodiments, the signal peptide is present on the N-terminus of some polypeptides.
  • the signal peptide is present on the N-terminus of all polypeptides. In some embodiments, the signal peptide is encoded by signal sequence. In some embodiments, the signal peptide is a golgi targeting signal peptide.
  • cleavage sequence as used herein means a sequence of nucleotides that encodes a cleavage peptide.
  • the cleavage peptide is a substrate for golgi specific proteases.
  • the cleavage peptide comprises one or more cleavage peptides, for example, cleavage peptide- 1, cleavage peptide-2 and so on.
  • the cleavage peptide optionally comprises a linker peptide between two cleavage peptides.
  • the cleavage peptide may self-cleave into individual polypeptides or may be cleaved by the action of cellular proteases.
  • one linker peptide connects cleavage peptide with the target peptide and another linker peptide connects target peptide with the self-assembling peptide in a polypeptide. In some embodiments, one linker peptide connects cleavage peptide with the target peptide and another linker peptide connects target peptide with the self-assembling peptide. In some embodiments, one linker peptide connects cleavage peptide with the target peptide and another linker peptide connects the signal peptide with the target peptide. In some embodiments, the linker peptide connects two cleavage peptides. In some embodiments, the linker peptide is an amino acid linker, a foldon, a scaffold or a combination thereof.
  • amino acid linker sequence means a sequence of nucleotides that encodes an amino acid linker.
  • amino acid linker means a sequence of amino acids that provides structural integrity to polypeptide such that the components of the polypeptide remain, as far as possible, in their native or stable conformation.
  • amino acid linker also helps in orientation of a polypeptide such that the domains or epitopes on the target peptide are exposed or displayed for interaction or communication with cells or biomolecules or immune system in the absence of foldon or scaffold.
  • the amino acid linker connects two cleavage peptides.
  • amino acid linkers includes, glycine serine linker, glycine proline linker, glycine threonine linker, alanine serine linker, any combination of two amino acids or a combination thereof. In some embodiments, amino acid linker is about 2-49 amino acid long.
  • glycine serine linker sequence as used herein means a sequence of nucleotides that encodes a glycine serine linker.
  • foldon sequence means a sequence of nucleotides that encodes a foldon.
  • scaffold sequence means a sequence of nucleotides that encodes a scaffold.
  • scaffold as used herein means a sequence of amino acids that provides structural and/or functional integrity or support to the target peptide and helps in orientation of target peptide such that the domains or epitopes of the target peptide are exposed or displayed for interaction or communication with cells or biomolecules or immune system.
  • self-assembling sequence means a sequence of nucleotides that encodes a self-assembling peptide.
  • self-assembling peptide means a sequence of amino acids that enables the polypeptides to self-assemble into polypeptide nanoparticle.
  • polypeptide nanoparticle as used herein means a nanoparticle formed by self-assembly of polypeptides.
  • the polypeptide nanoparticle comprises of two or more homologous polypeptides, or two or more heterologous polypeptides, or one or more oligomeric complexes or a combination thereof.
  • heterologous polypeptide as used herein means polypeptides in a multitarget peptide that have different target peptides. For example, if two polypeptides in the multitarget peptide have different or non-identical target peptides, they are considered to be heterologous polypeptides.
  • mutant as used herein, whether in the context of a nucleic acid, nucleotide, protein, polypeptide, or peptide sequence, means a sequence which is not a wild type sequence. A mutant is also understood to mean a nucleic acid, nucleotide, protein, polypeptide, or peptide sequence that carries a mutation.
  • mutant as used herein means, a change or modification in the sequence of nucleic acid or amino acid in comparison to a reference sequence and includes insertion, deletion, substitution, or a combination thereof.
  • a multitarget nucleic acid sequence includes two or more polynucleotide sequences wherein some or all polynucleotide sequences comprises either a target sequence, a linker sequence, and a self-assembling sequence, or a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence, wherein one polynucleotide sequence is connected to the another polynucleotide sequence by a cleavage sequence, wherein the multitarget nucleic acid sequence includes a signal sequence upstream of one or more polynucleotide sequences.
  • the signal sequence may be present upstream of all or some polynucleotide sequences.
  • the linker sequence connects signal sequence with the polynucleotide sequence. In some embodiments, one linker sequence connects cleavage sequence with the target sequence and another linker sequence connects the target sequence with the self-assembling sequence in a polynucleotide sequence.
  • RNA ribonucleic acid
  • RNA messenger RNA
  • the multitarget nucleic acid sequence is an mRNA.
  • the mRNA is circular. In other embodiments, the mRNA is linear.
  • the multitarget nucleic acid sequence and the multitarget peptide includes target sequence and target peptide respectively.
  • the target sequence is a DNA or an RNA.
  • the target sequence is an mRNA.
  • the target sequence may be modified or unmodified.
  • the target sequence includes codon optimized sequences, fragments, mutants, variants, or combination thereof.
  • Target sequence may be obtained from a prokaryote or a eukaryote, a unicellular organism or a multicellular organism, a virus, a bacterium, a fungus, a protozoan, a worm, a mycoplasma, an animal, a human, or a combination thereof.
  • the target sequence is a sequence of nucleotides that encodes a target peptide.
  • the target peptide is capable of performing one or more functions such as, but not limited to, immunostimulation, immunomodulation, etc.
  • the target peptide means a peptide of interest.
  • target peptide is an antigen.
  • the target peptide is identical in two or more polypeptides, for example homologous polypeptides.
  • the target peptide is different in two or more polypeptides, for example as in heterologous polypeptides.
  • a hapten is used in a composition of the disclosure, it may be attached to a carrier to form a hapten-carrier adduct.
  • the hapten-carrier adduct is capable of initiating a humoral immune response, whereas the hapten itself would not elicit antibody production.
  • Nonlimiting examples of haptens are aniline, urushiol (a toxin in poison ivy), hydralazine, fluorescein, biotin, digoxigenin and dinitrophenol.
  • the target sequence encoding the target peptide that may be incorporated in the multitarget peptide may be obtained from bacteria belonging to genera, for example, Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio, Yersinia, or a combination thereof
  • the target sequence encoding the target peptide is obtained or derived from coronaviruses comprising SARS-CoV-1, MERS-CoV, SARS-CoV-2, OC43, HKU1, bat coronavirus, other betacoronavirus, or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof of such target sequences.
  • the target sequence encoding the target peptide includes, but not limited to, spike protein, membrane protein, envelope protein or nucleocapsid protein of coronaviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the target sequence encoding the target peptide includes spike protein or its fragment from a betacoronavirus.
  • Betacoronavirus is a genus within the subfamily Othrocoronavirinae of family Coronaviridae.
  • the International Committee on Taxonomy of Viruses (ICTV) has classified the genus Betacoronavirus into 5 subgenera viz., Embecovirus, Sarbecovirus, Merbecovirus, Nobecovirus and Hibecovirus. The first four of these subgenera were formerly known as lineages or subgroups A, B, C and D, respectively.
  • Betacoronaviruses have been of greater clinical importance as they have been found to cause outbreaks, for example, the 2002-2003 SARS outbreak caused by SARS- CoV-1 or SARS-CoV, the 2012 MERS outbreak caused by Middle East Respiratory Virus, and more recently the 2019-2020 COVID-19 pandemic caused by SARS-CoV-2.
  • the other betacoronaviruses known to infect human beings are HKU1, and OC43.
  • the genome of betacoronaviruses encodes 4 main structural proteins viz spike(S), membrane(M), envelope(E), and nucleocapsid(N) proteins.
  • the spike protein is the immunodominant protein among the major structural proteins. It contains two subunits, SI and S2.
  • the former is further divided into an N-terminal domain (NTD) and a C-terminal domain (CTD). Both or one of the domains may act as receptor binding domains (RBD) interacting with host cell receptors.
  • the RBD contains the receptor-binding motif (RBM).
  • the SI subunits are organized to form a trimeric structure. S2 subunit helps the virus to enter the host cell through membrane fusion.
  • S2 subunit contains fusion peptide (FP), heptad repeat 1 (HR1), central helix (C -helix), connector domain (CD), stem helix (SH), heptad repeat 2 (HR2), transmembrane domain and cytoplasmic domain (Dacon, Cherrelle etal. 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).
  • FP fusion peptide
  • HR1 heptad repeat 1
  • C -helix central helix
  • connector domain CD
  • SH stem helix
  • HR2 heptad repeat 2
  • the 51 subunit of the betacoronaviruses contains the receptor binding domain either on the N-terminal region or the C-terminal region.
  • RBD interact with the host cell receptors.
  • the RBD includes full length SI subunit of the spike protein or fragment thereof of a betacoronavirus, including mutant, derivative, or variant thereof that retains the ability to interact with the host cell receptor.
  • the receptor binding domain interacts with angiotensin-converting enzyme 2 (ACE2) receptor or dipeptidyl peptidase 4 (DPP4) receptor, 9-O-acetylated sialic acid (9-O-Ac-Sia) receptor, or combination thereof.
  • ACE2 angiotensin-converting enzyme 2
  • DPP4 dipeptidyl peptidase 4
  • the target sequence encoding the target peptide is obtained or derived from HSV-1, HSV-2, HCMV, VZV, EBV, HHV-6A, HHV-6B, HHV-7, HHV- 8 or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof of such target sequences.
  • Exemplary target peptides includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof: HAEMQNPVY (SEQ ID NO: 80);
  • the target peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the target peptide includes, but not limited to, F9 membrane protein of a poxvirus, H3L protein of a poxvirus, A4 protein of a poxvirus, A27 protein of poxvirus, A33 protein of a poxvirus, A56 protein of a poxvirus, B5 protein of a poxvirus, or LI protein of a poxvirus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • Flaviviridae consists 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 within the flavivirus and hepacivirus genera respectively.
  • 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 codon optimized sequences, fragments, mutants, or variants thereof of such target sequences.
  • the target peptide is encoded by a target sequence which may be either a DNA, an RNA or an mRNA.
  • alphaviruses e.g., Chikungunya (CHIKV), Ross River (RRV), Mayaro (MAYV), Semliki Forest (SFV), Sindbis (SINV), and O'nyong-nyong (ONNV)
  • CHCV Chikungunya
  • RRV Ross River
  • MAYV Mayaro
  • SFV Semliki Forest
  • Sindbis Sindbis
  • O'nyong-nyong O'nyong-nyong
  • EEEV Eastern
  • WEEV Western
  • VEEV Venezuelan equine encephalitis viruses
  • the target peptide includes, but not limited to, domain A, domain B, or domain C of E2 protein of alphaviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the target peptide includes, but not limited to capsid protein, or envelope protein such as El, E2, and E3 of chikungunya virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • Exemplary target peptides includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the target peptide is encoded by a target sequence which may be either a DNA, an RNA or an mRNA.
  • Retroviridae consists of enveloped single stranded RNA viruses. It constitutes a large family of viruses that predominantly infect both human and animal vertebrate hosts and causes wide spectrum of diseases ranging from malignancies to immune deficiencies and neurologic disorders. Retroviriade family is organized into several genera viz., alpharetrovirus, betraretrovirus, gammaretrovirus, deltaretrovirus, epsilonretrovirus, lentivirus and spumavirus. Of these genera, lentiviruses have been the subject of considerable interest the most prominent among them being the human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • the target sequence encoding the target peptide is obtained or derived from alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus, epsilonretrovirus, lentivirus, spumavirus, or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof of such target sequences.
  • the target peptide includes, but not limited to, gag, pol or env proteins of retroviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the target peptide includes, pl7 Gag , p24 Gag , p7 Gag , p6 Gag , gpl2 Env , gp41 Env , or pol proteins from lentiviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the target peptide includes, pl7 Gag , p24 Gag , p7 Gag , p6 Gag , gpl2 Env , gp41 Env , or pol proteins from human immunodeficiency virus (HIV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • HIV human immunodeficiency virus
  • Exemplary target peptides includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the target peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the target peptide is encoded by a target sequence which may be either a DNA, an RNA or an mRNA.
  • paramyxoviridae family consists of enveloped, single stranded RNA viruses. Diseases caused by these viruses continue to produce high mortality and morbidity across the world.
  • the family Paramyxoviridae is classified into two subfamilies, the paramyxovirinae and the penumovirinae.
  • the former contains seven genera: respirovirus, rubulavirus, Moribilivirus, Henipavirus, Aqiaparamyxovirus, Avulavirus, and Ferlavirus, while the latter contains two genera: Penumovirus and Metapenumovirus.
  • Mumps virus Mumps virus
  • PAV5 Parainfluenza virus type 5
  • HPIV2/4a/4b Human parainfluenza virus type 2, types 4a and 4b
  • Newcastle disease virus NDV
  • HPIV1/3 NDV
  • Nipah virus NiV
  • Measles virus Measles virus
  • HRSV Human respiratory syncytial virus
  • HMPV Human metapneumovirus
  • the target sequence encoding the target peptide is obtained or derived from Mumps virus (MuV), Parainfluenza virus type 5 (PIV5), Human parainfluenza virus type 2, types 4a and 4b (HPIV2/4a/4b), Newcastle disease virus (NDV), Human parainfluenza virus type 1 and type 3 (HPIV1/3), Nipah virus (NiV), Measles virus (MeV), Human respiratory syncytial virus A2, Bl, S2, (HRSV), or Human metapneumovirus (HMPV), or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof of such target sequences.
  • the target peptide includes, but not limited to, nucleocapsid protein, P protein, V protein, W protein, D protein, I protein, C protein, L protein, M protein, H (hemagglutinin) protein, HN (hemagglutinin-neuraminidase) protein, G protein, F protein, or a combination thereof, of Mumps virus (MuV), Parainfluenza virus type 5 (PIV5), Human parainfluenza virus type 2, types 4a and 4b (HPIV2/4a/4b), Newcastle disease virus (NDV), Human parainfluenza virus type 1 and type 3 (HPIV1/3), Nipah virus (NiV), Measles virus (MeV), Human respiratory syncytial virus A2, Bl, S2, (HRSV), Human metapneumovirus (HMPV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • Mumps virus Mumps virus
  • PV5 Parainflu
  • the target peptide includes, but not limited to, nucleocapsid protein, P protein, V protein, W protein, D protein, I protein, C protein, L protein, M protein, H (hemagglutinin) protein, HN (hemagglutinin-neuraminidase) protein, G protein, F protein of human respiratory syncytial virus A2, Bl, S2 (HRSV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • 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 A2, Bl, S2 (HRSV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • Exemplary target peptides includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the target peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the target peptide is encoded by a target sequence which may be either a DNA, an RNA or an mRNA.
  • Papillomaviridae are small non-enveloped, double stranded DNA viruses.
  • the most common genus in the family papillomaviridae is papillomavirus.
  • Human papillomaviruses are the most prominent papillomaviruses which are known to cause papillomas or warts, and are responsible for causing cervical cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer, and oropharyngeal cancer.
  • the target sequence encoding the target peptide is obtained or derived from papillomavirus, preferably human papillomavirus, including codon optimized sequences, fragments, mutants, or variants thereof of such target sequences.
  • the target peptide includes, but not limited to, LI (major capsid) protein, L2 (minor capsid) protein, of papillomavirus, preferably human papillomavirus, or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof.
  • the target peptide includes, LI (major capsid) protein of papillomavirus, preferably human papillomavirus, that has lost the ability to self-assemble into a virus like particle.
  • LI protein is modified or mutated such that it loses its ability to self-assemble into a virus like particle.
  • the target peptide includes, L2 (minor capsid) protein of papillomavirus, preferably human papillomavirus, including codon optimized sequences, fragments, mutants, or variants thereof.
  • the target peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the target peptide is encoded by a target sequence which may be either a DNA, an RNA or an mRNA.
  • the multitarget nucleic acid sequence and multitarget peptide includes selfassembling sequence and self-assembling peptide respectively.
  • the self-assembling sequence comprises of a sequence of nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes a self-assembling peptide.
  • the self-assembling sequence includes codon optimized sequences, fragments, mutants, variants or a combination thereof.
  • the self-assembling sequence is a DNA or an RNA or an mRNA.
  • self-assembling peptide may be a full-length protein or its fragment, mutants, or variant thereof.
  • the self-assembling peptide includes, but not limited to, lumazine synthase from Aquifex species, hepatitis B surface antigen (HBsAg) from Hepatitis B Virus, hepatitis B core antigen (HBcAg) from Hepatitis B virus, human papillomavirus LI (HPV LI) protein, matrix protein Ml from influenza A virus, ferritin, riboflavin synthase, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • HBsAg hepatitis B surface antigen
  • HBcAg hepatitis B core antigen
  • HPV LI human papillomavirus LI
  • the self-assembling peptide is a ferritin peptide.
  • Ferritin is one of the ubiquitous proteins found in nature. It is produced by all living organisms including archaea, bacteria, algae, higher plants, and animals. Each ferritin protein is generally composed of 24 subunits or peptides which self-assembles into a ferritin nanoparticle.
  • the multitarget nucleic acid sequence and multitarget peptide includes ferritin sequence and ferritin peptide respectively.
  • the ferritin sequence comprises of a sequence of nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes a ferritin peptide.
  • the ferritin sequence is a DNA or an RNA or an mRNA.
  • the self-assembling peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the self-assembling peptide is encoded by the self-assembling sequence which may be either a DNA, an RNA or an mRNA.
  • the linker peptide is a foldon.
  • a foldon comprises of a sequence of amino acids encoded by a foldon sequence.
  • the foldon sequence includes codon optimized sequences, fragments, mutants, variants or a combination thereof.
  • a foldon enables two or more homologous polypeptides to organise to form an oligomeric complex.
  • foldon also helps in orientation of a polypeptide such that the domains or epitopes on the target peptide are exposed or displayed for interaction or communication with cells or biomolecules or immune system.
  • the resulting polypeptides may comprise either a target peptide, a linker peptide and a self-assembling peptide or a linker peptide, target peptide, linker peptide and a self-assembling peptide or a combination thereof.
  • the polypeptide in addition to these peptides, may also have some residues (amino acids) of cleavage peptide. Any cleavage peptide that is susceptible to the action of cellular proteases or a cleavage peptide that has the ability to undergo self-cleavage can be employed in accordance with the present disclosure.
  • the signal peptide shares at least 70 % identity with the sequence described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the signal peptide is encoded by a signal sequence which may be either a DNA, an RNA or an mRNA.
  • the multitarget nucleic acid sequence is obtained through a single IVT process. In some embodiments, the multitarget nucleic acid sequence obtained through single IVT process is an mRNA.
  • the mRNA may be unmodified or modified or a combination of both.
  • the modification may be in the nucleobase of the nucleotide, or sugar moiety of the nucleotide, or the phosphate of the nucleotide.
  • unmodified mRNA may comprise naturally occurring nucleosides, for example, adenosine, guanosine, cytidine, and uridine.
  • mRNA may comprise one or more modified nucleosides, for example, adenosine analog, guanosine analog, cytidine analog, or uridine analog.
  • the multitarget peptide comprises multiple repeats of polypeptide comprising either a target peptide, a linker peptide, and a self-assembling peptide or a linker peptide, target peptide, a linker peptide, and a self-assembling peptide, or a combination thereof, interspersed with cleavage peptide (see illustration in figures).
  • the total number of polypeptides present in a multitarget peptide may be up to 100 polypeptides.
  • one or more polypeptides in the multitarget peptide may have identical target peptides (homologous polypeptides).
  • one or more polypeptides in the multitarget peptide may have different target peptides (heterologous polypeptides).
  • a multitarget peptide as described herein is encoded by a multitarget nucleic acid sequence as described herein.
  • Each multitarget peptide comprises two or more polypeptides, wherein some or all polypeptides comprises either a target peptide, a linker peptide, and a self-assembling peptide or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide or a combination thereof.
  • the polypeptides are connected with each other through a cleavage peptide.
  • the multitarget peptide includes a signal peptide upstream (N-terminus) of one or more polypeptides.
  • the multitarget peptide includes a signal peptide upstream (N-terminus) of each of all or some polypeptides. In some embodiments, the multitarget peptide optionally includes a signal peptide upstream (N-terminus) of each polypeptide. In some embodiments, the multitarget peptide includes a signal peptide upstream (N-terminus) of some polypeptides. In some embodiments, the multitarget peptide includes a signal peptide upstream (N-terminus) of all polypeptides.
  • the signal peptide transports the multitarget peptide to golgi body or golgi apparatus.
  • the cellular proteases act on the cleavage sites present in the cleavage peptides or the cleavage peptide undergoes self-cleavage and cleaves the multitarget peptide into individual polypeptides comprising either a target peptide, a linker peptide, and a selfassembling peptide, or a linker peptide, a target peptide, a linker peptide, and a selfassembling peptide, or a signal peptide, a target peptide, a linker peptide, and a selfassembling peptide, or a signal peptide, a linker peptide, a target peptide, a linker peptide, and a selfassembling peptide, or a signal peptide, a linker peptide, a target peptide, a linker peptide, and a self-
  • the linker peptide may be an amino acid linker, a foldon, a scaffold, or a combination thereof.
  • polypeptides may be homologous polypeptides.
  • a polypeptide nanoparticle is formed by self-assembly of two or more homologous polypeptides, or two or more heterologous polypeptides, or one or more oligomeric complexes, or their combination.
  • a polypeptide nanoparticle comprises homologous polypeptides, heterologous polypeptides, oligomeric complex, or a combination thereof.
  • the polypeptide nanoparticles may be symmetrical, non- symmetrical, asymmetrical, or a combination thereof.
  • the polypeptide nanoparticles may be icosahedral, helical, spherical, rod-like or a combination thereof.
  • the polypeptide nanoparticles may be enveloped or nonenveloped or a combination thereof.
  • the polypeptide nanoparticles may be single layered or multi-layered or a combination thereof.
  • the polypeptide nanoparticle may comprise at least 2 or up to 100 polypeptides.
  • the polypeptide nanoparticle may comprise polypeptides between 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-99.
  • the polypeptide nanoparticle may comprise at least 2 or up to 100 homologous polypeptides.
  • the polypeptide nanoparticle may comprise two or more oligomeric complexes such that total number of polypeptides in the polypeptide nanoparticle are not more than 100.
  • the polypeptide nanoparticle may comprise some homologous polypeptides and some heterologous polypeptides such that the total number of polypeptides in the polypeptide nanoparticle are not more than 100.
  • the polypeptide nanoparticle may comprise some homologous polypeptides and some oligomeric complexes such that the total number of polypeptides in the polypeptide nanoparticle are not more than 100.
  • the polypeptide nanoparticle may comprise some heterologous polypeptides and some oligomeric complexes such that the total number of polypeptides in the polypeptide nanoparticle are not more than 100. In some embodiments, the polypeptide nanoparticle may comprise some homologous polypeptides, some heterologous polypeptides, some oligomeric complexes, or their combination such that the total number of polypeptides in the polypeptide nanoparticle are not more than 100.
  • Lipid nanoparticles (LNP) composition Lipid nanoparticles (LNP) composition
  • the multitarget nucleic acid sequences as described herein may be encapsulated in a lipid nanoparticle composition.
  • the lipid nanoparticle composition comprises lipid components, ionizable polymer, or a combination thereof and a multitarget nucleic acid sequence as described herein.
  • the lipid nanoparticle composition comprises lipid components such as a cationic lipid, a phospholipid, a sterol, a PEG-lipid and a multitarget nucleic acid sequence as described herein.
  • the lipid nanoparticle composition comprises an ionizable polymer, a cationic lipid, a phospholipid, a sterol, a PEG-lipid and a multitarget nucleic acid sequence as described herein.
  • Cationic lipid refers to a lipid that has a net positive charge at a selected pH.
  • Cationic lipids generally consist of a hydrophilic head group that carries the charge and a hydrophobic tail.
  • Exemplary cationic lipid for use in the lipid nanoparticle compositions include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N- dimethylammonium bromide(DDAB); N-(2,3dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP); 3-(N — (N',N'-dimethylaminoethane)- carbamoyl)cholesterol (DC-Chol), N-(l-(2, 3 -di oleoyloxy )propyl)N-2- (sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (DOSPA), dioctade
  • Cationic lipids with amine head group are preferred cationic lipids.
  • the amine group can be at primary, secondary or tertiary position.
  • the cationic lipid may comprise one (monoamine) or more (polyamine) such amine groups.
  • cationic lipid and/or ionizable lipid or imparting the cationic lipid the ability to behave as an ionizable lipid are well known in the art (W02005121348; W02009127060; W02009086558; W02010042877; W02010144740; WO2011075656; WO2017049245; WO2017075531; WO2018118102; WO2015199952; Reynier P. et al. Journal of Drug Targeting (2004) 12: 25-38; Sabnis, Staci et al. Molecular Therapy (2016) 26: 1509-1519).
  • the proportion of cationic lipid present in the lipid nanoparticle compositions is from about 25 mol % to about 70 mol % or any range therein.
  • Phospholipid includes a lipid containing a hydrophilic head with a phosphate group and a hydrophobic tail composed of fatty acid chains attached to a glycerol or sphingosine backbone.
  • Exemplary phospholipids for use in the lipid nanoparticle compositions include, but are not limited to, l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2- di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),
  • the proportion of phospholipid present in the lipid nanoparticle compositions is from about 2 mol % to about 30 mol %, from about 2 mol % to about 28 mol %, from about 2 mol % to about 26 mol %, from about 2 mol % to about 24 mol %, from about 2 mol % to about 22 mol %, from about 2 mol % to about 20 mol %, from about 3 mol % to about 19 mol %, from about 3 mol % to about 18 mol %, from about 3 mol % to about 17 mol %, from about 3 mol % to about 16 mol %, from about 3 mol % to about 15 mol %, from about 3 mol % to about 14 mol %, from about 3 mol % to about 13 mol %, from about 3 mol % to about 12 mol %, or any range therein.
  • Sterol Lipid nanoparticle composition disclosed herein may include sterol and/or sterol derivatives.
  • sterol as used herein include, but not limited to, cholesterol, sitosterol, fecosterol, ergosterol, campesterol, stigmasterol or their derivatives.
  • lipid nanoparticle composition comprises cholesterol and/or cholesterol derivatives.
  • PEG-lipid, pegylated lipid, PEG linked lipid, PEG conjugated lipid, PEG- lipid conjugate, PEG modified lipid have been used interchangeably to mean polyethylene glycol linked to a lipid moiety.
  • the lipid moiety may be linked directly to the PEG molecule or through a linker.
  • a PEG-lipid comprises a PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and/or PEG-modified cholesterol, and/or mixtures thereof.
  • the methods of making PEG-lipid are well known to persons skilled in the art, see for example, US20030077829; US2005008689; US5885613; US7404969; W02005026372;
  • PEG-lipid is selected from mPEG-Dimyristoyl glycerol (mPEG-DMG), mPEG-N,N-Ditetradecylacetamide (mPEG-DTA or ALC0159), mPEG- Cholesterol (mPEG-CLS), mPEG-DSPE, mPEG-DMPE, mPEG-DPPE, mPEG-DLPE, mPEG-DOPE, mPEG-DPPC, mPEG-DSPC, l,2-Distearoyl-sn-Glycero-3- Phosphoethanolamine with conjugated methoxyl polyethylene glycol) (mPEG-DSPE), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) or mixtures thereof.
