Compositions and methods for therapeutic or vaccine delivery

Abstract

Described herein are compositions for delivering therapeutic agents or vaccines. Also described herein are methods for using the compositions described herein for delivering therapeutic agents or vaccines.

Description

[Technical field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 271,675, filed October 25, 2021, and U.S. Provisional Application No. 63 / 413,188, filed October 4, 2022, the entireties of which are incorporated by reference herein.

[0002] Incorporated by reference

[0002] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, the specification is intended to supersede and / or take precedence over any such conflicting material. [Background technology]

[0003] The use of genetic material, such as viral vectors, to deliver therapeutic agents has emerged as one of the cornerstones of modern medicine. However, such genetic material can cause undesirable mitogenic effects. For example, retroviral vectors can integrate into the genome of cells, thus transforming such cells into cancer cells. One solution to such problems is to use RNA-based vehicles to deliver genetic material. Summary of the Invention

[0004]

[0004] The use of RNA-based vehicles can be complicated and cumbersome. In addition, RNA-based vehicles can be easily degraded and exhibit short half-life. Other DNA-based vehicles (e.g., non-viral DNA) for delivering therapeutic agents face problems such as the efficiency of transporting and targeting the vehicle to target cells, and the induction of immune response or toxicity in subjects. Therefore, there is still a need for vehicles for delivering therapeutic agents.