  • mPEG-DMG mPEG-Dimyristoyl glycerol
  • the PEG moiety of the PEG-lipid may comprise an average molecular weight ranging from 0.5 kDa to 10 kDa.
  • the PEG-lipid has an average molecular weight of about 0.5 kDa to 5 kDa, about 0.5 kDa to 4 kDa, 0.5 kDa to 3 kDa, 0.5 kDa to 2 kDa.
  • the PEG-lipid has an average molecular weight of about 0.5 kDa to about 2 kDa.
  • the proportion of PEG-lipid present in the lipid nanoparticle compositions may be from about 0.2 mol % to about 2.0 mol % or any range therein.
  • the proportion of cellulose derivatives or dialdehyde cellulose derivatives or combination thereof present in the lipid nanoparticle compositions is from about 1 mol % to about 25 mol %, preferably about 1 mol % to about 20 mol %, most preferably about 1 mol % to about 15 mol % or any range therein.
  • a nucleic acid comprising a plurality of polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises a target sequence, a linker sequence, and a self-assembling sequence, wherein each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequence of the plurality.
  • betacoronavirus is a SARS-CoV-1, a SARS-CoV-2, a MERS-CoV, an OC43, a HKUl, a bat coronavirus, other betacoronavirus, or a combination thereof.
  • lipid nanoparticle composition according to paragraph 63, wherein the sterol is present in an amount from 30 mol percent to about 65 mol percent.
  • a method of treating or preventing a disease comprising administrating to a subject in need thereof the nucleic acid according to any one of claims 1-62.
  • lipid nanoparticle composition according to any one of paragraphs 63-71 in the manufacture of a medicament for the treatment or prevention of a disease in a subject.
  • a multitarget peptide comprising two or more polypeptides, wherein some or all polypeptides comprises either a target peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide or a combination thereof, wherein one polypeptide is connected to another polypeptide by a cleavage peptide, wherein the multitarget peptide includes a signal peptide on the aminoterminus of one or more of the polypeptides.
  • the multitarget peptide according to paragraph 77, wherein the signal peptide may be present on the amino-terminus of all or some polypeptides.
  • the target peptide is obtained or derived from a prokaryote, a eukaryote, a unicellular organism, a multicellular organism, a virus, a bacterium, a fungus, a protozoan, a worm, a mycoplasma, an animal, a human or a combination thereof.
  • the multitarget peptide according to paragraph 86 wherein the virus is selected from the family comprising picomaviride, calciviridae, astroviridae, togaviridae, flaviviridae, coronaviridae, arteriviridae, rhabndoviridae, filoviridae, paramyxoviridae, bornaviridae, orthomyxoviridae, bunyaviridae, arenaviridae, reoviridae, retroviridae, polyomaviridae, herpesviridae, poxviridae, papillomaviridae, hepadnaviridae, adenoviridae, parvoviridae, hepeviridae, circoviridae or a combination thereof.
  • the multitarget peptide according to paragraph 93 wherein the target peptide is a receptor binding domain, a fusion peptide, a stem helix of the spike protein, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the multitarget peptide according to paragraph 77 wherein the target peptide is a glycoprotein B, a glycoprotein C, a glycoprotein D, a glycoprotein E, a glycoprotein K, a glycoprotein L, or a glycoprotein M of a herpes simplex virus 1 (HSV-1) or a herpes simplex virus 2 (HSV-2), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants or variants thereof.
  • HSV-1 herpes simplex virus 1
  • HSV-2 herpes simplex virus 2
  • the multitarget peptide according to paragraph 77 wherein the target peptide is a glycoprotein B, a glycoprotein H, a glycoprotein L, a glycoprotein M, or a glycoprotein N of a human cytomegalovirus (HCMV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • HCMV human cytomegalovirus
  • the multitarget peptide according to paragraph 77 wherein the target peptide is a glycoprotein B, a glycoprotein H, a glycoprotein L, a glycoprotein M, a glycoprotein N, a glycoprotein 42, a glycoprotein 350 of an Epstein-Barr virus (EBV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • EBV Epstein-Barr virus
  • the target peptide is a F9 membrane protein of a poxvirus, a H3L protein of a poxvirus, an A4 protein of a poxvirus, an A27 protein of poxvirus, an A33 protein of a poxvirus, an A56 protein of a poxvirus, a B5 protein of a poxvirus, or a LI protein of a poxvirus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the multitarget peptide according to paragraph 77 wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of flaviviruses, hepaciviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • a capsid protein a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of flaviviruses, hepaciviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a Japanese encephalitis virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the multitarget peptide according to paragraph 77 wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a zika virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • a capsid protein a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a zika virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the multitarget peptide according to paragraph 77 wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a yellow fever virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • a capsid protein a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a yellow fever virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the multitarget peptide according to paragraph 77 wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a hepatitis C virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • a capsid protein a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a hepatitis C virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • the multitarget peptide according to paragraph 77 wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a dengue virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • a capsid protein such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5
  • the multitarget peptide according to paragraph 77 wherein the target peptide is a capsid protein, or an envelope protein such as El, E2 and E3 protein of an alphaviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
  • a Polypeptide nanoparticle comprising at least 2 or up to 100 polypeptides according to any one of paragraphs 1 to 62 or 77-115.
  • IVT was performed following standard procedure described in Hi ScribeTM T7 High Yield RNA Synthesis Kit (Catalogue no. E2040S, New England BioLabs Inc.). Briefly, IVT mix containing four ribonucleotide triphosphates (ATP, UTP, GTP, CTP), NEB T7 buffer, NEB T7 enzyme mix, CleanCap® Reagent AG (3' OMe) (Catalogue no, N-7413, TriLink BioTechnologies), and linearized plasmid DNA was taken in a PCR tube containing nuclease free sterile water. The mixture was incubated for 37 °C for about 2 hours. This was followed by DNase I (Catalogue no.
  • Spent media was collected by aspiration, and cells were collected with ice-cold lx PBS (1 mL) with hard pipetting. The cells were centrifuged at 4000 g for 5 min at 4 °C. Excess PBS was aspirated. The cells were suspended gently in 300 pL of lx NETN lysis buffer with inhibitors and kept on ice for 20 min. The lysate was centrifuged at 16,000 g for 20 min at 4 °C. The supernatant was aspirated into microtubes.
  • the efficacy of the multitarget nucleic acid sequence is evaluated by pseudovirus neutralization assays well known to persons skilled in the art.
  • the method typically involves incubating the pseudovirus in the presence of different concentrations of immunised serum containing the antibody of interest (i.e., antibodies produced against the target peptide) and adding this mixture to the cells and incubating it further to measure luminescence to determine inhibitory or neutralization titre.
  • the second polynucleotide sequence consisted of a target sequence (encoding receptor binding domain (RBD) of SRAS-CoV-1), a linker sequence (glycine serine linker sequence encoding glycine serine linker), and a self-assembling sequence (ferritin sequence encoding ferritin).
  • the third polynucleotide sequence consisted of a target sequence (encoding receptor binding domain (RBD) of MERS-CoV), a linker sequence (glycine serine linker sequence encoding glycine serine linker), and a self-assembling (ferritin sequence encoding ferritin).
  • Table 2 shows sequence of the multitarget peptide encoded by the multitarget nucleic acid sequence - trivalent RBD construct of example 2.
  • Amino acid sequence of different peptides present in the multitarget peptide encoded by the multitarget nucleic acid sequence of example 1 is separately identified in:
  • a western blot was performed on cell lysate, obtained in the previous step, using Jess instrument (an automated western blot system Catalogue no. 004-650, ProteinSimple, Bio-Techne).
  • the cell lysate was diluted and combined with 1 part 5x fluorescent master mix (component of Separation Module compatible with Jess, Catalogue no. SM-W001, ProteinSimple, Bio-Techne) and heated for 5 min at 95 °C. After protein denaturation, the sample was mixed with luminol-S and peroxide as per manufacturer protocol (Separation Module, Catalogue no. SM-W001, ProteinSimple, Bio-Techne).

Abstract

The present disclosure relates generally to nucleic acids comprising a plurality of polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises a target sequence, a linker sequence, and a self-assembling sequence, or a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence, or a combination thereof, wherein each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequences of the plurality.

Description

Multitarget Vaccines and Therapeutics
RELATED APPLICATIONS
This application claims the benefit of priority to U.S. Provisional Patent Application serial number 63/435,977, filed December 29, 2022, the contents of which are hereby incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
The present disclosure relates to multitarget nucleic acid sequences, multitarget peptides, and polypeptide nanoparticle and their compositions for vaccine and therapeutic purpose.
BACKGROUND
Variety of diseases are caused by infectious agents, such as bacteria, viruses, fungi, protozoans, and worms. Despite the advances in vaccination and other therapeutic interventions, the economic burden of eight major diseases (HIV/AIDS, malaria, measles, hepatitis, dengue fever, rabies, tuberculosis, and yellow fever) alone has been estimated to be up to US$8 trillion, with more than 156 million life years lost for the year 2016 alone (Armitage, Catherine Nature (2021) 598: S9).
Many pathogens such as influenza virus, HIV, human papillomavirus, SARS-CoV- 2, Streptococcus pneumoniae, Neisseria meningitidis, Neisseria gonorrhoeae, Trypanosoma brucei etc., have been ever evolving and adapting to escape the currently available vaccines and therapeutics. Such evolutionary changes leading to antigenic drift or serotype shift renders the current interventions ineffective. Targeting multiple facets of a disease or multiple pathogens through a single drug or therapeutic has always been a challenge.
Combination vaccines have been traditionally used to target multiple pathogens or variations within a pathogen (Skibinski, David AG et al. Journal of Global Infectious Diseases (2011) 3:63-72; Alderson, MarkR. etal. Microorganisms (2021) 9: 771).
Displaying antigens or epitopes over virus like particles or nanoparticles have been tried to increase the depth and breadth of immune response against emerging strains or variants of a pathogen (Tretyakova, Irina et al. Virology (2013) 442: 67-73; Schellenbacher, Christina et al. Journal of Virology (2009) 83: 10085-10095; Liu, Xingjian et al. (available on the World Wide Web at doi.org/10.1101/2021.02.05.428685; Tumban, Ebenezer et al. PLoS ONE (2012) 7: e49715). These methods used conventional approach of expressing the particles recombinantly in bacterial, insect or mammalian expression systems and purifying the particles to be used for immunization.
More recently, mRNA based vaccines have emerged as promising alternative to classical approaches to vaccine development. However, current mRNA based vaccines are still inadequate to address the emerging strains or variants of a pathogen. For example, the antibody titres elicited by SARS-CoV-2 vaccine against the emerging and antigenically divergent SARS-CoV-2 variants have been found to be lower, and wane over time suggesting decreased effectiveness of the vaccines targeted against a single variant/strain of the virus. Bivalent vaccines encapsulating different mRNAs, each targeting a specific variant of SARS-CoV-2, are undergoing clinical trials to increase the breadth of coverage (Chalkias, Spyros et al. New England Journal of Medicine (2022) 387: 1279-1291; Chalkias, Spyros et al. Nature Medicine (2022) available on the World Wide Web at doi.org/10.1038/s41591-022-02031-7). Multivalent vaccines containing as many as eight different mRNAs have been co-encapsulated in lipid nanoparticle for delivery (Chivukula, Sudha et al. NPJ Vaccines (2021) 6: 153; WO2022264109). Such strategies require multiple in vitro transcription (IVT) process to be performed complicating the manufacturing process and quick deployment of vaccines during pandemics. Strategies that can simplify the manufacturing process and induce more potent, durable, and/or broader immune response are necessary to combat pandemics and evolving pathogens. Thus, it would be advantageous to develop vaccines and therapeutics that provide protection against infection over a broad range of diseases, including multiple facets of a single disease or different strains and/or variations of a pathogen.
SUMMARY
Accordingly, the present disclosure relates to a nucleic acid comprising a plurality of polynucleotide sequences, wherein some or all polynucleotide sequence of the plurality comprises either a target sequence, a linker sequence, and a self-assembling sequence or a linker sequence, a target sequence, a linker sequence and a self-assembling sequence or a combination thereof. In some embodiments, each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence. In some embodiments, the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequences of the plurality. In some embodiments, the target sequence, the linker sequence, and the self-assembling sequence or the linker sequence, the target sequence, the linker sequence and the self-assembling sequence are in 5' to 3' order.
In another aspect, provided herein is a nucleic acid comprising a plurality of polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises a target sequence, a linker sequence, and a self-assembling sequence. In some embodiments, each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence. In some embodiments, the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequences of the plurality. In some embodiments, the target sequence, the linker sequence, and the self-assembling sequence are in 5' to 3' order.
In another aspect, provided herein is a nucleic acid comprising a plurality of polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence. In some embodiments, each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence. In some embodiments, the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequences of the plurality. In some embodiments, the linker sequence, the target sequence, the linker sequence and the self-assembling sequence are in 5' to 3' order.
In another aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein some or all polypeptides of the plurality comprises either a target peptide, a linker peptide, and a self-assembling peptide or a linker peptide, a target peptide, a linker peptide and a self-assembling peptide or a combination thereof. In some embodiments, each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide. In some embodiments, the nucleic acid further encodes a signal peptide on the amino-terminus of one or more of the polypeptides of the plurality. In some embodiments, the target peptide, the linker peptide, and the self-assembling peptide or the linker peptide, the target peptide, the linker peptide and the self-assembling peptide are in N-terminus to C- terminus order. In another aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein each polypeptide of the plurality comprises a target peptide, a linker peptide, and a self-assembling peptide. In some embodiments, each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide. In some embodiments, the nucleic acid further encodes a signal peptide on the aminoterminus of one or more of the polypeptides of the plurality. In some embodiments, the target peptide, the linker peptide, and the self-assembling peptide are in N-terminus to C- terminus order.
In another aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein each polypeptide of the plurality comprises a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide. In some embodiments, each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide. In some embodiments, the nucleic acid further encodes a signal peptide on the amino-terminus of one or more of the polypeptides of the plurality. In some embodiments, the linker peptide, the target peptide, the linker peptide and the selfassembling peptide are in N-terminus to C- terminus order.
In some embodiments, total number of the polynucleotide sequences is not more than 100. In some embodiments, total number of the polynucleotide sequences is between 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-99. In some embodiments, the nucleic acid is a DNA or an RNA. In some embodiments, the RNA is an mRNA. In some embodiments, the linker sequence encodes a linker peptide. In some embodiments, the linker peptide is an amino acid linker, foldon, scaffold or a combination thereof. In some embodiments, the amino acid linker comprises 2 to 49 amino acids. In some embodiments, the amino acid linker is glycine serine linker, glycine proline linker, glycine threonine linker, alanine serine linker, any combination of two amino acids, or a combination thereof.
In some embodiments, the linker peptide has an amino acid sequence of any one of SEQ ID NOs: 262-299, 330, and 350. In some embodiments, the self-assembling sequence encodes a self-assembling peptide. In some embodiments, the self-assembling peptide is lumazine synthase from Aquifex species, hepatitis B surface antigen (HBsAg) from Hepatitis B Virus, hepatitis B core antigen (HBcAg) from Hepatitis B virus, human papillomavirus LI (HPV LI) protein, matrix protein Ml from influenza A virus, ferritin, riboflavin synthase, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the ferritin comprises of ferritin subunit or ferritin peptide. In some embodiments, the ferritin peptide is derived from Helicobacter pylori ferritin. In some embodiments, the self-assembling peptide has an amino acid sequence of any one of SEQ ID NOs: 254-261, 331 and 333.
In some embodiments, the cleavage sequence encodes one or more cleavage peptide. In some embodiments, the one or more cleavage peptides are optionally connected to each other by a linker. In some embodiments, the cleavage peptide is a golgi specific cleavage peptide or self-cleaving peptide. In some embodiments, the cleavage peptide has an amino acid sequence of any one of SEQ ID NOs: 300-311 and 347-349.
In some embodiments, the signal sequence encodes a signal peptide. In some embodiments, the signal peptide is present on the amino-terminus of the first polypeptide. In some embodiments, the nucleic acid further encodes a second signal peptide on the amino-terminus of all or some polypeptides. In some embodiments, the signal peptide has an amino acid sequence of any one of SEQ ID NOs: 312-329.
In some embodiments, the target sequence encodes a target peptide. In some embodiments, the target peptide is encoded by a codon optimized nucleic acid sequence, or fragments, mutants, or variants thereof. In some embodiments, the target peptide is obtained from a prokaryote, a eukaryote, a unicellular organism, a multicellular organism, a virus, a bacterium, a fungus, a protozoan, a worm, a mycoplasma, an animal, a human or a combination thereof. In some embodiments, the virus is selected from the family comprising picomaviride, calciviridae, astroviridae, togaviridae, flaviviridae, coronaviridae, arteriviridae, rhabndoviridae, filoviridae, paramyxoviridae, bomaviridae, orthomyxoviridae, bunyaviridae, arenaviridae, reoviridae, retroviridae, polyomaviridae, herpesviridae, poxviridae, papillomaviridae, hepadnaviridae, adenoviridae, parvoviridae, hepeviridae, circoviridae or a combination thereof. In some embodiments, the bacterium is selected from the genus comprising Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio, Yersinia, or a combination thereof In some embodiments, the virus is selected from the family consisting of coronaviridae, herpesviridae, poxviridae, flaviviridae, togaviridae, retroviridae, paramyxoviridae, or a combination thereof. In some embodiments, the virus is alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus, torovirus or a combination thereof.
In some embodiments, the betacoronavirus is SARS-CoV-1, SARS-CoV-2, MERS- CoV, OC43, HKU1, bat coronavirus, other betacoronavirus or a combination thereof. In some embodiments, the target peptide is a spike protein, a membrane protein, an envelope protein or a nucleocapsid protein of coronaviruses. In some embodiments, the target peptide is a receptor binding domain, fusion peptide, or stem helix of the spike protein, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants or variants thereof. In some embodiments, the target peptide is a receptor binding domain obtained or derived from a betacoronavirus comprising SARS-CoV-1, SARS- CoV-2, MERS-CoV, OC43, HKU1, bat coronavirus, other betacoronavirus, or a combination thereof. In some embodiments, the target peptide is glycoprotein B, glycoprotein C, glycoprotein D, glycoprotein E, glycoprotein K, glycoprotein L, and glycoprotein M, of herpes simplex virus 1 (HSV-1) or herpes simplex virus 2 (HSV-2), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants or variants thereof.
In some embodiments, the target peptide is glycoprotein B, glycoprotein H, glycoprotein L, glycoprotein M, or glycoprotein N of human cytomegalovirus (HCMV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is glycoprotein B, glycoprotein C, glycoprotein H, or glycoprotein L of varicella-zoster virus (VZV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is glycoprotein B, glycoprotein H, glycoprotein L, glycoprotein M, glycoprotein N, glycoprotein 42, or glycoprotein 350 of Epstein-Barr virus (EBV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is F9 membrane protein of a poxvirus, H3L protein of a poxvirus, A4 protein of a poxvirus, A27 protein of poxvirus, A33 protein of a poxvirus, A56 protein of a poxvirus, B5 protein of a poxvirus, or LI protein of a poxvirus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of flaviviruses or hepaciviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is capsid protein, membrane protein, envelope protein, or non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Japanese encephalitis virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is capsid protein, membrane protein, envelope protein, or non-structural proteins (such as NSl, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of zika virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is capsid protein, membrane protein, envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of yellow fever virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is capsid protein, membrane protein, envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of west nile virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is capsid protein, membrane protein, envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of hepatitis C virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is capsid protein, membrane protein, envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of dengue virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide is capsid protein, or envelope protein such as El, E2 and E3 protein of alphaviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is domain A, domain B, or domain C of the E2 protein of alphaviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is capsid protein, or envelope protein such as El, E2 and E3 protein of chikungunya virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is gag, pol and env proteins of retroviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide is pl7Gag, p24Gag, p7Gag, p6Gag, gpl2Env, gp41Env, or pol proteins from lentiviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is pl7Gag, p24Gag, p7Gag, p6Gag, gpl2Env, gp41Env, or pol proteins from human immunodeficiency virus (HIV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide is nucleocapsid protein, P protein, V protein, W protein, D protein, I protein, C protein, L protein, M protein, H (hemagglutinin) protein, HN (hemagglutinin-neuraminidase) protein, G protein or F protein of Mumps virus (MuV), Parainfluenza virus type 5 (PIV5), Human parainfluenza virus type 2, types 4a and 4b (HPIV2/4a/4b), Newcastle disease virus (NDV), Human parainfluenza virus type 1 and type 3 (HPIV1/3), Nipah virus (NiV), Measles virus (MeV), Human respiratory syncytial virus A2, Bl, S2, (HRSV), or Human metapneumovirus (HMPV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is nucleocapsid protein, P protein, V protein, W protein, D protein, I protein, C protein, L protein, M protein, H (hemagglutinin) protein, HN (hemagglutinin-neuraminidase) protein, G protein or F protein of human respiratory syncytial virus A2, Bl, S2 (HRSV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is early protein for example (El, E2, E4, E5, E6, or E7), or late protein LI (major capsid protein) or L2 (minor capsid protein) of human papillomavirus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants, thereof. In some embodiments, the target peptide is a major capsid protein (LI) of a human papillomavirus that has lost the ability to self-assemble, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide is a minor capsid protein (L2) of a human papillomavirus, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide has an amino acid sequence of any one of SEQ ID NOs: 1-253, 334-337, 338-346, and 353-388. In another aspect, provided herein is a lipid nanoparticle composition comprising cationic lipid, a phospholipid, a sterol, a PEG-lipid, and the nucleic acid according to any of the preceding embodiments or paragraphs. In some embodiments, the cationic lipid comprises an ionizable lipid. In some embodiments, the ionizable lipid is present in an amount from 25 mol percent to 70 mol percent. In some embodiments, the phospholipid is present in an amount from 2 mol percent to about 30 mol percent. In some embodiments, the sterol is present in an amount from 30 mol percent to about 65 mol percent. In some embodiments, the PEG-lipid is present in an amount from 0.2 mol percent to about 2.0 mol percent. In some embodiments, the lipid nanoparticle composition additionally comprises an ionizable polymer. In some embodiments, the ionizable polymer is present in an amount from 1 mol percent to 25 mol percent. In some embodiments, the ionizable polymer is selected from the group comprising a chitosan, a cellulose derivative, a poly-L-lysine, a poly-L-glutamic acid, and/or their derivatives or a combination thereof.
In another aspect, provided herein is a method of treating or preventing a disease, comprising administrating to a subject in need thereof the nucleic acid disclosed herein.
In another aspect, provided herein is a method of treating or preventing a disease, comprising administrating to a subject in need thereof the multitarget peptide disclosed herein.
In another aspect, provided herein is a method of treating or preventing a disease, comprising administrating to a subject in need thereof the lipid nanoparticle composition disclosed herein.
In another aspect, provided herein is use of the 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 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 is a multitarget peptide encoded by the nucleic acid disclosed herein.
In another aspect, provided herein is a multitarget peptide comprising two or more polypeptides, wherein some or all polypeptides comprises either a target peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide, or a combination thereof, wherein one polypeptide is connected to another polypeptide by a cleavage peptide, wherein the multitarget peptide includes a signal peptide upstream (amino-terminus) of one or more of the polypeptides. In some embodiments, the signal peptide may be present on the amino-terminus of the first polypeptide. In some embodiments, the signal peptide may be present on the aminoterminus of some or each of the polypeptides.
In another aspect, provided herein is a polypeptide nanoparticle comprising at least 2 or up to 100 polypeptides disclosed herein. In some embodiments, the polypeptides are homologous polypeptides, heterologous polypeptides, oligomeric complex or combination thereof. In some embodiments, the polypeptide nanoparticle is icosahedral, helical, spherical, rod-like or combination thereof.
In another aspect, provided herein is a nucleic acid sequence comprising a multitarget nucleic acid sequence comprising two or more polynucleotide sequences, wherein some or all polynucleotide sequences comprises either a target sequence, a linker sequence, and a self-assembling sequence, or a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence, or a combination thereof, wherein one polynucleotide sequence is connected to another polynucleotide sequence by a cleavage sequence, wherein the multitarget nucleic acid sequence includes a signal sequence upstream of one or more of the polynucleotide sequences. In some embodiments, the linker sequence connects signal sequence with the first polynucleotide sequence. In some embodiments, the signal sequence may be present upstream of all or some of the polynucleotide sequences. In some embodiments, signal sequence is present upstream of the first polynucleotide sequence. In some embodiments, signal sequence is present upstream of all polynucleotide sequences. In some embodiments, linker sequence connects target sequence with the self-assembling sequence in a polynucleotide sequence. In some embodiments, one linker sequence connects cleavage sequence with the target sequence and another linker sequence connects the target sequence with the self-assembling sequence in a polynucleotide sequence. In some embodiments, the multitarget nucleic acid sequence is a DNA or an RNA. In some embodiments, the multitarget nucleic acid sequence is an mRNA. In some embodiments, the multitarget nucleic acid sequence encodes a multitarget peptide. In some embodiments, the multitarget nucleic acid is encapsulated in a lipid nanoparticle composition. In some embodiments, the multitarget nucleic acid sequence is synthesized through a single in vitro transcription (IVT) process. In some embodiments, the disclosure relates to a nucleic acid sequence encoding a multitarget peptide described herein.
In some embodiments, the disclosure relates to a nucleic acid sequence encoding a multitarget peptide comprising two or more polypeptides, wherein some or all polypeptides comprises either a target peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide, or a combination thereof, wherein one polypeptide is connected to another polypeptide by a cleavage peptide, wherein the multitarget peptide includes a signal peptide upstream of one or more of the polypeptides. In some embodiments, signal peptide may be present upstream of all or some of the polypeptides. In some embodiments, signal peptide may be present upstream of the first polypeptide. In some embodiments, signal peptide may be present upstream of all polypeptides.
In some embodiments, the disclosure also relates to a multitarget peptide comprising two or more polypeptides, wherein some or all polypeptides comprises either a target peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide, or a combination thereof, wherein one polypeptide is connected to another polypeptide by a cleavage peptide, wherein the multitarget peptide includes a signal peptide upstream (amino-terminus) of one or more of the polypeptides. In some embodiments, signal peptide may be present upstream (aminoterminus) of all or some of the polypeptides. In some embodiments, signal peptide may be present upstream (amino-terminus) of the first polypeptide. In some embodiments, signal peptide may be present upstream (amino-terminus) of all the polypeptides.
In some embodiments, the linker peptide connects signal peptide with the first polypeptide in a multitarget peptide. In some embodiments, the linker peptide connects target peptide with the self-assembling peptide in a polypeptide. In some embodiments, one linker peptide connects cleavage peptide with the target peptide and another linker peptide connects the target peptide with the self-assembling peptide in a polypeptide. In some embodiments, the multitarget peptide comprise homologous polypeptides. In some other embodiments, the multitarget peptide comprise heterologous polypeptides. In some embodiments, multitarget peptide comprise homologous polypeptides, or heterologous polypeptides. In some embodiments, the disclosure relates to a polypeptide nanoparticle comprising one or more homologous polypeptides, one or more heterologous polypeptides, one or more oligomeric complex, or a combination thereof. In some embodiments, the homologous polypeptides, heterologous polypeptides, oligomeric complex may comprise either a target peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide, or a combination thereof. In some embodiments, the polypeptides in a polypeptide nanoparticle may also have some residues of cleavage peptide.
In some embodiments, the disclosure relates to a polypeptide nanoparticle formed from the self-assembly of two or more polypeptides, wherein some or all polypeptides comprises either a target peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide or a combination thereof. In some embodiments, the polypeptides in the polypeptide nanoparticle may also have some residues of cleavage peptide. In some embodiments, the polypeptide nanoparticle comprises of homologous polypeptides, heterologous polypeptides, oligomeric complexes, or a combination thereof.
In some aspects, provided herein is a multitarget nucleic acid sequences described herein, encapsulated in a lipid nanoparticle composition. In some aspects, the lipid nanoparticle composition comprises a cationic lipid, a phospholipid, a sterol, a PEG lipid and a multitarget nucleic acid sequence described herein.
In some other aspects, the lipid nanoparticle composition comprises an ionizable polymer, a cationic lipid, a phospholipid, a sterol, a PEG-lipid and the multitarget nucleic acid sequence described herein.
In some aspects, provided herein is a method of treating or preventing a disease, comprising administering to a subject in need thereof the multitarget nucleic acid sequence as described herein.
In some aspects, provided herein is a method of treating or preventing a disease, comprising administering to a subject in need thereof the lipid nanoparticle composition comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid and the multitarget nucleic acid sequence as described herein.
In some aspects, provided herein is a method of treating or preventing a disease, comprising administering to a subject in need thereof the lipid nanoparticle composition comprising an ionizable polymer, a cationic lipid, a phospholipid, a sterol, a PEG-lipid and the multitarget nucleic acid sequence as described herein.
In some aspects, the disclosure relates to use of a lipid nanoparticle composition comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and the multitarget nucleic acid sequence as described herein in the manufacture of a medicament for the treatment or prevention of a disease in a subject.
In some embodiments, the target sequence is obtained from a prokaryote or a eukaryote, a unicellular organism or a multicellular organism, a virus, a bacterium, a fungus, a protozoan, a worm, a mycoplasma, an animal, human, or a combination thereof, including the codon optimized sequences, fragments, variants, or mutants of such target sequences. In some embodiments, the target sequence may be modified or unmodified. In some embodiments, the target sequence is obtained from viruses belonging to the families, for example, picornaviride, calciviridae, astroviridae, togaviridae, flaviviridae, coronoviridae, arteriviridae, rhabndoviridae, filoviridae, paramyxoviridae, bomaviridae, orthomyxoviridae, bunyaviridae, arenaviridae, reoviridae, retroviridae, polyomaviridae, herpesviridae, poxviridae, papillomaviridae, hepadnaviridae, adenoviridae, parvoviridae, hepeviridae, circoviridae, or a combination thereof. In some embodiments, the target sequence is obtained from bacteria belonging to genera, for example, Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio, Yersinia, or a combination thereof In some embodiments, the target sequence encodes a target peptide. In some embodiments, the target peptide is an antigen, its fragment, or mutant thereof. In some embodiments, the target sequence or target peptide may be modified or unmodified. In some embodiments, the target peptide regulates or modulates cellular functions. In some embodiments, the target peptide may have immunostimulatory or immunomodulatory effect.
In some embodiments, the self-assembling sequence encodes a self-assembling peptide. In some embodiments, the self-assembling peptide includes, but not limited to, lumazine synthase from Aquifex species, hepatitis B surface antigen (HBsAg) from Hepatitis B Virus, hepatitis B core antigen (HBcAg) from hepatitis B virus, human papillomavirus LI (HPV LI) protein from human papillomavirus, matrix protein (Ml) from influenza A virus, ferritin peptide, or a combination thereof, including their fragments, mutants, or variants thereof. In some embodiments, the ferritin peptide is a Helicobacter pylori ferritin or its fragment, mutant, or variant thereof.
In some embodiments, the linker sequence encodes a linker peptide. In some embodiments, the linker peptide connects the target peptide with the self-assembling peptide in a polypeptide. In some embodiments, the linker peptide connects the signal peptide with the first polypeptide. In some embodiments, one linker peptide connects cleavage peptide with the target peptide and another linker peptide connects target peptide with the self-assembling peptide in a polypeptide. The linker peptide may be an amino acid linker, a foldon, a scaffold or a combination thereof.
In some embodiments, the cleavage sequence encodes a cleavage peptide. The cleavage peptide connects one polypeptide with another polypeptide, for example, adjacent polypeptide. The cleavage peptide carries a cleavage site. In some embodiments, the cleavage peptide facilitates the action of cellular proteases to cleave the multitarget peptide into individual polypeptides. In some embodiments, the cleavage peptide self cleaves into individual polypeptides. In some embodiments, the cleavage peptide comprises two or more cleavage peptides (for example, cleavage peptide- 1, cleavage peptide-2 and so on), optionally connected via a linker. In some embodiments, the cleavage peptide may 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 golgi specific proteases.
In some embodiments, the signal sequence encodes a signal peptide. The signal peptide is present upstream (amino-terminus) of one or more polypeptides in a multitarget peptide. In some embodiments, the signal peptide is present upstream (amino-terminus) of the first polypeptide. In some embodiments, the signal peptide is present upstream (aminoterminus) of some polypeptides. In some embodiments, the signal peptide is present upstream (amino-terminus) of all polypeptides. In some embodiments, the signal peptide transports the multitarget peptide to cell organelles. In some embodiments, the signal peptide transports the multitarget peptide to golgi body or golgi apparatus. In some embodiments, the signal peptide is a golgi targeting signal peptide.
In some aspects, the present disclosure also includes a method of transforming a cell with the multitarget nucleic acid sequence as described herein.
BRIEF DESCRITPITON OF THE DRAWINGS
Figure 1 - shows representative schematic illustration of multitarget nucleic acid sequence wherein each polynucleotide sequence (PS) comprises a target sequence (TS), a linker sequence (LS), and a self-assembling sequence (SAS) or a linker sequence (LS), a target sequence (TS), a linker sequence (LS), and a self-assembling sequence (SAS) . Multiple polynucleotide sequences are connected through a cleavage sequence (CS) such that between any two polynucleotide sequences there is present a cleavage sequence. The multitarget nucleic acid sequence has a signal sequence (SS) upstream of the first polynucleotide sequence. The letter ‘n’ in figure 1 represents any number between 1 to 98. The multitarget nucleic acid sequence may additionally have 5’ cap and 3’ poly(A) tail. This multitarget nucleic acid sequence encodes corresponding multitarget peptide depicted in Figure 2.
Figure 2 - shows representative schematic illustration of multitarget peptide wherein each polypeptide (PP) comprises a target peptide (TP), a linker peptide (LP), and a self-assembling peptide (SAP) or a linker peptide (LP), a target peptide (TP), a linker peptide (LP), and a self-assembling peptide (SAP). Multiple polypeptides are connected through a cleavage peptide (CP) such that between any two polypeptides there is present a cleavage peptide. The multitarget peptide has a signal peptide (SP) on the N-terminus of the first polypeptide. The letter ‘n’ represents any number between 1 to 98.
Figure 3a - shows a western blot of cell lysate (lanes 4 and 5) showing expression of a multitarget peptide using polyclonal anti-spike antibody against SARS-CoV-2.
Figure 3b - shows a western blot of supernatant (lane 5) confirming the cleavage of multitarget peptide using polyclonal anti-spike RBD antibody against SARS-CoV-2.
Figure 4 - shows estimation of polypeptide nanoparticles in cell lysate and supernatant by ELISA. There is a decrease in protein concentration in the cell lysate at 48 h with a corresponding increase in protein concentration of supernatant at 48 h. These results indicate increase in the amount of polypeptide nanoparticle with the progression of time in the supernatant.
Figure 5 - shows TEM image showing the formation of polypeptide nanoparticle.
Figure 6 - shows a western blot of cell lysate (lane 2) showing expression of target peptides encoded by multitarget nucleic acid sequence - trivalent RBD construct of example 2 using polyclonal anti-spike antibody against SARS-CoV-2. Lanes 1 and 3 are protein ladder and negative control respectively.
Figures 7a, 7b, and 7c - show ELISA data showing generation of antibodies (IgG) against the respective target peptides encoded by the multitarget nucleic acid sequence - trivalent RBD construct of example 2.
Figures 8a, 8b, and 8c - show pseudovirus neutralization of the antibodies generated against the respective target peptides encoded by the multitarget nucleic acid sequence - trivalent RBD construct of example 2. Figure 9 - shows a western blot of cell lysate (lane 3) showing expression of target peptides encoded by multitarget nucleic acid sequence - pentavalent RBD construct of example 3 using polyclonal anti-spike antibody against SARS-CoV-2. Lanes 1 and 2 are protein ladder and negative control respectively.
DESCRIPTION
Unless defined otherwise, technical, and scientific terms used herein have the same meaning as commonly understood by one of person skill in the art. Some of the terms are defined briefly here below; the definitions should not be construed in a limiting sense.
The singular forms “a”, “an” and “the” as used in the specification also include plural aspects unless the context dictates otherwise. Similarly, any singular term used in the specification also mean plural or vice versa unless the context dictates otherwise. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more.
It must be noted that the words “comprising” or any of its form such as “comprise” or “comprises”, “having” or any of its forms such as “have” or “has”, “including” or any of its forms such as “include” or “includes”, or “containing” or any of its forms such as “contains” or “contains” are open-ended and do not exclude additional unrecited elements or method steps.
Wherever any quantity or range is stated one skilled in the art will recognize that quantity or range within 10 or 20 percent of the stated values can also be expected to be appropriate and reasonable and included within the scope of the invention.
Unless otherwise defined herein, scientific, and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skilled in the art. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, protein, adjuvant, pharmaceutical biotechnology, and biopharmaceutical manufacturing described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. The term “composition” or “formulation” has been used interchangeably to mean a lipid nanoparticle composition comprising a multitarget nucleic acid sequence, and lipid components such as cationic lipid, phospholipid, sterol, and PEG-lipid. The composition may optionally contain an ionizable polymer. The composition may additionally contain pharmaceutical carriers or excipients, such as but not limited to, buffering agents, stabilizers, tonicity modifiers, surfactants, chelating agents, salts, anti-oxidants, diluents, and/or preservatives or combinations thereof.
The term "therapeutic", “therapeutic agent”, “prophylactic”, “prophylactic agent’, or drug has been used interchangeably to mean a compound (such as multitarget nucleic acid sequence) or composition (such as a lipid nanoparticle composition described herein) having a biological effect or a combination of biological effects that prevents, inhibits, eliminates or prevents the progression of a disease or other aberrant biological processes in a subject, for example, an animal or human.
The term “preventing” is art-recognized, and when used in relation to a condition, such as an infection is well understood in the art, and includes administration of a composition, which reduces the frequency or severity, or delays the onset, of one or more symptoms of the medical condition in a subject relative to a subject who does not receive the composition. Thus, the prevention of a condition, such as an infection, includes, for example, the reduction of the frequency or severity of one or more symptoms of the medical condition in a population of patients receiving a therapy relative to a control population that did not receive the therapy, e.g., by a statistically and/or clinically significant amount. Similarly, the prevention of an infection includes reducing the likelihood that a patient receiving a therapy will develop the infection or related symptoms, relative to a patient who does not receive the therapy.
The term “molar ratio”, “mol ratio”, “molar percent”, “mol percent”, “molar %”, or “mol %” have been used interchangeably to mean number of moles of a component expressed as percentage relative to total moles of all lipid components (such as cationic lipid, phospholipid, sterol and PEG-lipid) and, if present, ionizable polymer component(s) present in the lipid nanoparticle compositions described herein. For example, 50 mol % of cationic lipid means, 50 mol % of cationic lipid is present in the lipid nanoparticle composition and other lipids components constitute the remaining 50% such that the total amount of all the lipid components together constitute 100 mol %. Alternatively, 50 mol % of cationic lipid also means, 50 mol % of cationic lipid present in the lipid nanoparticle composition and other lipids components and ionizable polymer components together constitute the remaining 50 mol % such that the total amount of all the lipid components and ionizable polymer components constitute 100 mol %.
The terms “antibody” and “antibodies” have been used interchangeably herein and means any antibody or antibody fragment (whether produced naturally or recombinantly) which retains antigen binding activity. This includes a monoclonal or polyclonal antibody, a single chain antibody, a Fab fragment of a monoclonal or polyclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bispecific antibody, a multispecific antibody, or a nanobody.
The term “buffer” as used herein means those agents that maintains the pH of a solution in a desired range.
The term “cell” as used herein means a single cell or a population of cells or plurality of cells.
The term “biologically effective amount” or “therapeutically effective amount” as used herein means an amount of an agent, for example, a therapeutic, drug, therapeutic agent, prophylactic agent, diagnostic agent, composition, etc., that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, prevent, diagnose, improve symptoms of, and/or delay the onset of the infection, disease, disorder, and/or condition. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient.
As used herein, the term “treating” or “treatment” includes reducing, arresting, or reversing the symptoms, clinical signs, or underlying pathology of a condition to stabilize or improve a subject's condition or to reduce the likelihood that the subject’s condition will worsen as much as if the subject did not receive the treatment. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.
The term “subject” as used herein refers to a living mammal and may be interchangeably used with the term “patient”. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs, and the like. The term does not denote a particular age or gender.
As used herein, an individual “at risk” of developing a particular disease, disorder, or condition may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment methods described herein. “At risk” denotes that an individual has one or more risk factors, which are measurable parameters that correlate with development of a particular disease, disorder, or condition, as known in the art. An individual having one or more of these risk factors has a higher probability of developing a particular disease, disorder, or condition than an individual without one or more of these risk factors.
The term “disease” as used herein, means an interruption, cessation, or disorder of body function, system, or organ. Non limiting examples of disease include malignant diseases, autoimmune diseases, inherited diseases, metabolic disorders, or infectious diseases.
As used herein, administration “conjointly” with another compound or composition includes simultaneous administration and/or administration at different times. Conjoint administration also encompasses administration as a co-formulation or administration as separate compositions, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration.
The terms “multitarget nucleic acid sequence” or “multitargeted nucleic acid sequence” have been used interchangeably to mean two or more polynucleotide sequences wherein some or all polynucleotide sequences comprises either a target sequence, a linker sequence, and a self-assembling sequence, or a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence, or a combination thereof, wherein one polynucleotide sequence is connected to another polynucleotide sequence by a cleavage sequence, wherein the multitarget nucleic acid sequence includes a signal sequence upstream of one or more 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 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 cleavage sequence with the target sequence and another linker sequence connects the target sequence with the self-assembling sequence in a polynucleotide sequence. In some embodiments, the linker sequence connects signal sequence with the polynucleotide sequence. As illustrated in figure 1 multitarget nucleic acid sequence may comprise multiple repeats of polynucleotide sequences wherein each polynucleotide sequence may comprise either a target sequence, a linker sequence, and a self-assembling sequence, or a linker sequence, a target sequence, a linker sequence and a self-assembling sequence, or a combination thereof, such that total number of polynucleotide sequences in a multitarget nucleic acid sequence are not more than 100. In some embodiments, the linker sequence connects signal sequence with the first polynucleotide sequence. In some embodiments, the signal sequence is present upstream of each of some or all of the polynucleotide sequences. The multitarget nucleic acid sequence encodes multitarget peptide.
The terms “multitarget peptide” or “multitargeted peptide” has been used interchangeably to mean two or more polypeptides wherein some or all polypeptides comprises either a target peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a target peptide, a linker peptide and a self-assembling peptide, or a combination thereof, wherein one polypeptide is connected to another polypeptide by a cleavage peptide, wherein the multitarget peptide includes a signal peptide upstream (amino-terminus) of one or more polypeptides. In some embodiments, the signal peptide is present on the amino-terminus of the first polypeptide. In some embodiments, the signal peptide is present on the amino-terminus of some polypeptides. In some embodiments, the signal peptide is present on the amino-terminus of each of the polypeptides. In some embodiments, the polypeptide may comprise a target peptide, a linker peptide, and a selfassembling peptide. In some embodiments, the polypeptide may comprise a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide. Thus, in some embodiments, one linker peptide connects cleavage sequence with the target sequence and another linker peptide connects the target peptide with the self-assembling peptide in a multitarget peptide. In some embodiments, the multitarget peptide may either comprise a target peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide, or a combination thereof, such that the total number of polypeptides in a multitarget peptide are not more than 100. In some embodiments, the linker peptide connects signal peptide with the polypeptide. In some embodiments, the signal peptide is present on the amino-terminus of each of some or all polypeptides. The multitarget peptide may comprise homologous polypeptides or heterologous polypeptides.
The term “polynucleotide sequence” as used herein means a sequence of nucleotides that encodes a polypeptide.
The terms "protein" or "peptide" have been used interchangeably herein and mean a polymer of amino acids linked through peptide bonds, but does not imply any specific length. The term also includes fusion proteins, muteins, analogs or modified forms.
The term “polypeptide” as used herein means a sequence of amino acids that comprise either a target peptide, a linker peptide, and a self-assembling peptide or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide. In some embodiments, the polypeptide comprises a target peptide, a linker peptide, and a selfassembling peptide. In some embodiments, the polypeptide comprises a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide. In some embodiments, the polypeptide may have some residues (amino acids) of cleavage peptide. In some embodiments, the polypeptide may have signal peptide.
The term “target sequence” as used herein means a sequence of nucleotides that encodes a target peptide.
The term “target peptide” as used herein means a sequence of amino acids that has immunostimulatory or immunomodulatory effect. Target peptide also means a peptide of interest. In some embodiments, target peptide is an antigen. In some embodiments, the target peptide in two or more polypeptides may be identical i.e., homologous polypeptides. In some other embodiments, the target peptides in two or more polypeptides may be different i.e., heterologous polypeptides.
The term “signal sequence” as used herein means a sequence of nucleotides that encodes a signal peptide.
The term “signal peptide” as used herein means a sequence of amino acids that transports the multitarget peptide to specific cell organelles. In some embodiments the signal peptide transports the multitarget peptide to golgi apparatus or golgi body. The signal peptide is present on the N-terminus (amino-terminus) of one or more polypeptides. In some embodiments, the signal peptide is present on the N-terminus of some or all polypeptides. In some embodiments, the signal peptide is present on the N-terminus of the first polypeptide. In some embodiments, the signal peptide is present on the N-terminus of some polypeptides. In some embodiments, the signal peptide is present on the N-terminus of all polypeptides. In some embodiments, the signal peptide is encoded by signal sequence. In some embodiments, the signal peptide is a golgi targeting signal peptide.
The term “cleavage sequence” as used herein means a sequence of nucleotides that encodes a cleavage peptide.
The term “cleavage peptide” as used herein means a sequence of amino acids that facilitates the action of cellular proteases to cleave the multitarget peptide into individual polypeptides or self cleaves into individual polypeptides. The cleavage peptide is present between any two polypeptides. It connects one polypeptide with another polypeptide, for example, adjacent polypeptide. The cleavage peptide carries one or more cleavage sites. In some embodiments, the cleavage peptide is a substrate for proteases. In some embodiments, cleavage peptide undergoes self-cleavage to result in individual polypeptides. In some embodiments, the cleavage peptide is a substrate for golgi specific proteases. In some embodiments, the cleavage peptide comprises one or more cleavage peptides, for example, cleavage peptide- 1, cleavage peptide-2 and so on. In some embodiments, the cleavage peptide optionally comprises a linker peptide between two cleavage peptides. In some embodiments, the cleavage peptide may self-cleave into individual polypeptides or may be cleaved by the action of cellular proteases.
The term “linker sequence” as used herein means a sequence of nucleotides that encodes a linker peptide.
The term “linker peptide” or “peptide linker” have been used interchangeably to mean a sequence of amino acids that either connects the target peptide with the selfassembling peptide, connects the signal peptide with the target peptide, connects the cleavage peptide with the target peptide, connects signal peptide with the polypeptide, or connects two cleavage peptides. In some embodiments, the linker peptide connects the signal peptide with the polypeptide. In some embodiments, the linker peptide connects the target peptide with the self-assembling peptide in a polypeptide. In some embodiments, one linker peptide connects cleavage peptide with the target peptide and another linker peptide connects target peptide with the self-assembling peptide in a polypeptide. In some embodiments, one linker peptide connects cleavage peptide with the target peptide and another linker peptide connects target peptide with the self-assembling peptide. In some embodiments, one linker peptide connects cleavage peptide with the target peptide and another linker peptide connects the signal peptide with the target peptide. In some embodiments, the linker peptide connects two cleavage peptides. In some embodiments, the linker peptide is an amino acid linker, a foldon, a scaffold or a combination thereof.
The term “amino acid linker sequence” as used herein means a sequence of nucleotides that encodes an amino acid linker.
The term “amino acid linker” as used herein means a sequence of amino acids that provides structural integrity to polypeptide such that the components of the polypeptide remain, as far as possible, in their native or stable conformation. In some embodiments, amino acid linker also helps in orientation of a polypeptide such that the domains or epitopes on the target peptide are exposed or displayed for interaction or communication with cells or biomolecules or immune system in the absence of foldon or scaffold. In some embodiments, the amino acid linker connects two cleavage peptides. Some of the nonlimiting examples of amino acid linkers includes, glycine serine linker, glycine proline linker, glycine threonine linker, alanine serine linker, any combination of two amino acids or a combination thereof. In some embodiments, amino acid linker is about 2-49 amino acid long.
The term “glycine serine linker sequence” as used herein means a sequence of nucleotides that encodes a glycine serine linker.
The term “glycine serine linker” as used herein means a sequence of amino acid comprising one or more glycine (G) and serine (S) in any combinations without any preference of order or limitation on number of appearances of either glycine or serine. In some embodiments, the glycine serine linker is few amino acids in length to several amino acids in length.
The term “foldon sequence” as used herein means a sequence of nucleotides that encodes a foldon.
The term “foldon” as used herein means a sequence of amino acids that enables two or more homologous polypeptides to organise to form an oligomeric complex. In some embodiments, the foldon also helps in orientation of a polypeptide such that the domains or epitopes on the target peptide are exposed or displayed for interaction or communication with cells or biomolecules or immune system.
The term “scaffold sequence” as used herein means a sequence of nucleotides that encodes a scaffold. The term “scaffold” as used herein means a sequence of amino acids that provides structural and/or functional integrity or support to the target peptide and helps in orientation of target peptide such that the domains or epitopes of the target peptide are exposed or displayed for interaction or communication with cells or biomolecules or immune system.
The term “oligomeric complex” as used herein means a complex formed by two or more homologous polypeptides. In some embodiments, the oligomeric complex has at least two homologous polypeptides, at least three homologous polypeptides, at least four homologous polypeptides, at least five homologous polypeptides, or at least six homologous polypeptides and so on.
The term “self-assembling sequence” as used herein means a sequence of nucleotides that encodes a self-assembling peptide.
The term “self-assembling peptide” as used herein means a sequence of amino acids that enables the polypeptides to self-assemble into polypeptide nanoparticle.
The term "self-assembly” or “self-assemble” or “self-assembling” has been used interchangeably to means the ability of polypeptides to undergo multimerization to form a polypeptide nanoparticle. In some embodiments, the polypeptide nanoparticle may have at least two polypeptides (dimer or 2-mer), at least three polypeptides (trimer or 3-mer), at least four polypeptides (tetramer or 4-mer), at least five polypeptides (pentamer or 5-mer), at least six polypeptides (hexamer or 6-mer), at least seven polypeptides (heptamer or 7- mer), at least eight polypeptides (octamer or 8-mer), and so on. In some embodiments, the polypeptide nanoparticle is up to 100-mers. In some embodiments, hydrogen bonds, disulfide bonds, hydrophobic interactions, electrostatic interactions, and/or van der Walls forces combine to maintain self-assembled structure.
The term “multimerization” as used herein means association of two or more units of homologous polypeptides or heterologous polypeptides, or oligomeric complex or their combination.
The term “polypeptide nanoparticle” as used herein means a nanoparticle formed by self-assembly of polypeptides. In some embodiments, the polypeptide nanoparticle comprises of two or more homologous polypeptides, or two or more heterologous polypeptides, or one or more oligomeric complexes or a combination thereof.
The term “homologous polypeptides” as used herein means polypeptides in a multitarget peptide that have identical target peptides. For example, if two polypeptides in the multitarget peptide have identical target peptides they are considered to be homologous polypeptides.
The term “heterologous polypeptide” as used herein means polypeptides in a multitarget peptide that have different target peptides. For example, if two polypeptides in the multitarget peptide have different or non-identical target peptides, they are considered to be heterologous polypeptides.
The term “fragment” as used herein, whether in the context of a nucleic acid, nucleotide, protein, polypeptide, or peptide, means any length of the nucleic acid, protein, polypeptide, or peptide sequence except the full length of the respective nucleic acid, protein, polypeptide, or peptide sequence.
The term “variant” as used herein, whether in the context of a nucleic acid, nucleotide, protein, polypeptide, or peptide sequence, means homologs, orthologs, paralogs, mutants or analogs of respective nucleic acid, protein, polypeptide, or peptide sequence.
The term “mutant” as used herein, whether in the context of a nucleic acid, nucleotide, protein, polypeptide, or peptide sequence, means a sequence which is not a wild type sequence. A mutant is also understood to mean a nucleic acid, nucleotide, protein, polypeptide, or peptide sequence that carries a mutation. The term “mutation” as used herein means, a change or modification in the sequence of nucleic acid or amino acid in comparison to a reference sequence and includes insertion, deletion, substitution, or a combination thereof.