[0005] In some embodiments, a recombinant retroviral vector includes a first nucleic acid sequence encoding a mutant integrase and a second nucleic acid sequence encoding at least one payload, wherein the mutant integrase has a reduced Mg content in the catalytic core domain compared to a wild-type integrase. 2+Described herein is a recombinant retroviral vector comprising at least one mutation in a binding motif; and said at least one payload comprises an antigen. In some embodiments, the mutant integrase is deficient in retroviral integration activity. In some embodiments, the antigen comprises a pathogen polypeptide or fragment thereof or a cancer polypeptide or fragment thereof. In some embodiments, the cancer polypeptide or fragment thereof is associated with a cancer cell or tumor microenvironment. In some embodiments, the pathogen polypeptide or fragment thereof comprises a viral polypeptide or fragment thereof. In some embodiments, the pathogen polypeptide or fragment thereof comprises a bacterial polypeptide or fragment thereof. In some embodiments, the pathogen polypeptide or fragment thereof comprises a fungal polypeptide or fragment thereof. In some embodiments, the pathogen polypeptide or fragment thereof comprises a protozoan polypeptide or fragment thereof. In some embodiments, the pathogen polypeptide or fragment thereof comprises a protozoan polypeptide or fragment thereof. In some embodiments, the viral polypeptide or fragment thereof comprises a coronavirus polypeptide or fragment thereof. In some embodiments, the coronavirus polypeptide or fragment thereof comprises a severe acute respiratory syndrome (SARS-CoV) polypeptide or fragment thereof, a SARS-CoV-2 polypeptide or fragment thereof, or a Middle East Respiratory Syndrome (MERS-CoV) polypeptide or fragment thereof. In some embodiments, the coronavirus polypeptide or fragment thereof comprises a SARS-CoV-2 polypeptide or fragment thereof. In some embodiments, the SARS-Cov-2 polypeptide or fragment thereof comprises a spike protein or fragment thereof. In some embodiments, the spike protein or fragment thereof is a full-length spike protein. In some embodiments, the spike protein or fragment thereof is a truncated spike protein. In some embodiments, the truncated spike protein comprises the N-terminal domain of the spike protein or the S2 domain of the spike protein.In some embodiments, the truncated spike protein comprises an N-terminal domain of the spike protein and an S2 domain of the spike protein. In some embodiments, the spike protein or fragment thereof is a recombinant spike protein. In some embodiments, the spike protein or fragment thereof comprises at least one modification. In some embodiments, the at least one modification comprises codon optimization. In some embodiments, the codon optimization optimizes or increases expression of at least one payload in human cells. In some embodiments, the at least one modification comprises at least one amino acid mutation. In some embodiments, the at least one amino acid mutation eliminates a cleavage site in the spike protein. In some embodiments, the cleavage site is a furin cleavage site. In some embodiments, the furin cleavage site comprises amino acid residues 682-685, amino acid residues 679-682, or amino acid residue 811 in SEQ ID NO:21 or SEQ ID NO:22. In some embodiments, the cleavage site is a serine protease cleavage site. In some embodiments, the serine protease cleavage site comprises amino acid residues 986 and 987; or 983 and 984 in SEQ ID NO:21 or SEQ ID NO:22. In some embodiments, the at least one amino acid mutation comprises an amino acid substitution at amino acid residues 814, 889, 896, 939, 682-685, 679-682, 811, 986, 987, 983, 984, or combinations thereof in SEQ ID NO:21 or SEQ ID NO:22. In some embodiments, the at least one amino acid mutation comprises an amino acid substitution at amino acid residues 814, 889, 896, and 939 in SEQ ID NO:21 or SEQ ID NO:22. In some embodiments, the spike protein or fragment thereof comprises a signal peptide. In some embodiments, the signal peptide is a secretory peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO:13: MDAMKRGLCCVLLLCGAVFVSASQEIHARFRR. In some embodiments, the secretory peptide comprises an amino acid sequence at least 70% identical to IgE Fc receptor alpha.In some embodiments, the viral polypeptide or fragment thereof comprises an influenza polypeptide or fragment thereof. In some embodiments, the influenza polypeptide or fragment thereof comprises an influenza A polypeptide or fragment thereof, an influenza B polypeptide or fragment thereof, an influenza C polypeptide or fragment thereof, or an influenza D polypeptide or fragment thereof. In some embodiments, the influenza polypeptide or fragment thereof is an influenza A polypeptide or fragment thereof. In some embodiments, the influenza A polypeptide or fragment thereof comprises a neuraminidase (NA) or fragment thereof or a hemagglutinin (HA) or fragment thereof. In some embodiments, the influenza A polypeptide or fragment thereof comprises a hemagglutinin (HA) or fragment thereof. In some embodiments, the hemagglutinin (HA) or fragment thereof is a full length hemagglutinin (HA). In some embodiments, the hemagglutinin (HA) or fragment thereof is a truncated hemagglutinin (HA). In some embodiments, the truncated hemagglutinin (HA) comprises a Stalk domain. In some embodiments, the hemagglutinin (HA) or fragment thereof is a recombinant hemagglutinin (HA). In some embodiments, the hemagglutinin (HA) or fragment thereof comprises at least one modification. In some embodiments, the at least one modification comprises codon optimization. In some embodiments, the codon optimization optimizes or increases the expression of the at least one payload in human cells. In some embodiments, the at least one modification comprises at least one amino acid mutation. In some embodiments, the at least one modification comprises hemagglutinin (HA) or a fragment thereof, comprising the amino acid sequence of the extracellular domain of the M2 protein (M2e) of influenza A: SEQ ID NO: 14: MSLLTEVETPIRNEWGCRCNDSSD. In some embodiments, the recombinant retroviral vector encodes at least one envelope protein. In some embodiments, the at least one envelope protein comprises at least one alphavirus envelope protein.In some embodiments, the at least one alphavirus envelope protein comprises at least one Sindbis virus envelope protein. In some embodiments, the at least one Sindbis virus envelope protein comprises E3 protein, E2 protein, 6K protein, E1 protein, or a combination thereof. In some embodiments, the at least one Sindbis virus envelope protein comprises at least one mutation. In some embodiments, the at least one mutation increases the binding affinity between the at least one Sindbis virus envelope protein and a human cell. In some embodiments, the human cell is a dendritic cell. In some embodiments, the at least one mutation is E160G of the E2 protein. In some embodiments, the at least one mutation in the Mg2+ binding motif of the catalytic core domain comprises D125A, D184A, or a combination thereof. In some embodiments, the recombinant retroviral vector comprises at least one modified untranslated region (UTR). In some embodiments, the at least one modified UTR comprises a 5'-UTR. In some embodiments, the at least one modified UTR comprises a 3'-UTR. In some embodiments, the at least one modified UTR comprises a 5'-UTR and a 3'-UTR.