Multitarget nucleic acid sequence and multitarget peptide
In the present disclosure, a multitarget nucleic acid sequence includes two or more polynucleotide sequences wherein some or all polynucleotide sequences comprises either a target sequence, a linker sequence, and a self-assembling sequence, or a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence, wherein one polynucleotide sequence is connected to the another polynucleotide sequence by a cleavage sequence, wherein the multitarget nucleic acid sequence includes a signal sequence upstream of one or more polynucleotide sequences. In some embodiments, the signal sequence may be present upstream of all or some polynucleotide sequences. In some embodiments, signal sequence is present upstream of the first polynucleotide sequence. In some embodiments, the signal sequence is present upstream of some polynucleotide sequences. In some embodiments, signal sequence is present upstream of each of the polynucleotide sequences. In some embodiments, the polynucleotide sequence comprises a target sequence, a linker sequence, and a self-assembling sequence. In some embodiments, the polynucleotide sequence comprises a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence. In some embodiments, multitarget nucleic acid sequence may comprise one polynucleotide sequence comprising a target sequence, a linker sequence and a self-assembling sequence, and another polynucleotide sequence comprising a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence. In some embodiments, the multitarget nucleic acid sequence may either comprise a target sequence, a linker sequence and a self-assembling sequence, or a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence or a combination thereof, such that the total number of polynucleotide sequences in a multitarget nucleic acid sequences are not more than 100. In some embodiments, the linker sequence connects signal sequence with the polynucleotide sequence. In some embodiments, one linker sequence connects cleavage sequence with the target sequence and another linker sequence connects the target sequence with the self-assembling sequence in a polynucleotide sequence. Some exemplary illustrations of multitarget nucleic acid sequences are provided in figures 1 and their representative encoded multitarget peptides are provided in figures 2 respectively.
The term “nucleic acid” as used herein means a polymer comprising two or more nucleotides for example, deoxyribonucleotides or ribonucleotides, either in an unmodified or modified form. The nucleic acid may be either single stranded or double stranded, linear or circular. The term nucleic acid also encompasses fragments, variants, mutants or codon optimized sequences of deoxyribonucleotides or ribonucleotides.
The term "nucleotide” as used herein means a ribonucleotide or deoxyribonucleotide. If the term nucleotide is used in the context of RNA, it refers to ribonucleotide, and if it is used in the context of DNA, it refers to deoxyribonucleotide.
In some embodiments, the multitarget nucleic acid sequence is a DNA, an RNA or an mRNA. The multitarget nucleic acid sequence may be few nucleotides long to several thousand nucleotides long.
Deoxyribonucleic acid (DNA)
The term “deoxyribonucleic acid” or “DNA” has been used interchangeably herein and means a polymer of deoxyribonucleotides. The DNA may be either single stranded or double stranded, linear, or circular. In some embodiments, the multitarget nucleic acid sequence is a DNA. In some embodiments, the DNA encodes a multitarget peptide described herein.
Ribonucleic acid (RNA)
The term “ribonucleic acid” or “RNA” has been used interchangeably herein and means a polymer of ribonucleotides. The RNA may be either single stranded or double stranded, linear, or circular. The term RNA also includes messenger RNA (mRNA). In some embodiments, the multitarget nucleic acid sequence is an mRNA.
In some embodiments, the mRNA encodes a multitarget peptide as described herein.
In some embodiments, the mRNA may be unmodified or modified or a combination of both. The modification may be in the nucleobase of the nucleotide, or sugar moiety of the nucleotide, or the phosphate of the nucleotide.
In some embodiments, mRNA is produced using recombinant expression system, or chemically synthesized or obtained through in vitro transcription. In some embodiments, the mRNA is obtained through single in vitro transcription (IVT) process.
In some embodiments, the mRNA is circular. In other embodiments, the mRNA is linear.
In some embodiments, the mRNA is self-amplifying or self-replicating. Selfamplifying or self-replicating mRNA as used herein means an mRNA that self-replicate upon delivery into the cells. Such mRNAs typically contain a replicase sequence, usually derived from an alphavirus, which enables amplification of the original strand of mRNA encoding the protein of interest upon delivery into the cells (Beissert, Tim et al. Molecular Therapy (2020) 28: 119-128).
Target Sequence and target peptide
The multitarget nucleic acid sequence and the multitarget peptide includes target sequence and target peptide respectively. In some embodiments, the target sequence is a DNA or an RNA. In another embodiment, the target sequence is an mRNA. The target sequence may be modified or unmodified. The target sequence includes codon optimized sequences, fragments, mutants, variants, or combination thereof. Target sequence may be obtained from a prokaryote or a eukaryote, a unicellular organism or a multicellular organism, a virus, a bacterium, a fungus, a protozoan, a worm, a mycoplasma, an animal, a human, or a combination thereof. In some embodiments, the target sequence is a sequence of nucleotides that encodes a target peptide.
In some embodiments, the target peptide comprises of a sequence of amino acids that regulates or modulates cellular functions. The term “cellular functions” as used herein means various cellular or biological processes such as, but not limited to, biosynthesis, cell division, cell cycle regulation, cellular metabolism, ion transport, absorption, secretion, homeostasis, replication, transcription, translation, cell signalling, endocytosis, exocytosis, phagocytosis, trogocytosis, pyroptosis, apoptosis, DNA replication, DNA repair, protein synthesis, gene regulation, cell repair, cell growth, cell differentiation, cellular trafficking, cell proliferation, metabolic pathways etc.
The terms “regulate” or “modulate” or “regulation” or “modulation” have been used interchangeably herein and means an act of controlling a cellular or biological process or to exert a modifying or controlling influence on cellular or biological process.
In some aspects, the target peptide is capable of performing one or more functions such as, but not limited to, immunostimulation, immunomodulation, etc. In some embodiments, the target peptide means a peptide of interest. In some embodiments, target peptide is an antigen. In some embodiments, the target peptide is identical in two or more polypeptides, for example homologous polypeptides. In some embodiments, the target peptide is different in two or more polypeptides, for example as in heterologous polypeptides.
In some embodiments, the target peptide may be few amino acids long to several hundred amino acids long.
In some embodiments, the antigen may be, but not limited to, those derived from 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, alphafetoprotein, BCL-2, Bcr-Abl, BING-4, CEA, CPSF, CT, cyclin DIEp-CAM, EphA2, EphA3, ELF-2, FGF-5, G250, Gonadotropin Releasing Hormone, HER-2, intestinal carboxyl esterase (iCE), IL13Ralpha2, 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 1, TRAG-3, TRP-1, TRP-2, TERT, or WT1; those derived from a virus, such as Cowpoxvirus, Vaccinia virus, Pseudocowpox virus, Human herpesvirus 1, Human herpesvirus 2, Cytomegalovirus, Human adenovirus A-F, Polyomavirus, Human papillomavirus, Parvovirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Human immunodeficiency virus, Orthoreo virus, Rotavirus, Ebolavirus, parainfluenza virus, influenza virus (e.g., H5N1 influenza virus, influenza A virus, influenza B virus, influenza C virus), Measles virus, Mumps virus, Rubella virus, Pneumovirus, Human respiratory syncytial virus, Rabies virus, California encephalitis virus, Japanese encephalitis virus, Hantaan virus, Lymphocytic choriomeningitis virus, Epstein-Barr Virus (EBV), Coronavirus (e.g., MERS-CoV, SARS-CoV-1, SARS-CoV-2, OC43, HKU1, bat coronavirus, other betacoronavirus), Enterovirus, Rhino virus, Poliovirus, Norovirus, Flavivirus, Dengue virus, West Nile virus, Yellow fever virus and varicella; those derived from a bacterium, such as Anthrax (Bacillus anthracis), Brucella, Bordetella pertussis, Candida, Chlamydia pneumoniae, Chlamydia psittaci, Cholera, Clostridium botulinum, Cocci di oides immitis, Cryptococcus, Diphtheria, Escherichia coli 0151 : H7, Enterohemorrhagic Escherichia coli, Enterotoxigenic Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Legionella, Leptospira, Listeria, Meningococcus, Mycoplasma pneumoniae, Mycobacterium, Pertussis, Pneumonia, Salmonella, Shigella, Staphylococcus, Streptococcus pneumoniae and Yersinia enterocolitica,' or those derived from a protozoa, e.g., of the genus Plasmodium (Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi, or a combination thereof.
The antigen may be an allergen derived from, without limitation, cells, cell extracts, proteins, polypeptides, peptides, peptide mimics of polysaccharides and other molecules, such as small molecules, lipids, glycolipids, and carbohydrates of plants, animals, fungi, insects, food, drugs, dust, and mites. Allergens include but are not limited to environmental aeroallergens; plant pollens (e.g., ragweed/hayfever); weed pollen allergens; grass pollen allergens; Johnson grass; tree pollen allergens; ryegrass; arachnid allergens (e.g., house dust mite allergens); storage mite allergens; Japanese cedar pollen/hay fever; mold/fungal spore allergens; animal allergens (e.g., dog, guinea pig, hamster, gerbil, rat, mouse, etc., allergens); food allergens (e.g., crustaceans; nuts; citrus fruits; flour; coffee); insect allergens (e.g., fleas, cockroach); venoms: (Hymenoptera, yellow jacket, honey bee, wasp, hornet, fire ant); bacterial allergens (e.g., streptococcal antigens; parasite allergens such as Ascaris antigen); viral antigens; drug allergens; hormones (e.g., insulin); enzymes (e.g., streptokinase); and drugs or chemicals capable of acting as incomplete antigens or haptens (e.g., the acid anhydrides and the isocyanates). Where a hapten is used in a composition of the disclosure, it may be attached to a carrier to form a hapten-carrier adduct. The hapten-carrier adduct is capable of initiating a humoral immune response, whereas the hapten itself would not elicit antibody production. Nonlimiting examples of haptens are aniline, urushiol (a toxin in poison ivy), hydralazine, fluorescein, biotin, digoxigenin and dinitrophenol.
In other embodiments, the antigen is an antigen associated with a disease where it is desirable to sequester the antigen in circulation, such as for example an amyloid protein (e.g., Alzheimer's disease).
In some embodiments, the target sequence encoding the target peptide in the multitarget peptide may be obtained from viruses belonging to the families, for example, Coronaviridae, picornaviride, calciviridae, astroviridae, togaviridae, flaviviridae, , 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 target sequence encoding the target peptide that may be incorporated in the multitarget peptide may be obtained from bacteria belonging to genera, for example, Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio, Yersinia, or a combination thereof
Coronaviridae
Members of the family Coronaviridae are large, enveloped, single stranded RNA viruses with genomes ranging from 25 to 32 kb and virions of 118 to 140 nm in diameter. The family Coronaviridae include the following genera viz., alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus and torovirus. Most of the coronaviruses are responsible for causing mild respiratory infections. However, some of the coronaviruses have been responsible for deadly outbreaks, for example, severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1), Middle Eastern respiratory syndrome coronavirus (MERS-CoV), and most recently SARS-CoV-2. In some embodiments, the target sequence encoding the target peptide is obtained or derived from coronaviruses comprising SARS-CoV-1, MERS-CoV, SARS-CoV-2, OC43, HKU1, bat coronavirus, other betacoronavirus, or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof of such target sequences.
All coronaviruses encode four major structural proteins, the spike protein (S), the membrane protein (M), the envelope protein (E), and the nucleocapsid protein (N). These proteins or their fragments can serve as effective target peptides in accordance with the present disclosure.
In some embodiments, the target sequence encoding the target peptide includes, but not limited to, spike protein, membrane protein, envelope protein or nucleocapsid protein of coronaviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target sequence encoding the target peptide includes spike protein or its fragment from a betacoronavirus.
Betacoronavirus is a genus within the subfamily Othrocoronavirinae of family Coronaviridae. The International Committee on Taxonomy of Viruses (ICTV) has classified the genus Betacoronavirus into 5 subgenera viz., Embecovirus, Sarbecovirus, Merbecovirus, Nobecovirus and Hibecovirus. The first four of these subgenera were formerly known as lineages or subgroups A, B, C and D, respectively.
Betacoronaviruses have been of greater clinical importance as they have been found to cause outbreaks, for example, the 2002-2003 SARS outbreak caused by SARS- CoV-1 or SARS-CoV, the 2012 MERS outbreak caused by Middle East Respiratory Virus, and more recently the 2019-2020 COVID-19 pandemic caused by SARS-CoV-2. The other betacoronaviruses known to infect human beings are HKU1, and OC43.
The genome of betacoronaviruses encodes 4 main structural proteins viz spike(S), membrane(M), envelope(E), and nucleocapsid(N) proteins. The spike protein is the immunodominant protein among the major structural proteins. It contains two subunits, SI and S2. The former is further divided into an N-terminal domain (NTD) and a C-terminal domain (CTD). Both or one of the domains may act as receptor binding domains (RBD) interacting with host cell receptors. The RBD contains the receptor-binding motif (RBM). The SI subunits are organized to form a trimeric structure. S2 subunit helps the virus to enter the host cell through membrane fusion. S2 subunit contains fusion peptide (FP), heptad repeat 1 (HR1), central helix (C -helix), connector domain (CD), stem helix (SH), heptad repeat 2 (HR2), transmembrane domain and cytoplasmic domain (Dacon, Cherrelle etal. Cell Host & Microbe (2023) 31 : 1-15; Lan, Jun et al. Nature (2020) 581 : 215-220; Wang, Mei-Yue et al. Frontiers in Cellular and Infection Microbiology (2020) 10:587269).
Receptor Binding Domain (RBD)
51 subunit of the betacoronaviruses contains the receptor binding domain either on the N-terminal region or the C-terminal region. RBD interact with the host cell receptors. In one of the embodiments, the RBD includes full length SI subunit of the spike protein or fragment thereof of a betacoronavirus, including mutant, derivative, or variant thereof that retains the ability to interact with the host cell receptor. In some embodiments, the receptor binding domain interacts with angiotensin-converting enzyme 2 (ACE2) receptor or dipeptidyl peptidase 4 (DPP4) receptor, 9-O-acetylated sialic acid (9-O-Ac-Sia) receptor, or combination thereof. In some of the embodiments, the multitarget nucleic acid sequence encodes multitarget peptide comprising one or more receptor binding domains obtained or derived from one or more betacoronaviruses. In some of the embodiments, the multitarget nucleic acid sequence encodes multitarget peptide comprising one or more receptor binding domains obtained or derived from SARS-CoV-1, SARS-CoV-2, MERS-CoV, OC43, HKU1, bat coronavirus, other betacoronavirus, or a combination thereof.
Fusion Peptide
52 subunit of the spike protein of betacoronavirus contains a region called fusion peptide which facilitates membrane fusion. In some embodiments, the target sequence of the present disclosures encodes a fusion peptide of a betacoronavirus. The fusion peptide includes fragments, mutants, derivatives or variants of fusion peptide. In some of the embodiments, the multitarget nucleic acid sequence encodes multitarget peptide comprising one or more fusion peptides obtained or derived from one or more betacoronaviruses.
Stem Helix
S2 subunit of the spike protein of betacoronavirus contains a region called stem helix or S2 stem helix which enables heptad repeats to assume a configuration facilitating fusion pore formation and viral entry into the cell. In some embodiments, the target sequence of the present disclosure encodes a stem helix. The stem helix includes fragments, mutants, derivatives, or variants of fusion peptide. In some of the embodiments, the multitarget nucleic acid sequence encodes multitarget peptide comprising one or more stem helix obtained or derived from one or more betacoronaviruses.
Exemplary target peptides includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof:
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
In some embodiments, the target peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes the above target peptides. Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some embodiments, the target peptide is encoded by a target sequence which may be either a DNA, an RNA or an mRNA.
Herpesviridae
Members of the family Herpesviridae consists of large, enveloped, double stranded DNA viruses. They are known to infect variety of hosts, including mammals. Nine herpesviruses have been identified so far that have human as their primary host viz., herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), human cytomegalovirus (HCMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Human herpesviruses 6A (HHV-6A), 6B (HHV-6B), and 7 (HHV-7), and Kaposi’s sarcoma-associated herpesvirus (HHV-8) (Pellett, Philip E. and Roizman, Bernard, Chapter 59 - Herpesviridae, Editors(s): Knipe, David M. and Howley, Peter M., in Fields Virology, 6th edition, 2013 pages 1802-1822 ISBN 9781451105636). These viruses cause different clinical manifestations that range from asymptomatic to severe disease, depending on the host immune status.
In some embodiments, the target sequence encoding the target peptide is obtained or derived from HSV-1, HSV-2, HCMV, VZV, EBV, HHV-6A, HHV-6B, HHV-7, HHV- 8 or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof of such target sequences.
In some embodiments, the target peptide includes glycoprotein B, glycoprotein C, glycoprotein D, glycoprotein E, glycoprotein K, glycoprotein L, or glycoprotein M, of herpes simplex virus 1 (HSV-1) or herpes simplex virus 2 (HSV-2), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants or variants thereof.
In some embodiments, the target peptide includes glycoprotein B, glycoprotein H, glycoprotein L, glycoprotein M, or glycoprotein N of human cytomegalovirus (HCMV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes glycoprotein B, glycoprotein C, glycoprotein H, or glycoprotein L of varicella-zoster virus (VZV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes, but not limited to, glycoprotein B, glycoprotein H, glycoprotein L, glycoprotein M, glycoprotein N, glycoprotein 42, or glycoprotein 350 of Epstein-Barr virus (EBV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Exemplary target peptides includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof:
Figure imgf000038_0001
Figure imgf000039_0001
HAEMQNPVY (SEQ ID NO: 80);
In some embodiments, the target peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes the above target peptides. Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some embodiments, the target peptide is encoded by a target sequence which may be either a DNA, an RNA or an mRNA.
Poxviridae
Members of the family poxviridae consists of large, enveloped, double stranded, DNA viruses that replicates generally in the cytoplasm of the host cells. Smallpox (variola) virus, vaccinia virus, cowpox virus, and monkeypox virus are the most common viruses within the orthopoxvirus genus of poxviridae family.
In some embodiments, the target sequence encoding the target peptide is obtained or derived from poxviruses such as, but not limited to, smallpox virus, vaccina virus, cowpox virus or monkeypox virus, or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof of such target sequences.
In some embodiments, the target peptide includes, but not limited to, F9 membrane protein of a poxvirus, H3L protein of a poxvirus, A4 protein of a poxvirus, A27 protein of poxvirus, A33 protein of a poxvirus, A56 protein of a poxvirus, B5 protein of a poxvirus, or LI protein of a poxvirus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Exemplary target peptides includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof
Figure imgf000040_0001
Figure imgf000041_0001
In some embodiments, the target peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes the above target peptides. Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some embodiments, the target peptide is encoded by a target sequence which may be either a DNA, an RNA or an mRNA. Flaviviridae
Members of the family Flaviviridae consists 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 within the flavivirus and hepacivirus genera respectively.
In some embodiments, the target sequence encoding the target peptide is obtained or derived from dengue virus, Japanese encephalitis virus, zika virus, yellow fever virus, west nile virus, or hepatitis C virus, or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof of such target sequences.
In some embodiments, the target peptide includes, but not limited to, capsid protein, membrane protein, envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of flaviviruses or hepaciviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes capsid protein, membrane protein, envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of Japanese encephalitis virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes capsid protein, membrane protein, envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of zika virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes capsid protein, membrane protein, envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of yellow fever virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes capsid protein, membrane protein, envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of west nile virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes capsid protein, membrane protein, envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of hepatitis C virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes capsid protein, membrane protein, envelope protein, or non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of dengue virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Exemplary target peptides includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Figure imgf000043_0001
( Q );
Figure imgf000044_0001
In some embodiments, the target peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes the above target peptides. Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some embodiments, the target peptide is encoded by a target sequence which may be either a DNA, an RNA or an mRNA.
Togaviridae
Members of the family Togaviridae consists of enveloped, single stranded RNA viruses. The family togaviridae consists of two genera viz., the alphaviruses and the rubiviruses. Alphaviruses cause severe human illnesses including persistent arthritis and fatal encephalitis and are responsible for emerging human diseases. Some alphaviruses (e.g., Chikungunya (CHIKV), Ross River (RRV), Mayaro (MAYV), Semliki Forest (SFV), Sindbis (SINV), and O'nyong-nyong (ONNV)) cause acute inflammatory musculoskeletal and joint-associated syndromes, which can become chronic, whereas others (Eastern (EEEV), Western (WEEV), and Venezuelan (VEEV) equine encephalitis viruses) cause infection in the brain and neurological disease (Holmes, Autumn C. et al. 2020 PLoS Pathogens 16(10): el008876).
In some embodiments, the target sequence encoding the target peptide is obtained or derived from Chikungunya (CHIKV), Ross River (RRV), Mayaro (MAYV), Semliki Forest (SFV), Sindbis (SINV), and O'nyong-nyong (ONNV) viruses, Eastern (EEEV), Western (WEEV), or Venezuelan (VEEV) equine encephalitis viruses, or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof of such target sequences.
In some embodiments, the target peptide includes, but not limited to, capsid protein, or envelope protein such as El, E2 and E3 of alphaviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes, but not limited to, domain A, domain B, or domain C of E2 protein of alphaviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide includes, but not limited to capsid protein, or envelope protein such as El, E2, and E3 of chikungunya virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes, but not limited to, domain A, domain B, or domain C of E2 protein of chikungunya virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Exemplary target peptides includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
In some embodiments, the target peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes the above target peptides. Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some embodiments, the target peptide is encoded by a target sequence which may be either a DNA, an RNA or an mRNA.
Retroviridae
Members of the family retroviridae consists of enveloped single stranded RNA viruses. It constitutes a large family of viruses that predominantly infect both human and animal vertebrate hosts and causes wide spectrum of diseases ranging from malignancies to immune deficiencies and neurologic disorders. Retroviriade family is organized into several genera viz., alpharetrovirus, betraretrovirus, gammaretrovirus, deltaretrovirus, epsilonretrovirus, lentivirus and spumavirus. Of these genera, lentiviruses have been the subject of considerable interest the most prominent among them being the human immunodeficiency virus (HIV).
In some embodiments, the target sequence encoding the target peptide is obtained or derived from alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus, epsilonretrovirus, lentivirus, spumavirus, or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof of such target sequences.
In some embodiments, the target peptide includes, but not limited to, gag, pol or env proteins of retroviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. In some embodiments, the target peptide includes, pl7Gag, p24Gag, p7Gag, p6Gag, gpl2Env, gp41Env, or pol proteins from lentiviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes, pl7Gag, p24Gag, p7Gag, p6Gag, gpl2Env, gp41Env, or pol proteins from human immunodeficiency virus (HIV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Exemplary target peptides includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Figure imgf000049_0001
Figure imgf000050_0001
In some embodiments, the target peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes the above target peptides. Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some embodiments, the target peptide is encoded by a target sequence which may be either a DNA, an RNA or an mRNA.
Paramyxoviridae
Members of the paramyxoviridae family consists of enveloped, single stranded RNA viruses. Diseases caused by these viruses continue to produce high mortality and morbidity across the world. The family Paramyxoviridae is classified into two subfamilies, the paramyxovirinae and the penumovirinae. The former contains seven genera: respirovirus, rubulavirus, Moribilivirus, Henipavirus, Aqiaparamyxovirus, Avulavirus, and Ferlavirus, while the latter contains two genera: Penumovirus and Metapenumovirus.
Some of the important members of paramyxoviridae that are known to cause disease outbreaks are: Mumps virus (MuV), Parainfluenza virus type 5 (PIV5), Human parainfluenza virus type 2, types 4a and 4b (HPIV2/4a/4b), Newcastle disease virus (NDV), Human parainfluenza virus type 1 and type 3 (HPIV1/3), Nipah virus (NiV), Measles virus (MeV), Human respiratory syncytial virus A2, Bl, S2, (HRSV), and Human metapneumovirus (HMPV).
In some embodiments, the target sequence encoding the target peptide is obtained or derived from Mumps virus (MuV), Parainfluenza virus type 5 (PIV5), Human parainfluenza virus type 2, types 4a and 4b (HPIV2/4a/4b), Newcastle disease virus (NDV), Human parainfluenza virus type 1 and type 3 (HPIV1/3), Nipah virus (NiV), Measles virus (MeV), Human respiratory syncytial virus A2, Bl, S2, (HRSV), or Human metapneumovirus (HMPV), or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof of such target sequences. In some embodiments, the target peptide includes, but not limited to, nucleocapsid protein, P protein, V protein, W protein, D protein, I protein, C protein, L protein, M protein, H (hemagglutinin) protein, HN (hemagglutinin-neuraminidase) protein, G protein, F protein, or a combination thereof, of Mumps virus (MuV), Parainfluenza virus type 5 (PIV5), Human parainfluenza virus type 2, types 4a and 4b (HPIV2/4a/4b), Newcastle disease virus (NDV), Human parainfluenza virus type 1 and type 3 (HPIV1/3), Nipah virus (NiV), Measles virus (MeV), Human respiratory syncytial virus A2, Bl, S2, (HRSV), Human metapneumovirus (HMPV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes, but not limited to, 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 A2, Bl, S2 (HRSV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Exemplary target peptides includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Figure imgf000051_0001
In some embodiments, the target peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes the above target peptides. Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some embodiments, the target peptide is encoded by a target sequence which may be either a DNA, an RNA or an mRNA.
Papillomaviridae
Members of the family Papillomaviridae are small non-enveloped, double stranded DNA viruses. The most common genus in the family papillomaviridae is papillomavirus. Human papillomaviruses are the most prominent papillomaviruses which are known to cause papillomas or warts, and are responsible for causing cervical cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer, and oropharyngeal cancer.
In some embodiments, the target sequence encoding the target peptide is obtained or derived from papillomavirus, preferably human papillomavirus, including codon optimized sequences, fragments, mutants, or variants thereof of such target sequences.
In some embodiments, the target sequence encoding the target peptide is obtained or derived from human papillomaviruses selected from the group comprising HPV1, HPV2, HPV3, HPV4, HPV5, HPV6, HPV7, HPV8, HPV9, HPV10, HP V I 1, HPV 12, 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, HPV37, HPV38, HPV39, HPV40, HPV41, HPV42, HPV43, HPV44, HPV45, HPV46, HPV47, HPV48, HPV49, HPV50, HPV51, HPV52, HPV53, HPV54, HPV55, HPV56, HPV57, HPV58, HPV59, HPV60, HPV61, HPV62, HPV63, HPV64, HPV65, HPV66, HPV67, HPV68, HPV69, HPV70, HPV71, HPV72, HPV73, HPV74, HPV75, HPV76, HPV77, HPV78, HPV79, HPV80, HPV81, HPV82, HPV83, HPV84, HPV85, HPV86, HPV87, HPV88, HPV89, HPV90, HPV91, HPV92, HPV93, HPV94, HPV95, HPV96, HPV97, HPV98, HPV99, HPV100, HPV101, HPV102, HPV103, HPV104, HPV105, HPV106, HPV107, HPV108, HPV109, HPV110, HPV111, HPV112, HPV113, HPV114, HPV115, HPV116, HPV117, HPV118, HPV119, HPV120, or a combination thereof. In some embodiments, the target peptide includes, but not limited to, early (E) protein or late (L) protein or a combination thereof, of papillomavirus, preferably human papillomavirus, including codon optimized sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes, but not limited to, El, E2, E4, E5, E6, or E7 protein of papillomavirus, preferably human papillomavirus, or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes, but not limited to, LI (major capsid) protein, L2 (minor capsid) protein, of papillomavirus, preferably human papillomavirus, or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof.
In some embodiments, the target peptide includes, LI (major capsid) protein of papillomavirus, preferably human papillomavirus, that has lost the ability to self-assemble into a virus like particle. In some embodiments, the LI protein is modified or mutated such that it loses its ability to self-assemble into a virus like particle.
In some embodiments, the target peptide includes, L2 (minor capsid) protein of papillomavirus, preferably human papillomavirus, including codon optimized sequences, fragments, mutants, or variants thereof.
Exemplary target peptides includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
In some embodiments, the target peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the target peptides, a person skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes the above target peptides. Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some embodiments, the target peptide is encoded by a target sequence which may be either a DNA, an RNA or an mRNA.
Self-assembling sequence and self-assembling peptide
The multitarget nucleic acid sequence and multitarget peptide includes selfassembling sequence and self-assembling peptide respectively. The self-assembling sequence comprises of a sequence of nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes a self-assembling peptide. The self-assembling sequence includes codon optimized sequences, fragments, mutants, variants or a combination thereof. In some embodiments, the self-assembling sequence is a DNA or an RNA or an mRNA.
Any self-assembling peptide that is capable of self-assembling into a polypeptide nanoparticle can be employed in accordance with the present disclosure.
In some embodiments self-assembling peptide may be a full-length protein or its fragment, mutants, or variant thereof.
In some embodiments, the self-assembling peptide includes, but not limited to, lumazine synthase from Aquifex species, hepatitis B surface antigen (HBsAg) from Hepatitis B Virus, hepatitis B core antigen (HBcAg) from Hepatitis B virus, human papillomavirus LI (HPV LI) protein, matrix protein Ml from influenza A virus, ferritin, riboflavin synthase, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
In some embodiments, the self-assembling peptide is a ferritin peptide.
Ferritin is one of the ubiquitous proteins found in nature. It is produced by all living organisms including archaea, bacteria, algae, higher plants, and animals. Each ferritin protein is generally composed of 24 subunits or peptides which self-assembles into a ferritin nanoparticle.
In some aspects, the multitarget nucleic acid sequence and multitarget peptide includes ferritin sequence and ferritin peptide respectively. The ferritin sequence comprises of a sequence of nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes a ferritin peptide. In some embodiments, the ferritin sequence is a DNA or an RNA or an mRNA.
Any ferritin peptide that is capable of self-assembling into a nanoparticle can be employed in accordance with the present disclosure. In some embodiments, the ferritin peptide is obtained or derived from Helicobacter pylori ferritin, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Exemplary self-assembling peptides includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including codon optimized sequences, fragments, mutants, or variants thereof:
Figure imgf000059_0001
( )
Figure imgf000060_0001
Figure imgf000061_0001
In some embodiments, the self-assembling peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the self-assembling peptides, a person skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes the above self-assembling peptides. Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some embodiments, the self-assembling peptide is encoded by the self-assembling sequence which may be either a DNA, an RNA or an mRNA.
Linker sequence and linker peptide
The multitarget nucleic acid sequence and multitarget peptide includes linker sequence and linker peptide respectively. The linker sequence comprises of a sequence of nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes a linker peptide. The linker sequence includes codon optimized sequences, fragments, mutants, variants, or combination thereof. In some embodiments, the linker sequence is a DNA or an RNA or an mRNA.
In some embodiments, the linker peptide connects the target peptide with the selfassembling peptide in a polypeptide. In some embodiments, the linker peptide connects the signal peptide with a polypeptide. In some other embodiments, the linker peptide connects the signal sequence with the first polypeptide. In some embodiments, one linker peptide connects the cleavage peptide with the target peptide and another linker peptide connects the target peptide with the self-assembling peptide in a polypeptide. In some embodiments, the linker peptide connects two cleavage peptides. Any suitable linker peptides can be employed in accordance with the present disclosure. In some embodiments, linker peptide is an amino acid linker, a foldon, a scaffold or a combination thereof. In some embodiments, linker peptide consists of a combination of an amino acid linker and a foldon. In some embodiments, linker peptide consists of a combination of an amino acid linker and a scaffold. In some embodiments, linker peptide consists of a combination of a foldon and scaffold. In some embodiments, linker peptide consists of a combination of an amino acid linker, a foldon, and a scaffold.
In some embodiments, the amino acid linker comprises of about 2-49 amino acids, 2-40 amino acids, 2-40 amino acids, 2-20 amino acids, 2-15 amino acids, or 2-10 amino acids. In some embodiments, the amino acid linker may comprise a glycine serine linker, a glycine proline linker, a glycine threonine linker, an alanine serine linker etc.
The glycine proline linker comprises of glycine (G) and proline (P) amino acids consecutively without any preference of order of appearance of either glycine or proline. In some embodiments, the glycine proline linker is 2-49 amino acids in length.
The glycine threonine linker comprises of glycine (G) and threonine (T) amino acids consecutively without any preference of order of appearance of either glycine or threonine. In some embodiments, the glycine threonine linker is 2-49 amino acids in length.
The alanine serine linker comprises of alanine (A) and serine (S) amino acids consecutively without any preference of order of appearance of either alanine or serine. In some embodiments, the alanine serine linker is 2-49 amino acids in length.
The glycine serine linker comprises of glycine (G) and serine (Serine) amino acids consecutively without any preference of order of appearance of either glycine or serine. In some embodiments, the glycine serine linker is 2-49 amino acids in length.
Exemplary glycine serine linkers includes, but not limited to, the ones represented by the following amino acid sequences, or their combinations, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof: GSG (SEQ ID NO: 350) GSGG (SEQ ID NO: 330);
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);
Figure imgf000063_0001
In some embodiments, the glycine serine linkers share at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the glycine serine linkers, a person skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes the above glycine serine linkers. Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some embodiments, the glycine serine linker is encoded by a glycine serine linker sequence which may be either a DNA, an RNA or an mRNA.
In some embodiments, the linker peptide is a foldon. A foldon comprises of a sequence of amino acids encoded by a foldon sequence. The foldon sequence includes codon optimized sequences, fragments, mutants, variants or a combination thereof. A foldon enables two or more homologous polypeptides to organise to form an oligomeric complex. In some embodiments, foldon also helps in orientation of a polypeptide such that the domains or epitopes on the target peptide are exposed or displayed for interaction or communication with cells or biomolecules or immune system.
Exemplary foldons includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof:
Figure imgf000063_0002
In some embodiments, the foldon shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the foldons, a person skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes the above foldons. Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some embodiments, the foldon is encoded by a foldon sequence which may be either a DNA, an RNA or an mRNA.
In some embodiments, the linker peptide is a scaffold. A scaffold comprises of a sequence of amino acids encoded by a scaffold sequence. The scaffold sequence includes codon optimized sequences, fragments, mutants, variants, or combination thereof. A scaffold provides structural and/or functional integrity or support to the target peptide and may also helps in orientation of target peptide such that the domains or epitopes of the target peptide are exposed or displayed for interaction or communication with cells or biomolecules or immune system.
Exemplary scaffolds includes, but not limited to, the ones represented by the following amino acid sequences, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof:
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
In some embodiments, the scaffold peptide shares at least 70 % identity with the sequences described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the scaffold peptides, a person skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes the above scaffold peptides. Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some embodiments, the target peptide is encoded by the target sequence which may be either a DNA, an RNA, or an mRNA.
In some embodiments, the linker peptide consists of a glycine serine linker followed by a foldon. In another embodiment, the linker peptide consists of a foldon followed by a glycine serine linker. In some embodiments, the linker peptide consists of a glycine serine linker followed by a foldon and another glycine serine linker. In some embodiments, the linker peptide consists of a foldon followed by a glycine serine linker and another foldon.
Cleavage sequence and cleavage peptide
The multitarget nucleic acid sequence and multitarget peptide includes cleavage sequence and cleavage peptide respectively. The cleavage sequence comprises of a sequence of nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes a cleavage peptide. The cleavage sequence includes codon optimized sequences, fragments, mutants, variants or a combination thereof. In some embodiments, the cleavage sequence is a DNA or an RNA or an mRNA.
The cleavage peptide connects one polypeptide with another polypeptide, for example, the adjacent polypeptide. The cleavage peptide carries one or more cleavage sites. In some embodiments, the cleavage peptide comprises one or more cleavage peptides, for example cleavage peptide- 1, cleavage peptide-2 and so on. In some embodiments, the cleavage peptide optionally comprises a linker peptide between two cleavage peptides. In some embodiments, the cleavage peptide facilitates the action of cellular proteases to cleave the multitarget polypeptide into individual polypeptides or self cleaves into individual polypeptides. In some embodiments, the resulting polypeptides may comprise either a target peptide, a linker peptide and a self-assembling peptide or a linker peptide, target peptide, linker peptide and a self-assembling peptide or a combination thereof. In some embodiments, the polypeptide, in addition to these peptides, may also have some residues (amino acids) of cleavage peptide. Any cleavage peptide that is susceptible to the action of cellular proteases or a cleavage peptide that has the ability to undergo self-cleavage can be employed in accordance with the present disclosure.
In some embodiments, the cleavage peptide is a golgi specific cleavage peptide i.e., susceptible to action of golgi specific proteases.
Exemplary cleavage peptide includes, but not limited to, the one represented by the following amino acid sequence, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof:
Figure imgf000067_0001
In some embodiments, the cleavage peptide shares at least 70 % identity with the sequence described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. Given the above amino acid sequences of the cleavage peptide, a person skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes the above cleavage peptide. Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some embodiments, the cleavage peptide is encoded by a cleavage sequence which may be either a DNA, an RNA or an mRNA.
Signal sequence and signal peptide
The multitarget nucleic acid sequence and multitarget peptide includes signal sequence and signal peptide respectively. The signal sequence comprises of a sequence of nucleotides, either deoxyribonucleotides or ribonucleotides, that encodes a signal peptide. The signal sequence includes codon optimized sequences, fragments, mutants, variants, or a combination thereof. In some embodiments, the signal sequence is a DNA or an RNA or an mRNA.
The signal peptide is present upstream (N-terminus or amino-terminus) of one or more polypeptides. In some embodiments, the signal peptide may be present on the N- terminus of all or some polypeptides. In some embodiments, the signal peptide is present on the N-terminus of the first polypeptide. In some embodiments, the signal peptide is present upstream (N-terminus) of some polypeptides. In some embodiments, the signal peptide is present upstream (N-terminus) of each of the polypeptides.
In some embodiments, the signal peptide transports the multitarget peptide to cell organelles. In some embodiments, the signal peptide transports the multitarget peptide to golgi body or golgi apparatus. Any signal peptide that transports the multitarget peptide to golgi bodies can be employed in accordance with the present disclosure. In some embodiments, the signal peptide is a golgi targeting signal peptide i.e., directs the multitarget peptide to golgi complex.
Exemplary signal peptide includes, but not limited to, the one represented by the following amino acid sequence, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof:
Figure imgf000068_0001
Figure imgf000069_0001
In some embodiments, the signal peptide shares at least 70 % identity with the sequence described herein above, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
Given the above amino acid sequences of the signal peptide, a person skilled in the art would be able to deduce all possible DNA or RNA sequences that encodes the above signal peptide. Such DNA or RNA sequences are deemed to be incorporated in this disclosure. In some embodiments, the signal peptide is encoded by a signal sequence which may be either a DNA, an RNA or an mRNA.
Synthesis of multitarget nucleic acid sequences
Multitarget nucleic acid sequence according to the present disclosure can be either a DNA or an RNA or an mRNA. The multitarget nucleic acid sequence as described herein can be synthesized by molecular biology or genetic engineering techniques well known in the art, for example, using recombinant expression system, chemical synthesis, or in vitro transcription (IVT).
In some embodiments, the multitarget nucleic acid sequence is obtained through a single IVT process. In some embodiments, the multitarget nucleic acid sequence obtained through single IVT process is an mRNA.
In some embodiments, the multitarget nucleic acid sequence is a messenger RNA (mRNA). The mRNA encodes a multitarget peptide as described herein. Typically, an mRNA includes at least a coding region (which encodes the multitarget peptide), a 5’ UTR, a 3’ UTR, a 5’ cap and a 3’ poly(A) tail. UTR (untranslated regions) flanks the coding region or open reading frame (ORF). The 5’ UTR and the 3’ UTR are sections of the mRNA before the start codon and after the stop codon respectively. The 5’ UTR has a cap (5’ cap) consisting of altered nucleotides. mRNA also contains a polyadenylated region at its 3’ end having adenine nucleotides called poly(A) tail.
In some embodiments, the mRNA may be unmodified or modified or a combination of both. The modification may be in the nucleobase of the nucleotide, or sugar moiety of the nucleotide, or the phosphate of the nucleotide. In some embodiments, unmodified mRNA may comprise naturally occurring nucleosides, for example, adenosine, guanosine, cytidine, and uridine. mRNA may comprise one or more modified nucleosides, for example, adenosine analog, guanosine analog, cytidine analog, or uridine analog.
In some embodiments, the one or more modified nucleosides is a nucleoside analog selected from 2-aminoadenosine, 3-methyl adenosine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, or 8-oxoguanosine or a combination thereof.
In some embodiments, the one or more modified nucleosides is a uridine analog selected from propynyl-uridine, pseudouridine, C5-bromouridine, C5-fluorouridine, C5- 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, l-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-l-methyl-pseudouridine, 2 -thio- 1 -m ethylpseudouridine, 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 a combination thereof.
In some embodiments, the one or more modified nucleosides is a cytidine analog 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-m ethoxy- 1 -methyl- pseudoisocytidine or a combination thereof.
Methods for making modified nucleosides are well known in the art (WO 2020168466, US8278036; US8691966; US8748089; US8835108; US9750824; US10232055; W02007024708; WO2012135805; WO2013052523; WO2011012316)
In some embodiments, the modified nucleoside is pseudouridine, for example, 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-l-methyl-pseudouridine, dihydro-pseudouridine or a combination thereof.
In some embodiments, mRNA is produced using recombinant expression system, chemically synthesized, or obtained through in vitro transcription.
In some embodiments, the multitarget nucleic acid sequence is obtained through a single IVT process. mRNAs according to the present disclosure may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and/or RNase inhibitor. The exact conditions may vary according to the specific application. Methods of making mRNA through IVT reaction is well known in the art (see for example, Beckert, Bertrand andMasquida, 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 . 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; W02020185811; W02022082001).
In some embodiments, the in vitro transcription occurs in a single batch. In some embodiments, IVT reaction includes capping and tailing reactions either co- transcriptionally or separately. A cap analog is added to the in vitro transcription reaction and will be incorporated at the 5’ end of the mRNA during the reaction. Alternative method of capping involves adding the cap post-transcriptionally through an enzymatic reaction. The poly (A) tail can be incorporated into the DNA template sequence, and thus the poly (A) tail will be incorporated into the mRNA by T7 RNA polymerase during the in vitro transcription. Alternative method of tailing involves adding the poly (A) tail post- transcriptionally through an enzymatic reaction. In some embodiments, capping and tailing reactions are performed co-transcriptionally i.e., during the IVT reaction. In some embodiments, capping and tailing reactions are performed separately from IVT reaction i.e., post transcriptionally. mRNA produced as a result of IVT reaction may be purified using techniques well known in the art, such as, centrifugation, filtration and/or chromatographic techniques. The purification of mRNA may be accomplished before capping and tailing steps are performed or after capping and tailing. The synthesized mRNA may be purified by ethanol precipitation or filtration or chromatography methods. In some embodiments, tangential flow filtration is used to purify mRNA. In some embodiments, mRNA is purified by chromatographic step. In other embodiments, mRNA is purified by a combination of filtration and chromatography steps.
In some embodiments, a suitable mRNA sequence is an mRNA sequence encoding a protein, peptide, polypeptide. In some embodiments, a suitable mRNA sequence is codon optimized for efficient expression in a host cell or organism. Codon optimization typically includes modifying a naturally-occurring or wild-type nucleic acid sequence encoding a peptide, polypeptide or protein to achieve the highest possible expression of peptide, polypeptide, protein or an antibody without altering the amino acid sequence.
In some embodiments, the mRNA is circular. In other embodiments, the mRNA is linear.
In some embodiments, the mRNA is self-amplifying or self-replicating.
The multitarget nucleic acid sequence as described herein, express multitarget peptide.
Polypeptide nanoparticle
The multitarget peptide comprises multiple repeats of polypeptide comprising either a target peptide, a linker peptide, and a self-assembling peptide or a linker peptide, target peptide, a linker peptide, and a self-assembling peptide, or a combination thereof, interspersed with cleavage peptide (see illustration in figures). In some embodiments, the total number of polypeptides present in a multitarget peptide may be up to 100 polypeptides. In some embodiments, one or more polypeptides in the multitarget peptide may have identical target peptides (homologous polypeptides). In some embodiments, one or more polypeptides in the multitarget peptide may have different target peptides (heterologous polypeptides).
A multitarget peptide as described herein is encoded by a multitarget nucleic acid sequence as described herein. Each multitarget peptide comprises two or more polypeptides, wherein some or all polypeptides comprises either a target peptide, a linker peptide, and a self-assembling peptide or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide or a combination thereof. The polypeptides are connected with each other through a cleavage peptide. The multitarget peptide includes a signal peptide upstream (N-terminus) of one or more polypeptides. In some embodiments, the multitarget peptide includes a signal peptide upstream (N-terminus) of each of all or some polypeptides. In some embodiments, the multitarget peptide optionally includes a signal peptide upstream (N-terminus) of each polypeptide. In some embodiments, the multitarget peptide includes a signal peptide upstream (N-terminus) of some polypeptides. In some embodiments, the multitarget peptide includes a signal peptide upstream (N-terminus) of all polypeptides.
The signal peptide transports the multitarget peptide to golgi body or golgi apparatus. The cellular proteases act on the cleavage sites present in the cleavage peptides or the cleavage peptide undergoes self-cleavage and cleaves the multitarget peptide into individual polypeptides comprising either a target peptide, a linker peptide, and a selfassembling peptide, or a linker peptide, a target peptide, a linker peptide, and a selfassembling peptide, or a signal peptide, a target peptide, a linker peptide, and a selfassembling peptide, or a signal peptide, a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide, or a combination thereof. The polypeptides may additionally also have some residues (amino acids) of cleavage peptide.
In some embodiments, the linker peptide may be an amino acid linker, a foldon, a scaffold, or a combination thereof.
In some embodiments, the polypeptides may be homologous polypeptides.
In some embodiments, two or more homologous polypeptides may organise to form an oligomeric complex. In some embodiments, the oligomeric complex may comprise at least two homologous polypeptides, at least three homologous polypeptides, at least four homologous polypeptides, at least five homologous polypeptides, or at least six homologous polypeptides and so on. In some embodiments, the polypeptides may be heterologous polypeptides.
A polypeptide nanoparticle is formed by self-assembly of two or more homologous polypeptides, or two or more heterologous polypeptides, or one or more oligomeric complexes, or their combination.
In some embodiments, a polypeptide nanoparticle comprises homologous polypeptides, heterologous polypeptides, oligomeric complex, or a combination thereof.
In some embodiments, the polypeptide nanoparticles may be symmetrical, non- symmetrical, asymmetrical, or a combination thereof.
In some embodiments, the polypeptide nanoparticles may be icosahedral, helical, spherical, rod-like or a combination thereof.
In some embodiments, the polypeptide nanoparticles may be enveloped or nonenveloped or a combination thereof.
In some embodiments, the polypeptide nanoparticles may be single layered or multi-layered or a combination thereof.
In some of the embodiments, the polypeptide nanoparticle may comprise at least 2 or up to 100 polypeptides.
In some embodiments, the polypeptide nanoparticle may comprise polypeptides between 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-99.
In one of the embodiments, the polypeptide nanoparticle may comprise at least 2 or up to 100 homologous polypeptides.
In some embodiments, the polypeptide nanoparticle may comprise at least 2 or up to 100 heterologous polypeptides.
In some embodiments, the polypeptide nanoparticle may comprise two or more oligomeric complexes such that total number of polypeptides in the polypeptide nanoparticle are not more than 100.
In some embodiments, the polypeptide nanoparticle may comprise some homologous polypeptides and some heterologous polypeptides such that the total number of polypeptides in the polypeptide nanoparticle are not more than 100.
In some embodiments, the polypeptide nanoparticle may comprise some homologous polypeptides and some oligomeric complexes such that the total number of polypeptides in the polypeptide nanoparticle are not more than 100.
In some embodiments, the polypeptide nanoparticle may comprise some heterologous polypeptides and some oligomeric complexes such that the total number of polypeptides in the polypeptide nanoparticle are not more than 100. In some embodiments, the polypeptide nanoparticle may comprise some homologous polypeptides, some heterologous polypeptides, some oligomeric complexes, or their combination such that the total number of polypeptides in the polypeptide nanoparticle are not more than 100.
Lipid nanoparticles (LNP) composition
The multitarget nucleic acid sequences as described herein may be encapsulated in a lipid nanoparticle composition.
In some embodiments, the lipid nanoparticle composition comprises lipid components, ionizable polymer, or a combination thereof and a multitarget nucleic acid sequence as described herein.
In some embodiments, the lipid nanoparticle composition comprises lipid components such as a cationic lipid, a phospholipid, a sterol, a PEG-lipid and a multitarget nucleic acid sequence as described herein.
In another embodiment, the lipid nanoparticle composition comprises an ionizable polymer, a cationic lipid, a phospholipid, a sterol, a PEG-lipid and a multitarget nucleic acid sequence as described herein.
Lipid Components
Lipid components of the lipid nanoparticle compositions may include one or more lipids, such as a cationic lipid, a phospholipid, a sterol, and a PEG-lipid.
Cationic lipid
Cationic lipid refers to a lipid that has a net positive charge at a selected pH. Cationic lipids generally consist of a hydrophilic head group that carries the charge and a hydrophobic tail.
Exemplary cationic lipid for use in the lipid nanoparticle compositions include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N- dimethylammonium bromide(DDAB); N-(2,3dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP); 3-(N — (N',N'-dimethylaminoethane)- carbamoyl)cholesterol (DC-Chol), N-(l-(2, 3 -di oleoyloxy )propyl)N-2- (sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (DOSPA), dioctadecylamidoglycyl carboxy spermine (DOGS), 1,2-di oleoyl- 3 -dimethylammonium propane (DODAP), N, N-dimethyl-2, 3 -di oleoyloxy )propylamine (DODMA), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxy ethyl ammonium bromide (DMRIE), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 3- dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc- tadecadienoxy)propane (Clin-DMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3- dimethyl-l-(cis,cis-9', 12'-octadecadienoxy)propane (CpLin-DMA), 2,3-Dilinoleoyloxy- N,N-dimethylpropylamine (DLin-DAP), l,2-N,N'-Dilinoleylcarbamyl-3- dimethylaminopropane (DLincarb-DAP), l,2-Dilinoleoylcarbamyl-3- dimethylaminopropane (DLin-CDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]- dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen- 19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA), heptadecan-9-yl 8-[2-hydroxyethyl-(6- oxo-6-undecoxyhexyl)amino]octanoate (SM- 102), 6-[6-(2-hexyldecanoyloxy)hexyl-(4- hydroxybutyl)amino]hexyl 2-hexyldecanoate (ALC-0315), nonyl 8-[(8-heptadecan-9- yloxy-8-oxooctyl)-(2-hydroxyethyl)amino]octanoate (SLP-0001) or a combination thereof.
Cationic lipids with amine head group are preferred cationic lipids. The amine group can be at primary, secondary or tertiary position. The cationic lipid may comprise one (monoamine) or more (polyamine) such amine groups.
In some embodiments, the cationic lipids are positively charged at acidic pH i.e., pH 1.0 to pH 6.9. In certain embodiments, the cationic lipids are neutral at certain pH i.e., around physiological pH (pH 7.0 to pH 7.5). A cationic lipid that can exist in a positively charged or neutral form depending on the pH is referred to as ionizable lipid. Preferred cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH. For example, an ionizable lipid may be neutral around physiological pH (pH 7.0 to pH 7.5), and cationic around acidic pH (pH 1.0 to pH 6.9).
In some embodiments, cationic lipid present in the lipid nanoparticle compositions is an ionizable lipid.
Methods of making cationic lipid and/or ionizable lipid or imparting the cationic lipid the ability to behave as an ionizable lipid are well known in the art (W02005121348; W02009127060; W02009086558; W02010042877; W02010144740; WO2011075656; WO2017049245; WO2017075531; WO2018118102; WO2015199952; Reynier P. et al. Journal of Drug Targeting (2004) 12: 25-38; Sabnis, Staci et al. Molecular Therapy (2018) 26: 1509-1519). The proportion of cationic lipid present in the lipid nanoparticle compositions is from about 25 mol % to about 70 mol % or any range therein.
In some embodiments, the proportion of cationic lipid present in the lipid nanoparticle compositions is from about 25 mol % to about 70 mol %, from about 25 mol % to about 65 mol %, from about 25 mol % to about 60 mol %, from about 25 mol % to about 55 mol %, from about 25 mol % to about 50 mol %, from about 25 mol % to about 48 mol %, from about 25 mol % to about 46 mol %, from about 25 mol % to about 45 mol %, from about 25 mol % to about 44 mol %, from about 25 mol % to about 43 mol %, from about 25 mol % to about 42 mol %, from about 25 mol % to about 41 mol %, from about 25 mol % to about 40 mol %, or any range therein.
In some embodiments, the proportion of cationic lipid present in the lipid nanoparticle compositions is about 25 mol %, about 26 mol %, about 27 mol %, about 28 mol %, about 29 mol %, about 30 mol %, about 31 mol %, about 32 mol %, about 33 mol %, about 34 mol %, about 35 mol %, about 36 mol %, about 37 mol %, about 40 mol %, about 41 mol %, about 42 mol %, about 43 mol %, about 44 mol %, about 45 mol %, about 46 mol %, about 47 mol %, about 48 mol %, about 49 mol %, about 50 mol % about 51 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.
Phospholipids
Phospholipid includes a lipid containing a hydrophilic head with a phosphate group and a hydrophobic tail composed of fatty acid chains attached to a glycerol or sphingosine backbone.
Exemplary phospholipids for use in the lipid nanoparticle compositions include, but are not limited to, l,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2- dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3 -phosphocholine (POPC), 1,2- di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl -sn- glycero-3 -phosphocholine (Cl 6 Lyso PC), l,2-dilinolenoyl-sn-glycero-3 -phosphocholine, l,2-diarachidonoyl-sn-glycero-3 -phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,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, l,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1,2- dilinolenoyl-sn-glycero-3 -phosphoethanolamine, l,2-diarachidonoyl-sn-glycero-3- phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG), l-myristoyl-2- stearoyl-sn-glycero-3-phosphocholine (MSPC), l-palmitoyl-2-myristoyl-sn-glycero-3- phosphocholine (PMPC), l-palmitoyl-2-stearoyl-sn-glycero-3 -phosphocholine (PSPC), 1- stearoyl-2-myristoyl-sn-glycero-3-Phosphocholine (SMPC), l-Stearoyl-2-palmitoyl-sn- glycero-3 -phosphocholine (SPPC), l-stearoyl-2-oleoyl-sn -glycero-3 -phosphocholine (SOPC), l-stearoyl-2-docosahexaenoyl-sn-glycero-3 -phosphocholine (SDPC), sphingomyelin, or a combination thereof.