[0006]

[0006] In some aspects, a recombinant virus is described herein that includes a recombinant retroviral vector described herein. In some embodiments, the recombinant virus is a recombinant Sindbis virus. In some embodiments, the recombinant Sindbis virus includes an E160G mutation in the E2 protein.

[0007]

[0007] In some aspects, described herein are cells comprising a recombinant retroviral vector described herein or a recombinant virus described herein. In some embodiments, the cells express at least one payload. In some embodiments, the cells secrete at least one payload. In some embodiments, the cells express and secrete at least one payload. In some embodiments, the cells express at least one payload for at least 1 day, at least 3 days, at least 5 days, or at least 9 days. In some embodiments, the cells secrete at least one payload for at least 1 day, at least 3 days, at least 5 days, or at least 9 days. In some embodiments, the cells express and secrete at least one payload for at least 1 day, at least 3 days, at least 5 days, or at least 9 days.

[0008]

[0008] In some aspects, described herein are pharmaceutical compositions comprising a recombinant retroviral vector described herein, a recombinant virus described herein, or a cell comprising a recombinant retroviral vector described herein or a recombinant virus described herein. In some embodiments, the pharmaceutical composition comprises at least one additional active ingredient. In some embodiments, the at least one additional active ingredient comprises an adjuvant. In some embodiments, the pharmaceutical composition comprises at least one pharma- ceutical acceptable excipient.

[0009]

[0009] In some aspects, described herein are methods of treating or preventing a disease or condition in a subject, comprising administering to a subject a recombinant retroviral vector described herein, a recombinant virus described herein, a cell comprising a recombinant retroviral vector described herein or a recombinant virus described herein, or a pharmaceutical composition described herein, wherein at least one payload comprising an antigen induces an immune response in the subject, thereby treating or preventing the disease or condition in the subject. In some embodiments, the immune response comprises induction of neutralizing antibodies targeting the antigen, thereby generating immunity to the antigen in the subject. In some embodiments, the immune response comprises induction of immunoglobulin antibodies targeting the antigen, thereby generating immunity to the antigen in the subject. In some embodiments, the immunoglobulin antibodies comprise IgG antibodies, IgM antibodies, IgA antibodies, IgD antibodies, IgE antibodies, or combinations thereof. In some embodiments, the immunoglobulin antibodies comprise IgG antibodies. In some embodiments, the at least one payload is expressed in the subject for at least 1 day, at least 3 days, at least 5 days, or at least 9 days. In some embodiments, the at least one payload is secreted in the subject for at least 1 day, at least 3 days, at least 5 days, or at least 9 days. In some embodiments, the duration of the immune response induced by the at least one payload expressed for at least 1 day, at least 3 days, at least 5 days, or at least 9 days is increased by at least 10%, at least 20%, at least 50%, at least 100%, at least 5-fold, at least 10-fold, or more, compared to the duration of the immune response induced by a comparable payload expressed in less than 1 day, less than 3 days, less than 5 days, or less than 9 days.In some embodiments, the duration of an immune response induced by at least one payload secreted for at least 1 day, at least 3 days, at least 5 days, or at least 9 days is increased by at least 10%, at least 20%, at least 50%, at least 100%, at least 5-fold, at least 10-fold, or more, compared to the duration of an immune response induced by a comparable payload expressed in less than 1 day, less than 3 days, less than 5 days, or less than 9 days. In some embodiments, the immune response persists in the subject for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 12 months, or longer.

[0010] In some embodiments, a recombinant retroviral vector includes a nucleic acid construct comprising a polynucleotide sequence encoding a mutant integrase, wherein the mutant integrase has a reduced Mg content in the catalytic core domain compared to a wild-type integrase. 2+Described herein is a recombinant retroviral vector comprising a first mutation in a binding motif. In some embodiments, the first mutation comprises changing an aspartic acid (D) or glutamic acid (E) to an amino acid having a hydrophobic side chain. In some embodiments, the amino acid is selected from the group consisting of valine (V), alanine (A), leucine (L), isoleucine (I), and phenylalanine (F). In some embodiments, the amino acid comprises phenylalanine (F). In some embodiments, the amino acid comprises alanine (A). In some embodiments, the first mutation comprises changing an aspartic acid (D) or glutamic acid (E) to an amino acid having a positively charged side chain. In some embodiments, the amino acid comprises histidine (H). In some embodiments, the first mutation comprises changing an aspartic acid (D) or glutamic acid (E) to an amino acid having a polar side chain. In some embodiments, the amino acid comprises serine (S). In some embodiments, the first mutation comprises changing an aspartic acid (D) or glutamic acid (E) to a cysteine ​​(C). In some embodiments, the first mutation comprises a D125A mutation. In some embodiments, the first mutation comprises a D184A mutation. In some embodiments, the first mutation comprises an E220A mutation. In some embodiments, the mutant integrase comprises a Mg 2+The binding motif further comprises a second mutation. In some embodiments, the second mutation comprises changing an aspartic acid (D) or a glutamic acid (E) to an amino acid having a hydrophobic side chain. In some embodiments, the second mutation comprises an amino acid selected from the group consisting of valine (V), alanine (A), leucine (L), isoleucine (I), and phenylalanine (F). In some embodiments, the second mutation comprises phenylalanine (F). In some embodiments, the second mutation comprises alanine (A). In some embodiments, the second mutation comprises changing an aspartic acid (D) or a glutamic acid (E) to an amino acid having a positively charged side chain. In some embodiments, the second mutation comprises histidine (H). In some embodiments, the second mutation comprises changing an aspartic acid (D) or a glutamic acid (E) to an amino acid having a polar charged side chain. In some embodiments, the second mutation comprises serine (S). In some embodiments, the second mutation comprises changing an aspartic acid (D) or glutamic acid (E) to a cysteine ​​(C). In some embodiments, the first mutation comprises a D125A mutation and the second mutation comprises a D184A mutation. In some embodiments, the first mutation comprises a D184A mutation and the second mutation comprises an E220A mutation. In some embodiments, the first mutation comprises a D125A mutation and the second mutation comprises an E220A mutation. In some embodiments, the mutant integrase has a Mg residue in the catalytic core domain. 2+The binding motif further comprises a third mutation. In some embodiments, the third mutation comprises changing an aspartic acid (D) or a glutamic acid (E) to an amino acid having a hydrophobic side chain. In some embodiments, the third mutation comprises an amino acid selected from the group consisting of valine (V), alanine (A), leucine (L), isoleucine (I), and phenylalanine (F). In some embodiments, the third mutation comprises phenylalanine (F). In some embodiments, the third mutation comprises alanine (A). In some embodiments, the third mutation comprises changing an aspartic acid (D) or a glutamic acid (E) to an amino acid having a positively charged side chain. In some embodiments, the third mutation comprises histidine (H). In some embodiments, the third mutation comprises changing an aspartic acid (D) or a glutamic acid (E) to an amino acid having a polar charged side chain. In some embodiments, the third mutation comprises serine (S). In some embodiments, the third mutation comprises changing an aspartic acid (D) or a glutamic acid (E) to a cysteine ​​(C). In some embodiments, the first and second mutations each comprise changing an aspartic acid (D) to an amino acid having a hydrophobic side chain, and the third mutation comprises changing a glutamic acid (E) to an amino acid having a hydrophobic side chain. In some embodiments, the recombinant retroviral vector is a murine leukemia virus (MLV), and the first mutation is selected from the group consisting of D125A, D184A, and E220A. In some embodiments, the mutant integrase comprises changing an Mg residue in the catalytic core domain. 2+ In some embodiments, the mutated integrase further comprises a second mutation in the binding motif, the second mutation being selected from the group consisting of D125A, D184A, and E220A. 2+and a third mutation in the Mg2+-binding motif, wherein the first mutation comprises D125A, the second mutation comprises E220A, and the third mutation comprises D184A. In some embodiments, the mutant integrase further comprises a third mutation in the Mg2+-binding motif of the catalytic core domain, wherein the first mutation comprises D125A, and the second mutation comprises D184A.