The proportion of phospholipid present in the lipid nanoparticle compositions is from about 2 mol % to about 30 mol % or any range therein.
In some embodiments, the proportion of phospholipid present in the lipid nanoparticle compositions is from about 2 mol % to about 30 mol %, from about 2 mol % to about 28 mol %, from about 2 mol % to about 26 mol %, from about 2 mol % to about 24 mol %, from about 2 mol % to about 22 mol %, from about 2 mol % to about 20 mol %, from about 3 mol % to about 19 mol %, from about 3 mol % to about 18 mol %, from about 3 mol % to about 17 mol %, from about 3 mol % to about 16 mol %, from about 3 mol % to about 15 mol %, from about 3 mol % to about 14 mol %, from about 3 mol % to about 13 mol %, from about 3 mol % to about 12 mol %, or any range therein.
In some embodiments, the proportion of phospholipid present in the lipid nanoparticle compositions is about 2 mol %, about 3 mol %, about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about 10 mol %, about 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.
Sterol Lipid nanoparticle composition disclosed herein may include sterol and/or sterol derivatives. The term “sterol” as used herein include, but not limited to, cholesterol, sitosterol, fecosterol, ergosterol, campesterol, stigmasterol or their derivatives. In some embodiments, lipid nanoparticle composition comprises cholesterol and/or cholesterol derivatives. Non-limiting examples of cholesterol and cholesterol derivatives include 5a- cholestanol, 5P-coprostanol, cholesteryl-(2’-hydroxy)-ethyl ether, cholesteryl-(4’- hydroxy)-butyl ether, 6-ketocholestanol, 5a-cholestane, cholestenone, 5a-cholestanone, 5P-cholestanone, cholesteryl decanoate, or mixtures thereof. Methods of making cholesterol and cholesterol derivatives are well known in the art (W02009127060; WO2019152557).
The proportion of sterol present in the lipid nanoparticle compositions may be from about 30 mol % to about 65 mol % or any range therein.
In some embodiments, the proportion of sterol present in the lipid nanoparticle compositions is from about 30 mol % to about 65 mol %, from about 31 mol % to about 60 mol %, from about 32 mol % to about 60 mol %, from about 33 mol % to about 60 mol %, from about 34 mol % to about 60 mol %, from about 35 mol % to about 60 mol %, or any range therein.
In some embodiments, the proportion of sterol present in the lipid nanoparticle compositions is about 30 mol %, about 31 mol %, about 32 mol %, about 33 mol %, about 34 mol %, about 35 mol %, about 36 mol %, about 37 mol %, about 38 mol %, about 39 mol %, about 40 mol %, about 41 mol %, about 42 mol %, about 43 mol %, about 44 mol %, about 45 mol %, about 46 mol %, about 47 mol %, about 48 mol %, about 49 mol %, 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.
PEG-lipid
The term PEG-lipid, pegylated lipid, PEG linked lipid, PEG conjugated lipid, PEG- lipid conjugate, PEG modified lipid have been used interchangeably to mean polyethylene glycol linked to a lipid moiety. The lipid moiety may be linked directly to the PEG molecule or through a linker. In some embodiments, a PEG-lipid comprises a PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and/or PEG-modified cholesterol, and/or mixtures thereof. The methods of making PEG-lipid are well known to persons skilled in the art, see for example, US20030077829; US2005008689; US5885613; US7404969; W02005026372;
W02009086558).
In some embodiments, PEG-lipid is selected from mPEG-Dimyristoyl glycerol (mPEG-DMG), mPEG-N,N-Ditetradecylacetamide (mPEG-DTA or ALC0159), mPEG- Cholesterol (mPEG-CLS), mPEG-DSPE, mPEG-DMPE, mPEG-DPPE, mPEG-DLPE, mPEG-DOPE, mPEG-DPPC, mPEG-DSPC, l,2-Distearoyl-sn-Glycero-3- Phosphoethanolamine with conjugated methoxyl polyethylene glycol) (mPEG-DSPE), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) or mixtures thereof.
The PEG moiety of the PEG-lipid may comprise an average molecular 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, 0.5 kDa to 2 kDa. In preferred embodiments, the PEG-lipid has an average molecular weight of about 0.5 kDa to about 2 kDa.
The proportion of PEG-lipid present in the lipid nanoparticle compositions may be from about 0.2 mol % to about 2.0 mol % or any range therein.
In some embodiments, the proportion of PEG-lipid present in the lipid nanoparticle compositions is from about 0.2 mol % to about 2.0 mol %, from about 0.2 mol % to about 1.8 mol %, from about 0.2 mol % to about 1.5 mol %, or any range therein.
In some embodiments, the proportion of PEG-lipid present in the lipid nanoparticle compositions is about 0.2 mol %, about 0.3 mol %, about 0. 4 mol %, about 0.5 mol %, about 0.6 mol %, about 0.7 mol %, about 0.8 mol %, about 0.9 mol %, about 1.0 mol %, about 1.1 mol %, about 1.2 mol %, about 1.3 mol %, about 1.4 mol %, about 1.5 mol %, about 1.6 mol %, about 1.7 mol %, about 1.8 mol %, about 1.9 mol %, or about 2.0 mol % or any portion or fraction thereof.
In some embodiments, the lipid nanoparticle composition additionally may contain an ionizable polymer.
Ionizable polymer
As used herein the term “polymer” means a compound formed from a plurality of repeating units called monomers. Polymers are produced through a process called polymerization wherein two or more monomers are linked through chemical bonds to form the polymer. In some embodiments, the polymer is branched or unbranched. In some embodiments, the polymer may be homopolymer, i.e., consisting of same type of repeat units or monomers, or heteropolymer, i.e., consisting of more than one type of repeat units or monomers. The terms heteropolymer and copolymer have been used interchangeably herein.
The term “ionizable polymer” as used herein means, a polymer that can exist in a positively charged or neutral form depending on the pH of the solution or environment, for example, ionizable polymer will be cationic (positively charged) around acidic pH (pH 1.0 to pH 6.9) and neutral (no charge) around physiological pH (pH 7.0 to pH 7.5).
In some embodiments, the ionizable polymer is a biocompatible polymer or biodegradable polymer. The term “biocompatible polymer” and “biodegradable polymer” have been used interchangeably to mean a polymer that is substantially free from any deleterious effects when introduced into a living or biological system. Such polymers are capable of undergoing degradation when introduced into the living or biological systems and are not expected to produce significant toxicity or immunological response.
In some embodiments, the lipid nanoparticle compositions comprise an ionizable polymer. The ionizable polymer may be selected from chitosan, chitosan derivatives, cellulose derivatives, poly-L-lysine (PLL), poly-L-glutamic acid, protamine, polyethyleneimine, their derivatives, or a combinations thereof.
In some embodiments, the ionizable polymer is positively charged at acidic pH i.e., pH 1.0 to 6.9 and is neutral around physiological pH (pH 7.0 to 7.5).
The proportion of ionizable polymer present in the lipid nanoparticle compositions may be from about 1 mol % to about 25 mol %.
In some embodiments, the proportion of ionizable polymer present in the lipid nanoparticle compositions is from about 1 mol % to about 25 mol %, 1 mol % to about 25 mol %, from about 1 mol % to about 24 mol %, from about 1 mol % to about 23 mol %, from about 1 mol % to about 22 mol %, from about 1 mol % to about 21 mol %, from 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.
In some embodiment, the proportion of ionizable polymer present in the lipid nanoparticle compositions is from about 1 mol % to about 25 mol %, from about 1 mol % to about 24 mol %, from about 1 mol % to about 23 mol %, from about 1 mol % to about 22 mol %, from about 1 mol % to about 21 mol %, from about 1 mol % to about 20 mol %, from about 1 mol % to about 19 mol %, from about 1 mol % to about 18 mol %, from 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.
In some embodiments, the proportion of ionizable polymer present in the lipid nanoparticle compositions is about 1 mol %, is about 2 mol %, about 3 mol %, about 4 mol %, 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about 10 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.
In some embodiments, the preferred ionizable polymer comprises chitosan, chitosan derivatives, cellulose derivatives, or a combination thereof.
Chitosan
Chitosan is a natural polymer composed of glucosamine units. Chitosan is chemically poly-P-(l-4)-2-amino-2-deoxy -D-glucose. Chitosan is prepared by partial deacetylation of chitin, which is commonly carried out by alkaline hydrolysis. Thus, chitosan may contain acetylated units (N-acetyl-D-glucosamine) as well as deacetylated units (P-(l— >4)-linked D-glucosamine). Typically, chitosan molecule has greater than 60% degree of deacetylation when compared to chitin. The molecular weight of chitosan typically varies between 10 kDa to 1000 kDa. Chitosan nanoparticles have been used for drug delivery, including delivery of nucleic acids. However, chitosan nanoparticles alone are insufficient in effective delivery of nucleic acids (Ragelle, Helolse et al. Journal of Controlled Release (2013) 172: 207-218).
In some embodiments, the lipid nanoparticle composition comprises an ionizable polymer such as chitosan along with lipid components and nucleic acid.
In some embodiments, the ionizable polymer is chitosan or its derivatives. In some embodiments, the ionizable polymer includes chitosan derivatives or dialdehyde chitosan derivatives or a combination thereof.
The chitosan or chitosan derivatives employed in the present disclosure may have a molecular weight from about 25 kDa to about 400 kDa. In some embodiments, the molecular weight of chitosan or its derivatives is from about 25 kDa to 375 Kda, from about 30 kDa to about 350 kDa, from about from about 35 kDa to about 325 kDa, from about 40 kDa to about 300 kDa, from about 40 kDa to about 250 kDa, from about 40 kDa to about 225 kDa, from about 40 kDa to about 220 kDa, from about 40 kDa to about 210 kDa, or from about 40 kDa to about 200 kDa or any range therein.
The proportion of chitosan or its derivatives or a combination thereof present in the lipid nanoparticle compositions may be from about 1 mol % to about 25 mol %.
In some embodiments, the proportion of chitosan or its derivatives or a combination thereof present in the lipid nanoparticle compositions is from about 1 mol % to about 25 mol %, , from about 1 mol % to about 24 mol %, from about 1 mol % to about 23 mol %, from about 1 mol % to about 22 mol %, from about 1 mol % to about 21 mol %, from about 1 mol % to about 20 mol %, from about 1 mol % to about 19 mol %, or from about 1 mol % to about 18 mol %, from about 1 mol % to about 17 mol %, from about 1 mol % to about 16 mol %, from about 1 mol % to about 15 mol % or any range therein.
In some embodiments, the proportion of chitosan or its derivatives or a combination thereof present in the lipid nanoparticle compositions is from about 1 mol % to about 25 mol %, preferably about 1 mol % to 20 mol %, most preferably about 1 mol % to about 15 mol %.
In some embodiments, the proportion of chitosan or its derivatives or a combination thereof present in the lipid nanoparticle compositions is about 1 mol %, about 2 mol %, about 3 mol %, about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about 10 mol %, about 11 mol %, about 12 mol %, about 13 mol %, 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.
Cellulose
Cellulose is a polymer composed of a linear chain of d-glucose units linked via β- 1,4 glycosidic bonds. It typically contains repeating glucose units ranging from few hundreds to several thousands. Native cellulose is not ideal for mRNA delivery and therefore, requires modification in accordance with the present disclosure. In some embodiments, cellulose is modified (Jelkmann et al. Biomacromolecules (2018) 19: 4059- 4067; Lee, Hye Ji et al. International Journal of Biosciences Biochemistry and Bioinformatics (2019) 9: 134-140). Cellulose and cellulose derived materials have been used for drug delivery of small molecules (Amalin Kavitha, K. Thomas Paul, Parambath Anilkumar, Chapter 18 - Cellulose-derived materials for drug delivery applications, Editor(s): Faruq Mohammad, Hamad A. Al-Lohedan, Mohammad Jawaid, In Micro and Nano Technologies, Sustainable Nanocellulose and Nanohydrogels from Natural Sources, Elsevier, 2020, Pages 367-390, ISBN 9780128167892).
In some embodiments, the lipid nanoparticle composition comprises cellulose derivatives. Cellulose derivatives may be dialdehyde cellulose derivatives.
The proportion of cellulose derivatives or dialdehyde cellulose derivatives or a combination thereof present in the lipid nanoparticle compositions may be from about 1 mol % to about 25 mol %.
In some embodiments, the proportion of cellulose derivatives or dialdehyde cellulose derivatives or combination thereof present in the lipid nanoparticle compositions is 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.
In some embodiments, the proportion of cellulose derivatives or dialdehyde cellulose derivatives or combination thereof present in the lipid nanoparticle compositions is from about 1 mol % to about 25 mol %, preferably about 1 mol % to about 20 mol %, most preferably about 1 mol % to about 15 mol % or any range therein.
In some embodiments, the proportion of cellulose or its derivatives or combination thereof present in the lipid nanoparticle compositions is about 1 mol %, about 2 mol %, about 3 mol %, about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about 10 mol %, about 11 mol %, about 12 mol %, about 13 mol %, 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.
Method of treatment In some aspects, provided herein is a method of treating or preventing a disease, comprising administering to a subject in need thereof the multitarget nucleic acid sequence as described herein.
In some aspects, provided herein is a method of treating or preventing a disease, comprising administering to a subject in need thereof the lipid nanoparticle composition comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid and the multitarget nucleic acid sequence as described herein.
In some aspects, provided herein is a method of treating or preventing a disease, comprising administering to a subject in need thereof the lipid nanoparticle composition comprising an ionizable polymer, a cationic lipid, a phospholipid, a sterol, a PEG-lipid and the multitarget nucleic acid sequence as described herein.
In some aspects, the disclosure relates to use of a lipid nanoparticle composition comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and the multitarget nucleic acid sequence as described herein in the manufacture of a medicament for the treatment or prevention of a disease in a subject.
In some embodiments, the multitarget nucleic acid sequence is present in biologically effective amount or therapeutically effective amount. In some embodiments, the biologically effective amount of multitarget nucleic acid sequence is between 0.1 μg to 2000 μg, 0.1 μg to 1800 μg, 0.1 μg to 1600 μg, 0.1 μg to 1600 μg, 0.1 μg to 1400 μg, 0.1 μg to 1200 μg, 0.1 μg to 1000 μg, 0.1 μg to 950 μg, 0.1 μg to 900 μg, 0.1 μg to 850 μg, 0.1 μg to 800 μg, 0.1 μg to 750 μg, 0.1 μg to 700 μg, 0.1 μg to 650, 0.1 μg to 600, 0.1 μg to 550, 0.1 μg to 500 μg, 0.1 to 450 μg, 0.1 μg to 400 μg, 0.1 μg to 350 μg, 0.1 to 300 μg, 0.1 to 200 μg or any range therein. In some embodiments, the biologically effective amount of multitarget nucleic acid sequence is from about 0.1 μg to 1000 μg, 0.1 μg to 950 μg, 0.1 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.
In some embodiments, the biologically effective amount of multitarget nucleic acid sequence is 0.1 μg, 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.6 μg, 0.7, μg, 0.8 μg, 0.9 μg, 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 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, 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 portion or fraction thereof.
In one aspect, provided herein is a nucleic acid comprising a plurality of polynucleotide sequences, wherein some or all polynucleotide sequence of the plurality comprises either a target sequence, a linker sequence, and a self-assembling sequence or a linker sequence, a target sequence, a linker sequence and a self-assembling sequence or a combination thereof, wherein each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequences of the plurality. In another aspect, provided herein is a nucleic acid comprising a plurality of polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises a target sequence, a linker sequence, and a selfassembling sequence or a second linker sequence, a second target sequence, a third linker sequence, and a second self-assembling sequence, wherein each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequences of the plurality. In another aspect, provided herein a nucleic acid comprising a plurality of polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises a target sequence, a linker sequence, and a self-assembling sequence, wherein each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequences of the plurality. In yet another aspect, provided herein is a nucleic acid comprising a plurality of polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence, wherein each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequences of the plurality. In one aspect, provided herein is a nucleic acid comprising a first plurality of polynucleotide sequences and a second plurality of polynucleotide sequences, each polynucleotide sequence of the first plurality comprises a first target sequence, a first linker sequence, and a first self-assembling sequence, wherein each polynucleotide sequence of the second plurality comprises a second linker sequence, a second target sequence, a third linker sequence, and a second self-assembling sequence, wherein each polynucleotide sequence of the first plurality and each polynucleotide sequence of the second plurality is connected to an adjacent polynucleotide sequence of the first plurality or an adjacent polynucleotide sequence of the second plurality by a cleavage sequence, and wherein the first polynucleotide sequence in the nucleic acid is a polynucleotide sequence of the first plurality or a polynucleotide sequence of the second plurality, and wherein the nucleic acid further comprises a signal sequence upstream of the first polynucleotide sequence of the first plurality or the second polynucleotide sequence of the second plurality, or a combination thereof. In another aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein some or all polypeptides of the plurality comprises either a target peptide, a linker peptide, and a self-assembling peptide or a linker peptide, a target peptide, a linker peptide and a self-assembling peptide or a combination thereof, wherein each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide, and wherein the nucleic acid further comprises a signal peptide on the amino-terminus of one or more of the polypeptides of the plurality. In another aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein some or all polypeptides of the plurality comprises either a target peptide, a linker peptide, and a self-assembling peptide or a second linker peptide, a second target peptide, a third linker peptide and a second self-assembling peptide or combination thereof, wherein each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide, and wherein the nucleic acid further comprises a signal peptide on the amino-terminus of the first polypeptide of the plurality or the second polypeptide of the plurality or a combination thereof. In one aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein each polypeptide of the plurality comprises a target peptide, a linker peptide, and a self-assembling peptide, wherein each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide, and wherein the nucleic acid further encodes a signal peptide on the amino-terminus of one or more of the polypeptides of the plurality. In another aspect, provided herein is a nucleic acid encoding a plurality of polypeptides, wherein each polypeptide of the plurality comprises a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide, wherein each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide, and wherein the nucleic acid further encodes a signal peptide on the amino-terminus of one or more of the polypeptides of the plurality. In yet another aspect, provided herein is nucleic acid encoding a first plurality of polypeptides and a second plurality of polypeptides, wherein each polypeptide of the first plurality comprises a target peptide, a linker peptide, and a self-assembling peptide, wherein each polypeptide of the second plurality comprises a second linker peptide, a second target peptide, a third linker peptide, and a second self-assembling peptide, wherein each polypeptide of the first plurality and each polypeptide of the second plurality is connected to an adjacent polypeptide of the first plurality or an adjacent polypeptide of the second plurality by a cleavage peptide, and wherein the first polypeptide encoded by the nucleic acid is a polypeptide of the first plurality or a polypeptide of the second plurality, and wherein the nucleic acid further encodes a signal peptide on the amino-terminus of the first polypeptide of the first plurality or the second polypeptide of the second plurality or a combination thereof.
Embodiments
Some of the embodiments of the present disclosure are set out in the following numbered paragraphs:
1. A nucleic acid comprising a plurality of polynucleotide sequences, wherein some or all polynucleotide sequence of the plurality comprises either a target sequence, a linker sequence, and a self-assembling sequence or a linker sequence, a target sequence, a linker sequence and a self-assembling sequence or a combination thereof, wherein each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequence of the plurality.
2. A nucleic acid comprising a plurality of polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises a target sequence, a linker sequence, and a self-assembling sequence, wherein each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequence of the plurality. 3. A nucleic acid comprising a plurality of polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence, wherein each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequence of the plurality.
4. A nucleic acid encoding a plurality of polypeptides, wherein some or all polypeptides of the plurality comprises either a target peptide, a linker peptide, and a selfassembling peptide or a linker peptide, a target peptide, a linker peptide and a selfassembling peptide or a combination thereof, wherein each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide, and wherein the nucleic acid further comprises a signal peptide on the amino-terminus of one or more of the polypeptide of the plurality.
5. A nucleic acid encoding a plurality of polypeptides, wherein each polypeptide of the plurality comprises a target peptide, a linker peptide, and a self-assembling peptide, wherein each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide, and wherein the nucleic acid sequence further encodes a signal peptide on the amino-terminus of one or more of the polypeptide of the plurality.
6. A nucleic acid encoding a plurality of polypeptides, wherein each polypeptide of the plurality comprises a linker peptide, a target peptide, a linker peptide, and a selfassembling peptide, wherein each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide, and wherein the nucleic acid sequence further encodes a signal peptide on the amino-terminus of one or more of the polypeptide of the plurality.
7. The nucleic acid according to any of the preceding paragraphs, wherein total number of the polynucleotide sequences is not more than 100.
8. The nucleic acid according to any of the preceding paragraphs, wherein total number of the polynucleotide sequences is between 2-10, 10-20, 20-30, 30-40, 40-50, 50- 60, 60-70, 70-80, 80-90, or 90-99. 9. The nucleic acid according to any of the preceding paragraphs, wherein the nucleic acid is a DNA or an RNA.
10. The nucleic acid according to paragraph 9, wherein the RNA is an mRNA.
11. The nucleic acid according to any of the preceding paragraph wherein the linker sequence encodes a linker peptide.
12. The nucleic acid according to paragraph 11, wherein the linker peptide is an amino acid linker, a foldon, a scaffold or a combination thereof.
13. The nucleic acid according to paragraph 12, wherein the amino acid linker comprises 2 to 49 amino acids.
14. The nucleic acid according to paragraph 13, wherein the amino acid linker is a glycine serine linker, a glycine proline linker, a glycine threonine linker, an alanine serine linker, any combination of two amino acids, or a combination thereof.
15. The nucleic acid according to any of the preceding paragraphs, wherein the linker peptide has an amino acid sequence of any one of SEQ ID NOs: 262-299, 330, and 350.
16. The nucleic acid according to any of the preceding paragraphs, wherein the selfassembling sequence encodes a self-assembling peptide.
17. The nucleic acid according to paragraph 16, wherein the self-assembling peptide is a lumazine synthase from Aquifex species, a hepatitis B surface antigen (HBsAg) from Hepatitis B Virus, a hepatitis B core antigen (HBcAg) from Hepatitis B virus, a human papillomavirus LI (HPV LI) protein, a matrix protein Ml from influenza A virus, a ferritin, a riboflavin synthase, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
18. The nucleic acid according to paragraph 17, wherein the ferritin comprises of a ferritin subunit or a ferritin peptide.
19. The nucleic acid according to paragraph 18, wherein the ferritin peptide is derived from Helicobacter pylori ferritin. 20. The nucleic acid according to any of the preceding paragraphs, wherein the selfassembling peptide has an amino acid sequence of any one of SEQ ID NOs: 254-261, 331 and 333.
21. The nucleic acid according to any of the preceding paragraphs, wherein the cleavage sequence encodes one or more cleavage peptide.
22. The nucleic acid according to paragraph 21, wherein the one or more cleavage peptides are optionally connected to each other by a linker peptide.
23. The nucleic acid according paragraph 22, wherein the cleavage peptide is a golgi specific cleavage peptide or a self-cleaving peptide.
24. The nucleic acid according to any of the preceding paragraphs, wherein the cleavage peptide has an amino acid sequence of any one of SEQ ID NOs: 300-311 and 347-349.
25. The nucleic acid according to any of the preceding paragraphs, wherein the signal sequence encodes a signal peptide.
26. The nucleic acid according to paragraph 25, wherein the signal peptide is present on the amino-terminus of one or more of the polypeptides of the plurality.
27. The nucleic acid according paragraph 26, wherein the nucleic acid further encodes a second signal peptide on the amino-terminus of all or some polypeptides of the plurality.
28. The nucleic acid according to any of the preceding paragraphs, wherein the signal peptide has an amino acid sequence of any one of SEQ ID NOs: 312-329.
29. The nucleic acid according to any of the preceding paragraphs, wherein the target sequence encodes a target peptide.
30. The nucleic acid sequence according to paragraph 29, wherein the target peptide is encoded by a codon optimized nucleic acid sequence, or fragments, mutants, or variants thereof.
31. The nucleic acid according to paragraph 30, wherein the target peptide is obtained from a prokaryote, a eukaryote, a unicellular organism, a multicellular organism, a virus, a bacterium, a fungus, a protozoan, a worm, a mycoplasma, an animal, a human or a combination thereof.
32. The nucleic acid according to paragraph 31, wherein the virus is selected from the family comprising picornaviride, calciviridae, astroviridae, togaviridae, flaviviridae, coronaviridae, arteriviridae, rhabndoviridae, filoviridae, paramyxoviridae, bomaviridae, orthomyxoviridae, bunyaviridae, arenaviridae, reoviridae, retroviridae, polyomaviridae, herpesviridae, poxviridae, papillomaviridae, hepadnaviridae, adenoviridae, parvoviridae, hepeviridae, circoviridae or a combination thereof.
33. The nucleic acid according to paragraph 31, wherein the bacterium is selected from the genus comprising Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio, Yersinia, or a combination thereof
34. The nucleic acid according to paragraph 32, wherein the virus is selected from the family comprising coronaviridae, herpesviridae, poxviridae, flaviviridae, togaviridae, retroviridae, paramyxoviridae, or a combination thereof.
35. The nucleic acid according to paragraph 31, wherein the virus is alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus, torovirus or a combination thereof.
36. The nucleic acid according to paragraph 35, wherein the betacoronavirus is a SARS-CoV-1, a SARS-CoV-2, a MERS-CoV, an OC43, a HKUl, a bat coronavirus, other betacoronavirus, or a combination thereof.
37. The nucleic acid according to 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 according to paragraph 37, wherein the target peptide is a spike protein, or its fragment thereof.
39. The nucleic acid according to paragraph 38, wherein the target peptide is a receptor binding domain, a fusion peptide, a stem helix of the spike protein, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants or variants thereof.
40. The nucleic acid according to paragraph 39, wherein the target peptide is a receptor binding domain obtained or derived from a betacoronavirus comprising SARS-CoV-1, SARS-CoV-2, MERS-CoV, OC43, HKU1, bat coronavirus, other betacoronavirus or a combination thereof.
41. The nucleic acid according to paragraph 30, wherein the target peptide is a glycoprotein B, a glycoprotein C, a glycoprotein D, a glycoprotein E, a glycoprotein K, a glycoprotein L, or a glycoprotein M, of a herpes simplex virus 1 (HSV-1) or a herpes simplex virus 2 (HSV-2), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants or variants thereof.