[0011] In some embodiments, a recombinant retroviral vector includes a nucleic acid construct comprising a polynucleotide sequence encoding a mutant integrase, wherein the mutant integrase has a reduced Mg content in the catalytic core domain compared to a wild-type integrase. 2+Described herein are recombinant retroviral vectors that consist of a single mutation in a binding motif. In some embodiments, the single mutation consists of changing an aspartic acid (D) or glutamic acid (E) to an amino acid with a hydrophobic side chain. In some embodiments, the single mutation comprises an amino acid selected from the group consisting of valine (V), alanine (A), leucine (L), isoleucine (I), and phenylalanine (F). In some embodiments, the single mutation comprises phenylalanine (F). In some embodiments, the single mutation comprises alanine (A). In some embodiments, the single mutation comprises changing an aspartic acid (D) or glutamic acid (E) to an amino acid with a positively charged side chain. In some embodiments, the single mutation comprises histidine (H). In some embodiments, the single mutation comprises changing an aspartic acid (D) or glutamic acid (E) to an amino acid with a polar charged side chain. In some embodiments, the single mutation comprises serine (S). In some embodiments, the single mutation comprises changing an aspartic acid (D) or glutamic acid (E) to a cysteine ​​(C). In some embodiments, the mutant integrase comprises a peptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:1. In some embodiments, the single mutation comprises a D125A mutation. In some embodiments, the mutant integrase comprises a peptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:2. In some embodiments, the single mutation comprises a D184A mutation. In some embodiments, the mutant integrase comprises a peptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:3. In some embodiments, the single mutation comprises an E220A mutation.In some embodiments, the mutant integrase comprises a peptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to SEQ ID NO:4.

[0012] In some embodiments, a recombinant retroviral vector includes a nucleic acid construct comprising a polynucleotide sequence encoding a mutant integrase, wherein the mutant integrase has a MgA domain in the catalytic core domain compared to a wild-type integrase. 2+Described herein is a recombinant retroviral vector consisting of two mutations in a binding motif. In some embodiments, at least one of the two mutations consists of changing an aspartic acid (D) or a glutamic acid (E) to an amino acid having a hydrophobic side chain. In some embodiments, at least one of the two mutations comprises an amino acid selected from the group consisting of valine (V), alanine (A), leucine (L), isoleucine (I), and phenylalanine (F). In some embodiments, at least one of the two mutations comprises phenylalanine (F). In some embodiments, at least one of the two mutations comprises alanine (A). In some embodiments, at least one of the two mutations comprises changing an aspartic acid (D) or a glutamic acid (E) to an amino acid having a positively charged side chain. In some embodiments, at least one of the two mutations comprises histidine (H). In some embodiments, at least one of the two mutations comprises changing an aspartic acid (D) or a glutamic acid (E) to an amino acid having a polarly charged side chain. In some embodiments, at least one of the two mutations comprises a serine (S). In some embodiments, at least one of the two mutations comprises changing an aspartic acid (D) or a glutamic acid (E) to a cysteine ​​(C). In some embodiments, the mutant integrase comprises a peptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to SEQ ID NO:1. In some embodiments, at least one of the two mutations comprises a D125A mutation, a D184A mutation, or an E220A mutation. In some embodiments, the two mutations consist of a D125A mutation and a D184A mutation. In some embodiments, the mutant integrase comprises a peptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to SEQ ID NO:5.In some embodiments, the two mutations consist of a D125A mutation and an E220A mutation. In some embodiments, the mutant integrase comprises a peptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:6. In some embodiments, the two mutations comprise a D184A mutation and an E220A mutation. In some embodiments, the mutant integrase comprises a peptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or more identical to SEQ ID NO:7.

[0013] In some embodiments, a recombinant retroviral vector includes a nucleic acid construct comprising a polynucleotide sequence encoding a mutant integrase, wherein the mutant integrase has a reduced Mg content in the catalytic core domain compared to a wild-type integrase. 2+Described herein is a recombinant retroviral vector consisting of three mutations in a binding motif. In some embodiments, at least one of the three mutations consists of changing an aspartic acid (D) or a glutamic acid (E) to an amino acid having a hydrophobic side chain. In some embodiments, at least one of the three mutations comprises an amino acid selected from the group consisting of valine (V), alanine (A), leucine (L), isoleucine (I), and phenylalanine (F). In some embodiments, at least one of the three mutations comprises phenylalanine (F). In some embodiments, at least one of the three mutations comprises alanine (A). In some embodiments, at least one of the three mutations comprises changing an aspartic acid (D) or a glutamic acid (E) to an amino acid having a positively charged side chain. In some embodiments, at least one of the three mutations comprises histidine (H). In some embodiments, at least one of the three mutations comprises changing an aspartic acid (D) or a glutamic acid (E) to an amino acid having a polarly charged side chain. In some embodiments, at least one of the three mutations comprises a serine (S). In some embodiments, at least one of the three mutations comprises changing an aspartic acid (D) or a glutamic acid (E) to a cysteine ​​(C). In some embodiments, the mutant integrase comprises a peptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to SEQ ID NO:1. In some embodiments, at least one of the three mutations comprises a D125A mutation, a D184A mutation, or an E220A mutation. In some embodiments, the three mutations comprise a D125A mutation, a D184A mutation, or an E220A mutation. In some embodiments, the three mutations comprise a D125A mutation, a D184A mutation, and an E220A mutation.In some embodiments, the mutant integrase comprises a peptide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to SEQ ID NO:8. In some embodiments, the nucleic acid construct further encodes a payload. In some embodiments, the payload comprises a cytokine. In some embodiments, the cytokine comprises interleukin-7. In some embodiments, the cytokine comprises interleukin-12. In some embodiments, the cytokine comprises an interferon. In some embodiments, the interferon comprises IFN-α. In some embodiments, the payload comprises a thymidine kinase. In some embodiments, the thymidine kinase comprises a mutant thymidine kinase. In some embodiments, the payload comprises an antigen. In some embodiments, the antigen comprises a viral protein. In some embodiments, the viral protein comprises a SARS-CoV-2 protein. In some embodiments, the viral protein comprises an influenza protein. In some embodiments, the antigen comprises a pathogen protein.

[0014]

[0014] In some aspects, a method for treating a disease or condition in a subject is described herein, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a recombinant retroviral vector described herein.

[0015]

[0015] In some aspects, a method of vaccinating a subject is described herein, the method comprising administering to the subject a pharmaceutical composition comprising a recombinant retroviral vector described herein.

[0016] This patent application contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0017] [Figure 1]

[0017] Figure 1 illustrates a schematic diagram of MLV integrase highlighting the triad of D125, D184, and E220 that form the Mg2+-binding motif in the catalytic core domain. The HH-CC motif contains the zinc-binding domain. The HH-CC motif is a specific type of Zn2+ ion chelating site found in zinc finger proteins. [Diagram 2]

[0018] FIG. 2 illustrates the sequencing results of the D184A mutant. [Diagram 3]

[0019] FIG. 3 illustrates the sequencing results of the D125A / E220A mutant. [Figure 4]

[0020] FIG. 4 illustrates the physical titers of vectors generated with seven different mutants. [Diagram 5]

[0021] FIG. 5 illustrates the killing activity of vTK / GCV cells carrying seven different mutants. [Figure 6]

[0022] FIG. 6A illustrates early vTK expression detected by Western blotting.

[0023] FIG. 6B illustrates an exemplary Western blot expression analysis from days 2 to 14 for the D184A mutant. [Figure 7]

[0024] FIG. 7 illustrates qPCR of relative integration with seven different mutants at days 2, 4, and 7. [Figure 8]

[0025] FIG. 8 illustrates qPCR of relative integration with seven different mutants at days 10, 14, and 21. [Figure 9]

[0026] FIG. 9 illustrates the vTK / GCV cell killing activity of retroviral vectors produced from an integrase-deficient gagpol cell line. [Figure 10]

[0027] FIG. 10 illustrates the killing activity of vTK / GCV cells of integrase-deficient vector cell lines. [Figure 11]

[0028] FIG. 11 illustrates viral vector titers and vTK / GCV cell killing of integrase-deficient BC6 vector clones. [Figure 12]

[0029] Figures 12A-C illustrate the lethal activity of vTK / GCV cells in various cancer cell lines between wtGP (Figure 12A) and integrase-deficient mutants (Figure 12B). Figure 12C illustrates the relative integration of vectors by qPCR between wtGP and integrase-deficient mutants. [Figure 13]