42. The nucleic acid according to paragraph 30, wherein the target peptide is a glycoprotein B, a glycoprotein H, a glycoprotein L, a glycoprotein M, or a glycoprotein N of a human cytomegalovirus (HCMV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
43. The nucleic acid according to paragraph 30, wherein the target peptide is a a glycoprotein B, a glycoprotein C, a glycoprotein H, or a glycoprotein L of a varicellazoster virus (VZV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
44. The nucleic acid according to paragraph 30, wherein the target peptide is a glycoprotein B, a glycoprotein H, a glycoprotein L, a glycoprotein M, a glycoprotein N, a glycoprotein 42, or a glycoprotein 350 of an Epstein-Barr virus (EBV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
45. The nucleic acid according to paragraph 30, wherein the target peptide is a F9 membrane protein of a poxvirus, a H3L protein of a poxvirus, an A4 protein of a poxvirus, an A27 protein of poxvirus, an A33 protein of a poxvirus, an A56 protein of a poxvirus, a B5 protein of a poxvirus, or a LI protein of a poxvirus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. 46. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of flaviviruses, hepaciviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
47. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a Japanese encephalitis virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
48. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a zika virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
49. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a yellow fever virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
50. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a awest nile virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
51. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a hepatitis C virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. 52. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a dengue virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
53. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid protein, or an envelope protein such as El, E2 and E3 protein of an alphaviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
54. The nucleic acid according to paragraph 30, wherein the target peptide is a domain A, a domain B, or a domain C of the E2 protein of an alphaviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
55. The nucleic acid according to paragraph 30, wherein the target peptide is a capsid protein, or an envelope protein such as El, E2 and E3 protein of a chikungunya virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
56. The nucleic acid according to paragraph 30, wherein the target peptide is a gag, a pol or an env proteins of retroviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
57. The nucleic acid according to paragraph 30, wherein the target peptide is a pl7Gag, a p24Gag, a p7Gag, a p6Gag, a gpl2Env, a gp41Env, or a pol proteins from lentiviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
58. The nucleic acid according to paragraph 30, wherein the target peptide is a pl7Gag, a p24Gag, a p7Gag, a p6Gag, a gpl2Env, a gp41Env, or a pol proteins from a human immunodeficiency virus (HIV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. 59. The nucleic acid according to paragraph 30, wherein the target peptide is a nucleocapsid protein, a P protein, a V protein, a W protein, a D protein, an I protein, a C protein, a L protein, a M protein, a H (hemagglutinin) protein, a HN (hemagglutininneuraminidase) protein, a G protein or a F protein, or a combination thereof, of a Mumps virus (MuV), a Parainfluenza virus type 5 (PIV5), a Human parainfluenza virus type 2, types 4a and 4b (HPIV2/4a/4b), a Newcastle disease virus (NDV), a Human parainfluenza virus type 1 and type 3 (HPIV1/3), a Nipah virus (NiV), a Measles virus (MeV), a Human respiratory syncytial virus A2, Bl, S2, (HRSV), or a Human metapneumovirus (HMPV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
60. The nucleic acid according to paragraph 30, wherein the target peptide is a nucleocapsid protein, a P protein, a V protein, a W protein, a D protein, an I protein, a C protein, a L protein, a M protein, a H (hemagglutinin) protein, a HN (hemagglutininneuraminidase) protein, a G protein or a F protein of human respiratory syncytial virus A2, Bl, S2 (HRSV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
61. The nucleic acid according to paragraph 30, wherein the target peptide is an El, an E2, an E4, an E5, an E6, an E7, a LI or a L2 protein of papillomavirus, preferably a human papillomavirus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
62. The nucleic acid according to any of the preceding paragraphs, wherein the target peptide has an amino acid sequence of any one of SEQ ID NOs: 1-253, 334-337, 338-346, and 353-388.
63. A lipid nanoparticle composition comprising a cationic lipid, a phospholipid, a sterol, a PEG-lipid, and the nucleic acid according to any of the preceding paragraphs.
64. The lipid nanoparticle composition according to paragraph 63, wherein the cationic lipid comprises an ionizable lipid.
65. The lipid nanoparticle composition according to paragraph 64, wherein the ionizable lipid is present in an amount from 25 mol percent to 70 mol percent. 66. The lipid nanoparticle composition according to paragraph 63, wherein the phospholipid is present in an amount from 2 mol percent to about 30 mol percent.
67. The lipid nanoparticle composition according to paragraph 63, wherein the sterol is present in an amount from 30 mol percent to about 65 mol percent.
68. The lipid nanoparticle composition according to paragraph 63, wherein the PEG- lipid is present in an amount from 0.2 mol percent to about 2.0 mol percent.
69. The lipid nanoparticle composition according to paragraph 63, additionally comprising an ionizable polymer.
70. The lipid nanoparticle composition according to paragraph 69, wherein the ionizable polymer is present in an amount from 1 mol percent to 25 mol percent.
71. The lipid nanoparticle composition according to paragraph 70, wherein the ionizable polymer is selected from the group comprising a chitosan, a cellulose derivative, a poly-L-lysine, a poly-L-glutamic acid, and/or their derivatives or a combination thereof.
72. A method of treating or preventing a disease, comprising administrating to a subject in need thereof the nucleic acid according to any one of claims 1-62.
73. A method of treating or preventing a disease, comprising administrating to a subject in need thereof the lipid nanoparticle composition according to any one of paragraphs 63-71.
74. Use of the nucleic acid sequence according to any one of paragraphs 1-62, in the manufacture of a medicament for the treatment or prevention of a disease in a subject.
75. Use of a lipid nanoparticle composition according to any one of paragraphs 63-71 in the manufacture of a medicament for the treatment or prevention of a disease in a subject.
76. A multitarget peptide encoded by the nucleic acid of any one of paragraphs 1-62.
77. A multitarget peptide comprising two or more polypeptides, wherein some or all polypeptides comprises either a target peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide or a combination thereof, wherein one polypeptide is connected to another polypeptide by a cleavage peptide, wherein the multitarget peptide includes a signal peptide on the aminoterminus of one or more of the polypeptides.
78. The multitarget peptide according to paragraph 77, wherein the linker peptide is an amino acid linker, a foldon, a scaffold or a combination thereof.
79. The multitarget peptide according to paragraph 78, wherein the amino acid linker comprises 2-49 amino acids.
80. The multitarget peptide according to paragraph 78, wherein the amino acid linker is selected form the group comprising a glycine serine linker, a glycine proline linker, a glycine threonine linker, an alanine serine linker, any combination of two amino acids, or a combination thereof.
81. The multitarget peptide according to paragraph 77, wherein the self-assembling peptide is selected from the group comprising a lumazine synthase from Aquifex species, a hepatitis B surface antigen (HBsAg) from Hepatitis B Virus, a hepatitis B core antigen (HBcAg) from Hepatitis B virus, a human papillomavirus LI (HPV LI) protein, a matrix protein Ml from influenza A virus, a ferritin, a riboflavin synthase, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
82. The multitarget peptide according to paragraph 81, wherein the ferritin comprises a ferritin subunit or a ferritin peptide.
83. The multitarget peptide according to paragraph 82, wherein the ferritin peptide is derived from Helicobacter pylori ferritin.
84. The multitarget peptide according to paragraph 77, wherein the cleavage peptide is a golgi specific cleavage peptide or a self-cleaving cleavage peptide or a combination thereof.
85. The multitarget peptide according to paragraph 77, wherein the signal peptide may be present on the amino-terminus of all or some polypeptides. 86. The multitarget peptide according to paragraph 77, wherein the target peptide is obtained or derived from a prokaryote, a eukaryote, a unicellular organism, a multicellular organism, a virus, a bacterium, a fungus, a protozoan, a worm, a mycoplasma, an animal, a human or a combination thereof.
87. The multitarget peptide according to paragraph 86, wherein the virus is selected from the family comprising picomaviride, calciviridae, astroviridae, togaviridae, flaviviridae, coronaviridae, arteriviridae, rhabndoviridae, filoviridae, paramyxoviridae, bornaviridae, orthomyxoviridae, bunyaviridae, arenaviridae, reoviridae, retroviridae, polyomaviridae, herpesviridae, poxviridae, papillomaviridae, hepadnaviridae, adenoviridae, parvoviridae, hepeviridae, circoviridae or a combination thereof.
88. The multitarget peptide according to paragraph 86, wherein the bacterium is selected from the genus comprising Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio, Yersinia, or a combination thereof
89. The multitarget peptide according to paragraph 87, wherein the virus is selected from the family comprising coronaviridae, herpesviridae, poxviridae, flaviviridae, togaviridae, retroviridae, paramyxoviridae, or a combination thereof.
90. The multitarget peptide according to paragraph 89, wherein the virus is alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus, torovirus or a combination thereof.
91. The multitarget peptide according to paragraph 90, wherein the betacoronavirus is selected from the group comprising a SARS-CoV-1, a SARS-CoV-2, a MERS-CoV, an OC43, a HKU1, a bat coronavirus, other betacoronavirus, or a combination thereof.
92. The multitarget peptide according to paragraph 77, wherein the target peptide is a spike protein, a membrane protein, an envelope protein or a nucleocapsid protein of coronaviruses, or a combination thereof, including their codon optimized nucleic acid sequences fragments, mutants or variants thereof. 93. The multitarget peptide according to paragraph 92, wherein the target peptide is a spike protein or its fragment thereof.
94. The multitarget peptide according to paragraph 93, wherein the target peptide is a receptor binding domain, a fusion peptide, a stem helix of the spike protein, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
95. The multitarget peptide according to paragraph 77, wherein the target peptide is a glycoprotein B, a glycoprotein C, a glycoprotein D, a glycoprotein E, a glycoprotein K, a glycoprotein L, or a glycoprotein M of a herpes simplex virus 1 (HSV-1) or a herpes simplex virus 2 (HSV-2), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants or variants thereof.
96. The multitarget peptide according to paragraph 77, wherein the target peptide is a glycoprotein B, a glycoprotein H, a glycoprotein L, a glycoprotein M, or a glycoprotein N of a human cytomegalovirus (HCMV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
97. The multitarget peptide according to paragraph 77, wherein the target peptide is a glycoprotein B, a glycoprotein C, a glycoprotein H, or a glycoprotein L of a varicellazoster virus (VZV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
98. The multitarget peptide according to paragraph 77, wherein the target peptide is a glycoprotein B, a glycoprotein H, a glycoprotein L, a glycoprotein M, a glycoprotein N, a glycoprotein 42, a glycoprotein 350 of an Epstein-Barr virus (EBV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
99. The multitarget peptide according to paragraph 77, wherein the target peptide is a F9 membrane protein of a poxvirus, a H3L protein of a poxvirus, an A4 protein of a poxvirus, an A27 protein of poxvirus, an A33 protein of a poxvirus, an A56 protein of a poxvirus, a B5 protein of a poxvirus, or a LI protein of a poxvirus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. 100. The multitarget peptide according to paragraph 77, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of flaviviruses, hepaciviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
101. The multitarget peptide according to paragraph 77, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a Japanese encephalitis virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
102. The multitarget peptide according to paragraph 77, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a zika virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
103. The multitarget peptide according to paragraph 77, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a yellow fever virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
104. The multitarget peptide according to paragraph 77, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a west nile virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
105. The multitarget peptide according to paragraph 77, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a hepatitis C virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. 106. The multitarget peptide according to paragraph 77, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non- structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a dengue virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
107. The multitarget peptide according to paragraph 77, wherein the target peptide is a capsid protein, or an envelope protein such as El, E2 and E3 protein of an alphaviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
108. The multitarget peptide according to paragraph 77, wherein the target peptide is a domain A, a domain B, a domain C of the E2 protein of an alphaviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
109. The multitarget peptide according to paragraph 77, wherein the target peptide is a capsid protein, or an envelope protein such as El, E2 and E3 protein of a chikungunya virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
110. The multitarget peptide according to paragraph 77, wherein the target peptide is a gag, a pol and an env proteins of retroviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
111. The multitarget peptide according to paragraph 77, wherein the target peptide is a pl7Gag, a p24Gag, a p7Gag, a p6Gag, a gpl2Env, a agp41Env, or a pol proteins from lentiviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
112. The multitarget peptide according to paragraph 77, wherein the target peptide is a pl7Gag, a p24Gag, a p7Gag, a p6Gag, a gpl2Env, a gp41Env, or a pol proteins from a human immunodeficiency virus (HIV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof. 113. The multitarget peptide according to paragraph 77, wherein the target peptide is a nucleocapsid protein, a P protein, a V protein, a W protein, a D protein, an I protein, a C protein, a L protein, a M protein, a H (hemagglutinin) protein, a HN (hemagglutininneuraminidase) protein, a G protein, a F protein, or a combination thereof, of a Mumps virus (MuV), a Parainfluenza virus type 5 (PIV5), a Human parainfluenza virus type 2, types 4a and 4b (HPIV2/4a/4b), a Newcastle disease virus (NDV), a Human parainfluenza virus type 1 and type 3 (HPIV1/3), a Nipah virus (NiV), a Measles virus (MeV), a Human respiratory syncytial virus A2, Bl, S2, (HRSV), or a Human metapneumovirus (HMPV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
114. The multitarget peptide according to paragraph 77, wherein the target peptide is a nucleocapsid protein, a P protein, a V protein, a W protein, a D protein, an I protein, a C protein, a L protein, a M protein, a H (hemagglutinin) protein, a HN (hemagglutininneuraminidase) protein, a G protein or a F protein of a human respiratory syncytial virus A2, Bl, S2 (HRSV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
115. The multitarget peptide according to paragraph 77, wherein the target peptide is an El, an E2, an E4, an E5, an E6, an E7, a LI, or a L2 protein of a papillomavirus, preferably a human papillomavirus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
116. The multitarget peptide according to any one of paragraphs 77-115, wherein total number of the polypeptides are not more than 100.
117. The multitarget peptide according to paragraph 116, wherein total number of the polypeptides is between 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-99.
118. A Polypeptide nanoparticle comprising at least 2 or up to 100 polypeptides according to any one of paragraphs 1 to 62 or 77-115.
119. The polypeptide nanoparticle according to paragraph 118, wherein the polypeptides are homologous polypeptides, heterologous polypeptides, oligomeric complex or combination thereof. 120. The polypeptide nanoparticle according to paragraph 118, wherein the polypeptide nanoparticle is icosahedral, helical, spherical, rod-like or a combination thereof.
Examples
Example 1: Synthesis of multitarget nucleic acid sequence (mRNA)
Plasmid DNA construction
The multitarget nucleic acid sequence comprising of signal sequence, three repeats of polynucleotide sequences, each separated by cleavage sequence, was codon optimized for human expression. The multitarget nucleic acid sequence was synthesised by Twist BioSciences, USA. Each polynucleotide sequence consisted of a different target sequence. The first polynucleotide sequence consisted of a target sequence (encoding receptor binding domain (RBD) of SARS-CoV-2), linker sequence (glycine serine linker sequence encoding glycine serine linker, foldon sequence encoding foldon, & glycine serine linker sequence encoding glycine serine linker) and a self-assembling sequence (ferritin sequence encoding ferritin). The second polynucleotide sequence consisted of a linker sequence (scaffold sequence encoding scaffold, and glycine serine linker sequence encoding glycine serine linker), a target sequence (encoding fusion peptide of SARS-CoV-2), a linker sequence (glycine serine linker sequence encoding glycine serine linker, scaffold sequence encoding scaffold, and glycine serine linker sequence encoding glycine serine linker), and a self-assembling sequence (ferritin sequence encoding ferritin). The third polynucleotide sequence consisted of a linker sequence (scaffold sequence encoding scaffold, and glycine serine linker sequence encoding glycine serine linker), a target sequence (encoding stem helix of SARS-CoV-2), a linker sequence (glycine serine linker sequence encoding glycine serine linker, scaffold sequence encoding scaffold, and glycine serine linker sequence encoding glycine serine linker) and a self-assembling sequence (ferritin sequence encoding ferritin). A signal sequence (golgi target signal sequence) was included upstream of the first polynucleotide sequence. 5’ UTR, cap, and 3’ UTR sequences were also included in the multitarget nucleic acid sequence. The multitarget nucleic acid sequence construct was inserted between Hindlll and BamHI restriction sites of pTwist Kan High Copy (Twist BioScience, USA). Sequence of the multitarget peptide encoded by the above multitarget nucleic acid sequence, is provided Table 1. Table 1 shows Sequence of the multitarget peptide encoded by the above multitarget nucleic acid sequence.
Figure imgf000105_0001
Amino acid sequence of different peptides present in the multitarget peptide encoded by the multitarget nucleic acid sequence of example 1 is separately identified in: Complete sequence of the multitarget peptide encoded by the multitarget nucleic acid of example 1
Figure imgf000106_0001
*The different bolded, underlined, and italicized sequences correspond to the different sequences in Table 1.
Plasmid DNA isolation and linearization
Plasmid DNA was obtained by standard techniques. Briefly, the plasmid DNA was introduced into E. coli DH5 alpha cells (Stellar™ Competent Cells Catalogue no. 636763, Takara Bio) by giving heat shock for 60-90 seconds at 42 °C, followed by cold shock for 5 min on ice. The cells were transferred to LB (Lauria-Bertani) medium in a tube and incubated for 1 h at 37 °C on a thermal shaker at 600-900 rpm. The cells were recovered and centrifuged at 4000 g for 2 min, pellet was collected and resuspended in the same medium. Resuspended cells were transferred to agar plate containing LB medium supplemented with kanamycin and cells were spread evenly using sterile glass beads and incubated for 14-16 h at 37 °C. Well grown colonies were picked and resuspended in LB medium supplemented with kanamycin and colony PCR was performed to check the presence of pDNA. The colonies were further cultured overnight and NucleoSpin® Plasmid Miniprep kit (catalogue no. 740588, Takara Bio Inc.) was used to isolate the pDNA. The pDNA was linearized by digestion with BbsI-HF restriction enzyme (catalogue no. R3539L, New England Biolabs Inc,). pDNA was quantified using NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer (Catalogue no. ND- ONE-W, Thermo Scientific).
Invitro transcription (IVT)
IVT was performed following standard procedure described in Hi Scribe™ T7 High Yield RNA Synthesis Kit (Catalogue no. E2040S, New England BioLabs Inc.). Briefly, IVT mix containing four ribonucleotide triphosphates (ATP, UTP, GTP, CTP), NEB T7 buffer, NEB T7 enzyme mix, CleanCap® Reagent AG (3' OMe) (Catalogue no, N-7413, TriLink BioTechnologies), and linearized plasmid DNA was taken in a PCR tube containing nuclease free sterile water. The mixture was incubated for 37 °C for about 2 hours. This was followed by DNase I (Catalogue no. M0303S, New England BioLabs Inc.) treatment to degrade any plasmid DNA. The mRNA was purified as per instructions given in Monarch® RNA Cleanup Kit (Catalogue no. T2050L, New England BioLabs Inc.). Poly A tail was added to purified mRNA following standard post tailing procedure using E. coli Poly(A) Polymerase (Catalogue no. M0276L, New England BioLabs). The mRNA with poly A tail was purified using Monarch® RNA Cleanup Kit (Catalogue no. T2050L, New England BioLabs Inc.). The RNA was quantified by Qubit™ RNA BR, Assay Kits (Catalogue no. QI 0210, Thermo Scientific) using Qubit 4 Fluorometer (Thermo Scientific).
Transfection
HEK293T cells (Catalogue no. CRL3216, ATCC) were seeded at 0.25 - 1 x 106 cells in a 6 well plate containing Gibco DMEM medium, high glucose, pyruvate (Catalogue no. 11995065, ThermoFisher Scientific) supplemented with 10% fetal bovine serum and 100 units/mL penicillin-streptomycin, and cultured to achieve 70-80% confluency. Transfection mix containing multitarget nucleic acid (mRNA, 3 ug of example 1) and Lipofectamine™ 2000 (9 uL) in 100 uL serum free medium was incubated for 15 min at room temperature and added to the cells and left for incubation for 24 and 48 hours. Spent media was collected by aspiration, and cells were collected with ice-cold lx PBS (1 mL) with hard pipetting. The cells were centrifuged at 4000 g for 5 min at 4 °C. Excess PBS was aspirated. The cells were suspended gently in 300 pL of lx NETN lysis buffer with inhibitors and kept on ice for 20 min. The lysate was centrifuged at 16,000 g for 20 min at 4 °C. The supernatant was aspirated into microtubes. Western Blot
6x Laemmli buffer was added to each sample (supernatant or cell lysate) to obtain a final concentration of lx. The samples were heated at 95 °C for 5 min and centrifuged at 10,000 g for 5 min. About 21 pL of cell lysate and 42 pL of supernatant along with molecular weight standard were added to the gel (NuPAGE 4-12% Bis-Tris Gel) and the gel was run using a mops-SDS running buffer. The proteins were transferred onto the PVDF membrane using iBlot 2 blotting system. The blots were blocked using 5% skimmed milk in lx TBST for 1 h and incubated with primary antibody (SARS-CoV-2 (2019-nCoV) Spike Antibody, Rabbit PAb Catalogue no. 40589-T62, Sino Biological Inc., or SARS-CoV-2 spike RBD polyclonal antibody (Catalogue no. E-AB-V-1006 Elab Sciences) for 1 h at room temperature. The membrane was washed thrice, and incubated with secondary antibody (anti rabbit IgG HRP Catalogue no. 7074S, manufacturer) for 1 h. The membrane was washed thrice and 50 pL luminol and 50 pL peroxide solution was used for signal development. The image was captured using ChemiDoc™ Imaging System (Bio-Rad). The results are shown in figures 3a and 3b.
ELISA
ELISA was performed on cell lysate and supernatant by following instructions as provided in SARS-CoV-2 (2019-nCoV) Spike Detection ELISA Kit (Catalogue no. KIT40591, Sino Biological, Inc.). The results are shown in figures 4a and 4b.
Transmission Electron Microscopy (TEM)
Post 48 h of transfection, 1 mL of the spent media is concentrated to 0.5 mL with ultrafiltration using a 100 kDa concentrator (Catalogue no. UFC210024, Millipore Amicon Ultra-2 mL) at 3000 g for 3 min. The sample was buffer exchanged 10 times with lx PBS using centricon filter. Finally, the sample was further concentrated to 200-250 pL by ultrafiltration. 10 pL of the sample was placed on a glow-discharged carbon-coated copper grid of 400 mesh and incubated for 1 min. The excess sample was removed by careful soaking with blotting paper. 10 pL of UranyLess staining reagent (Catalogue no. 22409, Electron Microscopy Sciences) was added and incubated for 1 min. Excess sample was removed as above. The sample was air-dried by incubating at room temperature for 1 h. TEM images were captured by Talos™ L120C TEM (ThermoFisher Scientific). The results are shown in figure 5.
Analytical or Immunogenicity assays
The testing of multitarget nucleic acid sequence comprises immunizing animals (typically mice) with multitarget nucleic acid sequence in appropriate formulation following prime-boost immunization strategy at pre-determined dosage amounts. The serum is collected at appropriate intervals and antibody response against the target peptide is measured by ELISA.
The efficacy of the multitarget nucleic acid sequence is evaluated by pseudovirus neutralization assays well known to persons skilled in the art. The method typically involves incubating the pseudovirus in the presence of different concentrations of immunised serum containing the antibody of interest (i.e., antibodies produced against the target peptide) and adding this mixture to the cells and incubating it further to measure luminescence to determine inhibitory or neutralization titre.
Example 2: synthesis of multitarget nucleic sequence (mRNA) - trivalent RBD construct
Plasmid DNA construction
The multitarget nucleic acid sequence of example 2 comprising three signal sequences, three repeats of polynucleotide sequences, each separated by cleavage sequence was codon optimized for human expression. The multitarget nucleic acid sequence was synthesized by Twist BioSciences, USA. Each polynucleotide sequence consisted of a different target sequence. The first polynucleotide sequence consisted of a target sequence (encoding receptor binding domain (RBD) of SARS-CoV-2 SBB.1.5 variant), a linker sequence (glycine serine linker sequence encoding glycine serine linker) and a selfassembling sequence (ferritin sequence encoding ferritin). The second polynucleotide sequence consisted of a target sequence (encoding receptor binding domain (RBD) of SRAS-CoV-1), a linker sequence (glycine serine linker sequence encoding glycine serine linker), and a self-assembling sequence (ferritin sequence encoding ferritin). The third polynucleotide sequence consisted of a target sequence (encoding receptor binding domain (RBD) of MERS-CoV), a linker sequence (glycine serine linker sequence encoding glycine serine linker), and a self-assembling (ferritin sequence encoding ferritin). The polynucleotide sequences were separated by a cleavage sequence encoding two cleavage peptides (cleavage peptide- 1 and cleavage peptide-2) connected by a linker sequence (glycine serine linker sequence encoding glycine serine linker). A signal sequence was included upstream of each of the three polynucleotide sequences. 5’ UTR, cap, and 3’ UTR sequences were also included in the multitarget nucleic acid sequence. The multitarget nucleic acid sequence construct was inserted between Hindlll and BamHI restriction sites of pTwist Kan High Copy (Twist BioScience, USA). Sequence of the multitarget peptide encoded by the above multitarget nucleic acid sequence of example 2, is provided in Table 2.
Table 2: shows sequence of the multitarget peptide encoded by the multitarget nucleic acid sequence - trivalent RBD construct of example 2.
Figure imgf000110_0001
Figure imgf000111_0001
Amino acid sequence of different peptides present in the multitarget peptide encoded by the multitarget nucleic acid sequence of example 1 is separately identified in:
Complete sequence of the multitarget peptide encoded by the multitarget nucleic acid of example 2
Figure imgf000111_0002
*The different bolded, underlined, and italicized sequences correspond to the different sequences in Table 2. Plasmid DNA isolation and linearization, and Invitro transcription (IVT) of the multitarget nucleic acid sequence of example 2 has been performed as described in example 1.
Transfection
HEK293T cells (Catalogue no. CRL3216, ATCC) were seeded at 0.05 xlO6 cells in a 24 well plate containing Gibco DMEM medium, high glucose, pyruvate (Catalogue no. 11995065, ThermoFisher Scientific) supplemented with 10% fetal bovine serum and 100 units/mL penicillin-streptomycin, and cultured to achieve 70-80% confluency. Transfection mix containing multitarget nucleic acid sequence of example 2 (mRNA, 0.5-1 ug of example 2) and Lipofectamine™ 2000 (2.5 uL) in 100 uL serum free medium was incubated for 15 min at room temperature and added to the cells and left for incubation for 24 and 48 hours. Spent media was collected by aspiration, and cells were collected with ice-cold lx PBS (1 mL) with hard pipetting. The cells were centrifuged at 4000 g for 5 min at 4 °C. Excess PBS was aspirated. The cells were suspended gently in 100 pL of lx NETN lysis buffer (100 mM NaCl, 20 mM Tris-Cl (pH 8.0), 0.5 mM EDTA, 0.5 % (v/v) Nonidet P-40 (NP-40)) with inhibitors and kept on ice for 20 min. The lysate was centrifuged at 16,000 g for 20 min at 4 °C. The supernatant was aspirated into microtubes.