[0030] Figure 13 illustrates an exemplary schematic of a recombinant retroviral vector comprising a nucleic acid sequence encoding the spike protein of SARS-CoV-2 or a fragment thereof. Top: Diagram of a retroviral vector encoding the SARS-CoV-2 full-length spike protein (Wuhan sequence with modifications). Center: Diagram of a retroviral vector encoding the SARS-CoV-2 spike protein fragments of the N-terminal domain (NTD) and S2 domain (Wuhan sequence with modifications). Bottom: Diagram of a retroviral vector encoding the SARS-CoV-2 full-length spike protein (Omicron BA.2 sequence with modifications). [Figure 14A]

[0031] FIG. 14A illustrates the presence of a full-length spike transgene as the payload of a retroviral vector or lentivector described herein. [Figure 14B]

[0032] FIG. 14B illustrates the presence of the NTD and S2 transgenes as payloads of the retroviral or lentivectors described herein. [Figure 14C]

[0033] FIG. 14C illustrates the presence of the omicronBA.2 transgene (full length spike) as the payload of a retroviral vector or lentivector described herein. [Figure 15]

[0034] FIG. 15 illustrates the presence of the E160G mutation in the Sindbis virus envelope. [Figure 16A]

[0035] Figure 16A illustrates Western blotting of spike protein secreted from retroviral vector-transduced A375 cells. "IDRV1" refers to the retroviral vector encoding a mutant integrase (integrase with a D184A mutation). Accumulated protein on each day is presented. [Figure 16B]

[0036] Figure 16B illustrates Western blotting of S2 protein secreted from retroviral vector-transduced A375 cells. "IDRV1" refers to the retroviral vector encoding a mutant integrase (integrase with the D184A mutation). Accumulated protein on each day is presented. [Figure 16C]

[0037] Figure 16C illustrates Western blotting of full-length spike and S2 proteins secreted from A375 cells transduced with retroviral vectors. The retroviral vectors also expressed mutant integrases (IDRV2: D125A and D184A mutations) described herein. Accumulated proteins for each day are presented. [Figure 17A]

[0038] FIG. 17A illustrates western blotting of intracellular full-length spike protein from A375 cells transduced with retroviral vectors expressing the omicron variant sequence with modifications and wild-type integrase or IDRV2 (IDRV2: D125A and D184A mutations). [Figure 17B]

[0039] FIG. 17B illustrates Western blotting of intracellular and secreted full-length spike protein from A375 cells transduced with retroviral vectors produced from clonal manufacturing cell lines. [Figure 18A]

[0040] FIG. 18A illustrates the expression of omicron spike protein detected by Western blotting after transduction of test cells with the retroviral vectors described herein. [Figure 18B]

[0041] Figure 18B illustrates the expression of omicron spike protein as detected by immunocytochemistry (ICC) staining: bright spots indicate nuclear staining, and grey indicates specific perinuclear and plasma membrane staining of omicron spike protein. [Figure 18C]

[0042] Figure 18C illustrates Western blotting of both reduced and non-reduced spike protein detected by anti-S2 antibody in plasma membrane fractions. Also shown is a Western blot of the same samples probed for the plasma membrane marker cadherin. [Figure 19]

[0043] FIG. 19 illustrates a schematic of a retroviral vector (HA Stalk with modifications) containing nucleic acids encoding M2e and HA4900 as a universal influenza vaccine. [Figure 20]

[0044] FIG. 20 illustrates the presence of M2e and HA4900 (influenza) transgenes as payloads of retroviral or lentivectors described herein. [Figure 21A]

[0045] FIG. 21A illustrates the expression of HA protein detected by Western blotting after transduction of test cells with the retroviral vectors described herein. [Figure 21B]

[0046] Figure 21B illustrates the expression of HA protein detected by immunocytochemistry (ICC) staining. Bright spots indicate nuclear staining, and grey indicates specific staining of HA protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018]

[0047] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments.