Western Blot
A western blot was performed on cell lysate, obtained in the previous step, using Jess instrument (an automated western blot system Catalogue no. 004-650, ProteinSimple, Bio-Techne). The cell lysate was diluted and combined with 1 part 5x fluorescent master mix (component of Separation Module compatible with Jess, Catalogue no. SM-W001, ProteinSimple, Bio-Techne) and heated for 5 min at 95 °C. After protein denaturation, the sample was mixed with luminol-S and peroxide as per manufacturer protocol (Separation Module, Catalogue no. SM-W001, ProteinSimple, Bio-Techne). The primary antibody (SARS-CoV-2 spike RBD polyclonal antibody (Catalogue no. E-AB-V-1006 Elab Sciences), diluted at a 1 :200 ratio along with a secondary -HRP ready to use anti-rabbit antibody and chemiluminescent substrate (both components of Detection Module compatible with Jess, Catalogue no. DM-001, ProteinSimple, Bio-Techne), were dispensed into assigned wells within a provided microplate by the manufacturer. This plate was then inserted into the Jess instrument where samples were drawn into individual capillaries
Ill located on a 25-capillary cassette (12-230 kDa Separation Module Catalogue no. SM- W001, ProteinSimple, Bio-Techne). Electrophoresis and immunodetection were conducted in automated manner by the Jess instrument. The data was obtained by using the compass software associated with Jess system. The results are shown in Figure 6.
Immunogenicity Assay
Immunogenicity of multitarget nucleic acid encoding multitarget peptide of the example 2 was assessed by ELISA. Mice were immunized with the multitarget nucleic acid sequence (mRNA) - trivalent RBD construct encapsulated in a lipid nanoparticle and sera collected on day 28 was used to perform the ELISA to find out titre against each of the target peptides encoded by the construct of example 2.
Plate preparation: Three ELISA plates were coated, each with SI subunit of spike protein either from SARS-CoV-2 (XBB 1.5 variant), SARS-CoV-1, or MERS-CoV at a concentration of 100 ng in 100 pL coating buffer (IX PBS, pH 7.4) per well. The ELISA plates were gently tapped to ensure the SI subunit of spike protein is evenly coated on the bottom of every well of the three ELISA plates. The ELISA plates were covered with a plate sealer and incubated overnight at 4 °C. After overnight incubation, coating buffer was discarded and the ELISA plates were washed using 300 pL wash buffer (IX PBS supplemented with 0.05% (v/v) Tween20) thrice. The ELISA plates were blotted on paper towel to remove residual liquid from the wells. 200 pL of blocking buffer (lx PBS with 3% NFDM) was added to each well and the plates were incubated at room temperature for 2 h.
Sample preparation: Mice sera samples were 3-fold serially diluted from 1 : 100 up to 1 :218700 in lx PBS containing 1% NFDM in a dilution plate.
Testing: Blocking solution from the ELISA plates was discarded and residual liquid was removed from the wells using paper towel. ELISA plates were washed thrice using wash buffer (composition as previously mentioned). 100 pL of the diluted sera sample from dilution plate was transferred to ELISA plates (in duplicates) as per the predefined plate layout. ELISA plates were covered with plate sealer and incubated at room temperature for 1 h. After incubation, sera sample was discarded from the ELISA plates and the plates were washed thrice using wash buffer (composition as previously mentioned).100 pL of diluted secondary antibody solution (1 :5000 in IX PBS) was added to each well and the ELISA plates were covered with plate sealer and incubated at room temperature for 1 h. ELISA plates were then washed thrice using wash buffer (composition as previously mentioned). 100 μL of TMB substrate was added to each well and incubated for 15 min. The enzyme substrate reaction was stopped by adding 50 pL of IM HCL to each well. The absorbance was recorded after 5 min using Spark multimode microplate reader (Tecan Trading AG) at the dual wavelength of 450 nm and 630 nm. End point titre was calculated in the following manner:
Cutoff = (Mean of negative control (NC) of all plates) x 4
NC = wells devoid of any sample i.e., containing PBS (phosphate buffered saline).
Endpoint Titre = (Respective Dilution/Cutoff value) x Respective Mean Absorbance Value
Endpoint Titre was considered as the highest sample dilution sample above the cutoff. The results are shown in figure 7.
Pseudovirus Generation and Pseudovirus Neutralization Assay
Lentiviral HIV- 1 -based pseudoviruses harbouring spike glycoprotein of SARS- CoV-2 XBB.1.5), SARS-CoV-1 and MERS-CoV were generated by transfecting Lenti- X™ 293T cells (Catalogue no. 632180, Takara Bio) with 2nd generation packaging plasmid psPAX2 vector (Catalogue no. V010353, Novopro Labs), spike expressing plasmids (Twist Biosciences), and lentiviral firefly luciferase reporter plasmid (Catalogue no. LR151, Alstem Inc.,) with Lipofectamine™ 3000 Transfection Reagent (Catalogue no. L3000150, Invitrogen). After 12-16 hours of incubation at 37° C and 5% CO2, the transfection media was replaced with DMEM+10% FBS. The supernatants containing pseudovirus particles were collected 72 hours post-transfection, centrifuged, filtered, aliquoted, and stored at -80 °C.
A pseudovirus neutralization assay was performed to evaluate neutralizing antibodies in pooled sera from the groups of six mice immunized with multitarget nucleic acid sequence - trivalent RBD construct of example 2. Briefly, in tissue culture-treated white opaque 96 well plates, IxlO4 ACE2 stably expressing HEK293 cells (Catalogue no. 79951, BPS Biosciences) were seeded per well in growth media (DMEM+10% FBS+ 1% Pen-Strep) and incubated overnight at 37 °C and 5% CO2. Next, 3-fold serially diluted pooled mouse sera starting from 1 : 10 initial dilution was prepared in growth media and mixed in a 1 : 1 ratio with respective pseudoviruses that yield Relative Luminescence Units (RLU) values ranging from 0.5x105 RLU to 5x105 RLU. The mixture is incubated at 37 °C for one hour, added to the HEK293-ACE cells, and incubated at 37° C and 5% CO2. After 72 hours, luminescence activity was measured using Bright-Glo™ Luciferase Assay System (Catalogue no. E2650, Promega) on Tecan Spark multimode Reader via Spark control Magellan 3.1 software (Tecan Trading AG). Percentage neutralization was calculated by normalizing the test RLU with virus control and cell-only control RLU. The pseudovirus neutralization reciprocal IC50 ((half-maximal inhibitory concentration) titres were calculated using a non-linear regression curve fit Tog(inhibitor) vs. response — Variable slope (four parameters)’ in GraphPad Prism 10. The results are shown in figures 8a, 8b and 8c.
Example 3: synthesis of multitarget nucleic sequence (mRNA) - pentavalent RBD construct
Plasmid DNA construction
The multitarget nucleic acid sequence of example 3 comprising five signal sequences, five repeats of polynucleotide sequences, each separated by cleavage sequence was codon optimized for human expression. The multitarget nucleic acid sequence was synthesized by Twist BioSciences, USA. Each polynucleotide sequence consisted of a different target sequence. The first polynucleotide sequence consisted of a target sequence (encoding receptor binding domain (RBD) of SARS-CoV-2 SBB.1.5 variant), a linker sequence (glycine serine linker sequence encoding glycine serine linker) and a selfassembling sequence (ferritin sequence encoding ferritin). The second polynucleotide sequence consisted of a target sequence (encoding receptor binding domain (RBD) of SRAS-CoV-1), a linker sequence (glycine serine linker sequence encoding glycine serine linker), and a self-assembling sequence (ferritin sequence encoding ferritin). The third polynucleotide sequence consisted of a target sequence (encoding receptor binding domain (RBD) of MERS-CoV), a linker sequence (glycine serine linker sequence encoding glycine serine linker), and a self-assembling sequence (ferritin sequence encoding ferritin). The fourth polynucleotide sequence consisted of a target sequence (encoding receptor binding domain (RBD) of OC43), a linker sequence (glycine serine linker sequence encoding glycine serine linker), and a self-assembling sequence (ferritin sequence encoding ferritin). The fifth polynucleotide sequence consisted of a target sequence (encoding receptor binding domain (RBD) of HKU1), a linker sequence (glycine serine linker sequence encoding glycine serine linker), a self-assembling sequence (ferritin sequence encoding ferritin). The polynucleotide sequences were separated by a cleavage sequence encoding two cleavage peptides (cleavage peptide- 1 and cleavage peptide-2) connected by a linker sequence (glycine serine linker sequence encoding glycine serine linker). A signal sequence was included upstream of each of the five polynucleotide sequences. 5’ UTR, cap, and 3’ UTR sequences were also included in the multi target nucleic acid sequence. The multitarget nucleic acid sequence construct was inserted between Hindlll and BamHI restriction sites of pTwist Kan High Copy (Twist BioScience, USA). Sequence of the multitarget peptide encoded by the multitarget nucleic acid sequence - pentavalent RBD construct of example 3, is provided in Table 3.
Table 3: shows sequence of the multitarget peptide encoded by the multitarget nucleic acid sequence - pentavalent RBD construct of example 3.
Figure imgf000116_0001
Figure imgf000117_0001
Amino acid sequence of different peptides present in the multitarget peptide encoded by the multitarget nucleic acid sequence - pentavalent RBD construct of example 3 is separately identified in:
Complete sequence of the multitarget peptide encoded by the multitarget nucleic acid of example 3
Figure imgf000118_0001
Figure imgf000119_0001
*The different bolded, underlined, and italicized sequences correspond to the different sequences in Table 3,
Plasmid DNA isolation and linearization, and Invitro transcription (IVT) of the multitarget nucleic acid sequence of example 3 has been done as described in example 1.
Transfection
HEK293T cells (Catalogue no. CRL3216, ATCC) were seeded at 0.05 xlO6 cells in a 24 well plate containing Gibco DMEM medium, high glucose, pyruvate (Catalogue no. 11995065, ThermoFisher Scientific) supplemented with 10% fetal bovine serum and 100 units/mL penicillin-streptomycin, and cultured to achieve 70-80% confluency. Transfection mix containing multitarget nucleic acid of example 3 (mRNA, 0.5-1 ug of example 3) and Lipofectamine™ 2000 (2.5 uL) in 100 uL serum free medium was incubated for 15 min at room temperature and added to the cells and left for incubation for 24 and 48 hours. Spent media was collected by aspiration, and cells were collected with ice-cold lx PBS (1 mL) with hard pipetting. The cells were centrifuged at 4000 g for 5 min at 4 °C. Excess PBS was aspirated. The cells were suspended gently in 100 pL of lx NETN lysis buffer (100 mM NaCl, 20 mM Tris-Cl (pH 8.0), 0.5 mM EDTA, 0.5 % (v/v) Nonidet P-40 (NP-40)) with inhibitors and kept on ice for 20 min. The lysate was centrifuged at 16,000 g for 20 min at 4 °C. The supernatant was aspirated into microtubes.
Western Blot
A western blot was performed on cell lysate, obtained in the previous step, using Jess instrument (an automated western blot system Catalogue no. 004-650, ProteinSimple, Bio-Techne). The cell lysate was diluted and combined with 1 part 5x fluorescent master mix (component of Separation Module compatible with Jess, Catalogue no. SM-W001, ProteinSimple, Bio-Techne) and heated for 5 min at 95 °C. After protein denaturation, the sample was mixed with luminol-S and peroxide as per manufacturer protocol (Separation Module, Catalogue no. SM-W001, ProteinSimple, Bio-Techne). The primary antibody (SARS-CoV-2 spike RBD polyclonal antibody (Catalogue no. E-AB-V-1006 Elab Sciences), diluted at 1 :200 ratio along with a secondary -HRP ready to use anti-rabbit antibody and chemiluminescent substrate (both components of Detection Module compatible with Jess, Catalogue no. DM-001, ProteinSimple, Bio-Techne), were dispensed into assigned wells within a provided microplate by the manufacturer. This plate was then inserted into the Jess instrument where samples were drawn into individual capillaries located on a 25-capillary cassette (12-230 kDa Separation Module Catalogue no. SM- W001, ProteinSimple, Bio-Techne). Electrophoresis and immunodetection were conducted in automated manner by the Jess instrument. The data was obtained by using the compass software associated with Jess system. The results are shown in figure 9.
INCORPORATION BY REFERENCE
Each of the patents, published patent applications, and non-patent references cited herein are hereby incorporated by reference in their entirety.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed:
1. A nucleic acid comprising a plurality of polynucleotide sequences, wherein some or all polynucleotide sequence of the plurality comprises either a target sequence, a linker sequence, and a self-assembling sequence or a linker sequence, a target sequence, a linker sequence and a self-assembling sequence or a combination thereof, wherein each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequence of the plurality.
2. A nucleic acid comprising a plurality of polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises a target sequence, a linker sequence, and a self-assembling sequence, wherein each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequence of the plurality.
3. A nucleic acid comprising a plurality of polynucleotide sequences, wherein each polynucleotide sequence of the plurality comprises a linker sequence, a target sequence, a linker sequence, and a self-assembling sequence, wherein each polynucleotide sequence of the plurality is connected to an adjacent polynucleotide sequence of the plurality by a cleavage sequence, and wherein the nucleic acid further comprises a signal sequence upstream of one or more of the polynucleotide sequence of the plurality.
4. A nucleic acid encoding a plurality of polypeptides, wherein some or all polypeptides of the plurality comprises either a target peptide, a linker peptide, and a selfassembling peptide or a linker peptide, a target peptide, a linker peptide and a selfassembling peptide or a combination thereof, wherein each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide, and wherein the nucleic acid further comprises a signal peptide on the amino-terminus of one or more of the polypeptide of the plurality.
5. A nucleic acid encoding a plurality of polypeptides, wherein each polypeptide of the plurality comprises a target peptide, a linker peptide, and a self-assembling peptide, wherein each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide, and wherein the nucleic acid sequence further encodes a signal peptide on the amino-terminus of one or more of the polypeptide of the plurality.
6. A nucleic acid encoding a plurality of polypeptides, wherein each polypeptide of the plurality comprises a linker peptide, a target peptide, a linker peptide, and a selfassembling peptide, wherein each polypeptide of the plurality is connected to an adjacent polypeptide of the plurality by a cleavage peptide, and wherein the nucleic acid sequence further encodes a signal peptide on the amino-terminus of one or more of the polypeptide of the plurality.
7. The nucleic acid according to any of the preceding claims, wherein total number of the polynucleotide sequences is not more than 100.
8. The nucleic acid according to any of the preceding claims, wherein total number of the polynucleotide sequences is between 2-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-99.
9. The nucleic acid according to any of the preceding claims, wherein the nucleic acid is a DNA or an RNA.
10. The nucleic acid according to claim 9, wherein the RNA is an mRNA.
11. The nucleic acid according to any of the preceding claims wherein the linker sequence encodes a linker peptide.
12. The nucleic acid according to claim 11, wherein the linker peptide is an amino acid linker, a foldon, a scaffold or a combination thereof.
13. The nucleic acid according to claim 12, wherein the amino acid linker comprises 2 to 49 amino acids.
14. The nucleic acid according to claim 13, wherein the amino acid linker is a glycine serine linker, a glycine proline linker, a glycine threonine linker, an alanine serine linker, any combination of two amino acids, or a combination thereof.
15. The nucleic acid according to any of the preceding claims, wherein the linker peptide has an amino acid sequence of any one of SEQ ID NOs: 262-299, 330, and 350.
16. The nucleic acid according to any of the preceding claims, wherein the self- assembling sequence encodes a self-assembling peptide.
17. The nucleic acid according to claim 16, wherein the self-assembling peptide is a lumazine synthase from Aquifex species, a hepatitis B surface antigen (HBsAg) from Hepatitis B Virus, a hepatitis B core antigen (HBcAg) from Hepatitis B virus, a human papillomavirus LI (HPV L1) protein, a matrix protein Ml from influenza A virus, a ferritin, a riboflavin synthase, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
18. The nucleic acid according to claim 17, wherein the ferritin comprises of a ferritin subunit or a ferritin peptide.
19. The nucleic acid according to claim 18, wherein the ferritin peptide is derived from Helicobacter pylori ferritin.
20. The nucleic acid according to any of the preceding claims, wherein the selfassembling peptide has an amino acid sequence of any one of SEQ ID NOs: 254-261, 331 and 333.
21. The nucleic acid according to any of the preceding claims, wherein the cleavage sequence encodes one or more cleavage peptide.
22. The nucleic acid according to claim 21, wherein the one or more cleavage peptides are optionally connected to each other by a linker peptide.
23. The nucleic acid according to claim 22, wherein the cleavage peptide is a golgi specific cleavage peptide or a self-cleaving peptide.
24. The nucleic acid according to any of the preceding claims, wherein the cleavage peptide has an amino acid sequence of any one of SEQ ID NOs: 300-311 and 347-349.
25. The nucleic acid according to any of the preceding claims, wherein the signal sequence encodes a signal peptide.
26. The nucleic acid according to claim 25, wherein the signal peptide is present on the amino-terminus of one or more of the polypeptides of the plurality.
27. The nucleic acid according to claim 26, wherein the nucleic acid further encodes a second signal peptide on the amino-terminus of all or some polypeptides of the plurality.
28. The nucleic acid according to any of the preceding claims, wherein the signal peptide has an amino acid sequence of any one of SEQ ID NOs: 312-329.
29. The nucleic acid according to any of the preceding claims, wherein the target sequence encodes a target peptide.
30. The nucleic acid sequence according to claim 29, wherein the target peptide is encoded by a codon optimized nucleic acid sequence, or fragments, mutants, or variants thereof.
31. The nucleic acid according to claim 30, wherein the target peptide is obtained from a prokaryote, a eukaryote, a unicellular organism, a multicellular organism, a virus, a bacterium, a fungus, a protozoan, a worm, a mycoplasma, an animal, a human or combination thereof.
32. The nucleic acid according to claim 31, wherein the virus is selected from the family comprising picornaviride, calciviridae, astroviridae, togaviridae, flaviviridae, coronaviridae, arteriviridae, rhabndoviridae, filoviridae, paramyxoviridae, bornaviridae, orthomyxoviridae, bunyaviridae, arenaviridae, reoviridae, retroviridae, polyomaviridae, herpesviridae, poxviridae, papillomaviridae, hepadnaviridae, adenoviridae, parvoviridae, hepeviridae, circoviridae or a combination thereof.
33. The nucleic acid according to claim 31, wherein the bacterium is selected from the genus comprising Bacillus, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Escherichia, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Vibrio, Yersinia, or a combination thereof.
34. The nucleic acid according to claim 32, wherein the virus is selected from the family consisting of coronaviridae, herpesviridae, poxviridae, flaviviridae, togaviridae, retroviridae, paramyxoviridae, and a combination thereof.
35. The nucleic acid according to claim 31, wherein the virus is alphacoronavirus, betacoronavirus, deltacoronavirus, gammacoronavirus, torovirus or a combination thereof.
36. The nucleic acid according to claim 35, wherein the betacoronavirus is a SARS- CoV-1, a SARS-CoV-2, a MERS-CoV, an OC43, a HKU1, a bat coronavirus, other betacoronavirus, or a combination thereof.
37. The nucleic acid according to claim 30, wherein the target peptide is a spike protein, a membrane protein, an envelope protein or a nucleocapsid protein of coronaviruses.
38. The nucleic acid according to claim 30, wherein the target peptide is a glycoprotein B, a glycoprotein C, a glycoprotein D, a glycoprotein E, a glycoprotein K, a glycoprotein L, or a glycoprotein M of a herpes simplex virus 1 (HSV-1) or a herpes simplex virus 2 (HSV- 2), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants or variants thereof.
39. The nucleic acid according to claim 30, wherein the target peptide is a glycoprotein B, a glycoprotein H, a glycoprotein L, a glycoprotein M, or a glycoprotein N of a human cytomegalovirus (HCMV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
40. The nucleic acid according to claim 30, wherein the target peptide is a glycoprotein B, a glycoprotein C, a glycoprotein H, or a glycoprotein L of a varicella-zoster virus (VZV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
41. The nucleic acid according to claim 30, wherein the target peptide is a glycoprotein B, a glycoprotein H, a glycoprotein L, a glycoprotein M, a glycoprotein N, a glycoprotein
42. a glycoprotein 350 of an Epstein-Barr virus (EBV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
42. The nucleic acid according to claim 30, wherein the target peptide is a F9 membrane protein of a poxvirus, a H3L protein of a poxvirus, an A4 protein of a poxvirus, an A27 protein of poxvirus, an A33 protein of a poxvirus, an A56 protein of a poxvirus, a B5 protein of a poxvirus, or a LI protein of a poxvirus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
43. The nucleic acid according to claim 30, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of flaviviruses or hepaciviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
44. The nucleic acid according to claim 30, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a Japanese encephalitis virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
45. The nucleic acid according to claim 30, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a zika virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
46. The nucleic acid according to claim 30, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a yellow fever virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
47. The nucleic acid according to claim 30, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a west nile virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
48. The nucleic acid according to claim 30, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a hepatitis C virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
49. The nucleic acid according to claim 30, wherein the target peptide is a capsid protein, a membrane protein, an envelope protein, or a non-structural proteins (such as NS1, NS2A, NS2B, NS3, NS4A, NS4B, or NS5) of a dengue virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
50. The nucleic acid according to claim 30, wherein the target peptide is a capsid protein, or an envelope protein such as El, E2 and E3 protein of alphaviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
51. The nucleic acid according to claim 30, wherein the target peptide is a domain A, a domain B, or a domain C of the E2 protein of alphaviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
52. The nucleic acid according to claim 30, wherein the target peptide is a capsid protein, or an envelope protein such as El, E2 and E3 protein of a chikungunya virus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
53. The nucleic acid according to claim 30, wherein the target peptide is a gag, a pol and an env proteins of retroviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
54. The nucleic acid according to claim 30, wherein the target peptide is a pl7Gag, a p24Gag, a p7Gag, a p6Gag, a gpl2Env, a gp41Env, or a pol proteins from lentiviruses, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
55. The nucleic acid according to claim 30, wherein the target peptide is a pl7Gag, a p24Gag, a p7Gag, a p6Gag, a gpl2Env, a gp41Env, or a pol proteins from a human immunodeficiency virus (HIV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
56. The nucleic acid according to claim 30, wherein the target peptide is a nucleocapsid protein, a P protein, a V protein, a W protein, a D protein, an I protein, a C protein, a L protein, a M protein, a H (hemagglutinin) protein, a HN (hemagglutinin-neuraminidase) protein, a G protein or a F protein of a Mumps virus (MuV), a Parainfluenza virus type 5 (PIV5), a Human parainfluenza virus type 2, types 4a and 4b (HPIV2/4a/4b), a Newcastle disease virus (NDV), a Human parainfluenza virus type 1 and type 3 (HPIV1/3), a Nipah virus (NiV), a Measles virus (MeV), a Human respiratory syncytial virus A2, Bl, S2, (HRSV), or a Human metapneumovirus (HMPV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
57. The nucleic acid according to claim 30, wherein the target peptide is a nucleocapsid protein, a P protein, a V protein, a W protein, a D protein, an I protein, a C protein, a L protein, a M protein, a H (hemagglutinin) protein, a HN (hemagglutinin-neuraminidase) protein, a G protein or a F protein of a human respiratory syncytial virus A2, B 1 , S2 (HRSV), or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
58. The nucleic acid according to claim 30, wherein the target peptide is an aEl, an E2, an E4, an aE5, an E6, an E7, a LI, or a L2 protein of papillomavirus, preferably a human papillomavirus, or a combination thereof, including their codon optimized nucleic acid sequences, fragments, mutants, or variants thereof.
59. The nucleic acid according to any of the preceding claims, wherein the target peptide has an amino acid sequence of any one of SEQ ID NOs: 1-253, 334-337, 338-346, and 353- 388.
60. A lipid nanoparticle composition comprising cationic lipid, a phospholipid, a sterol, a PEG-lipid, and the nucleic acid according to any of the preceding claims.
61. The lipid nanoparticle composition according to claim 60, wherein the cationic lipid comprises an ionizable lipid.
62. The lipid nanoparticle composition according to claim 61, wherein the ionizable lipid is present in an amount from 25 mol percent to 70 mol percent.
63. The lipid nanoparticle composition according to claim 60, wherein the phospholipid is present in an amount from 2 mol percent to about 30 mol percent.
64. The lipid nanoparticle composition according to claim 60, wherein the sterol is present in an amount from 30 mol percent to about 65 mol percent.
65. The lipid nanoparticle composition according to claim 60, wherein the PEG-lipid is present in an amount from 0.2 mol percent to about 2.0 mol percent.
66. The lipid nanoparticle composition according to claim 60, additionally comprising an ionizable polymer.
67. The lipid nanoparticle composition according to claim 66, wherein the ionizable polymer is present in an amount from 1 mol percent to 25 mol percent.
68. The lipid nanoparticle composition according to claim 66, wherein the ionizable polymer is selected from the group comprising a chitosan, a cellulose derivative, a poly-L- lysine, a poly-L-glutamic acid, and/or their derivatives or a combination thereof.
69. A method of treating or preventing a disease, comprising administrating to a subject in need thereof the nucleic acid according to any one of claims 1-59.
70. A method of treating of preventing a disease, comprising administrating to a subject in need thereof the lipid nanoparticle composition according to any one of claims 60-68.
71. Use of the nucleic acid sequence according to any one of claims 1-59, in the manufacture of a medicament for the treatment or prevention of a disease in a subject.
72. Use of a lipid nanoparticle composition according to any one of claims 60-68 in the manufacture of a medicament for the treatment or prevention of a disease in a subject.
73. A multitarget peptide encoded by the nucleic acid of any one of claims 1-59.
74. A multitarget peptide comprising two or more polypeptides, wherein some or all polypeptides comprises either a target peptide, a linker peptide, and a self-assembling peptide, or a linker peptide, a target peptide, a linker peptide, and a self-assembling peptide, or combination thereof, wherein one polypeptide is connected to another polypeptide by a cleavage peptide, wherein the multitarget peptide includes a signal peptide upstream of one or more of the polypeptides.
75. A polypeptide nanoparticle comprising at least 2 or up to 100 polypeptides according to any one of claims 1 to 59.
76. The polypeptide nanoparticle according to claim 75, wherein the polypeptides are homologous polypeptides, heterologous polypeptides, oligomeric complex or a combination thereof.
77. The polypeptide nanoparticle according to claim 76, wherein the polypeptide nanoparticle is icosahedral, helical, spherical, rod-like or a combination thereof.
PCT/IB2023/000787 2022-12-29 2023-12-28 Multitarget vaccines and therapeutics WO2024141786A2 (en)

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