[0019]

[0048] In some embodiments, a vector comprising at least one amino acid mutation is described herein. In some embodiments, the vector is a recombinant retroviral vector (or retrovector) comprising at least one amino acid mutation, wherein the at least one amino acid mutation is in the integrase encoded by the recombinant retroviral vector, thus creating a mutant integrase. In some embodiments, the mutant integrase comprising at least one amino acid mutation is dysfunctional and can no longer integrate the recombinant retroviral vector into the genome of a host cell. In some embodiments, the vector comprises a nucleic acid construct comprising at least one polynucleotide sequence encoding a mutant reverse transcriptase. In some embodiments, the mutant reverse transcriptase comprises at least one amino acid mutation, wherein the mutant reverse transcriptase can no longer convert the vector into DNA for insertion into the genome of a host cell. In some embodiments, the vector comprises a nucleic acid construct comprising at least one polynucleotide sequence encoding both a mutant integrase and a mutant reverse transcriptase.

[0020]

[0049] In some embodiments, a recombinant retroviral vector comprising a nucleic acid construct comprising a polynucleotide sequence encoding a mutant integrase, wherein the mutant integrase has an Mg domain in the catalytic core domain of the mutant integrase compared to a wild-type integrase. 2+ A recombinant retroviral vector comprising at least one amino acid mutation in the binding motif. 2+1 illustrates an exemplary vector diagram showing the placement of at least one mutation in a binding motif. In an embodiment, the mutant integrase comprises a Mg binding motif in the catalytic core domain of the mutant integrase. 2+ The binding motif comprises one, two, three, or more amino acid mutations. In some aspects, the vector comprising the mutant integrase may encode a payload. In some embodiments, the payload comprises a therapeutic agent. In some embodiments, the vector described herein comprises a recombinant retroviral vector comprising a payload encoding an antigen, the antigen being capable of stimulating innate immunity (e.g., as a vaccine). In some embodiments, the antigen comprises a pathogen polypeptide or fragment thereof or a cancer polypeptide or fragment thereof. In some embodiments, the cancer polypeptide or fragment thereof is associated with a cancer cell or tumor microenvironment. In some embodiments, the pathogen polypeptide or fragment thereof comprises a viral polypeptide or fragment thereof. In some embodiments, the viral polypeptide or fragment thereof comprises a coronavirus polypeptide or fragment thereof. In some embodiments, the viral polypeptide or fragment thereof comprises an influenza polypeptide or fragment thereof.

[0021]

[0050] In some embodiments, the pathogen polypeptide or fragment thereof comprises a bacterial polypeptide or fragment thereof. In some embodiments, the pathogen polypeptide or fragment thereof comprises a fungal polypeptide or fragment thereof. In some embodiments, the pathogen polypeptide or fragment thereof comprises a protozoan polypeptide or fragment thereof. In some embodiments, the pathogen polypeptide or fragment thereof comprises a protozoan polypeptide or fragment thereof.

[0022]

[0051] In some embodiments, the payload comprises an antigen, which can elicit an immune response and thus vaccinate the subject to which the vector is administered. In some aspects, described herein is a method of treating a disease or condition in a subject by administering a vector (e.g., a recombinant retroviral vector as described herein) to the subject, where the vector delivers a therapeutic agent as the payload of the vector. In some aspects, described herein is a method of vaccinating a subject by administering a vector (e.g., a recombinant retroviral vector as described herein) to the subject, where the vector delivers an antigen as the payload. The antigen can then initiate a natural immune response against the antigen and thus vaccinate the subject.

[0023] vector

[0052] In some aspects, vectors such as recombinant retroviral vectors are described herein, which comprise a nucleic acid construct comprising at least one polynucleotide sequence encoding a mutant integrase.In some embodiments, the vector comprises a nucleic acid construct comprising at least one polynucleotide sequence encoding a mutant reverse transcriptase.In some embodiments, the vector comprises a nucleic acid construct comprising at least one polynucleotide sequence encoding both a mutant integrase and a mutant reverse transcriptase.

[0024]

[0053] In some embodiments, the mutant integrase comprises at least one amino acid mutation compared to a wild-type integrase comprising the polypeptide sequence of SEQ ID NO:1. In some embodiments, the mutant integrase comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to SEQ ID NO:1. Figures 2 and 3 illustrate alignments of the polypeptide sequences of exemplary mutant integrases described herein to the wild-type integrase polypeptide sequence. Figure 4 illustrates that the mutant integrases described herein were dysfunctional with respect to integrating vectors into the host genome, but did not alter viral titers. Figures 5 and 9-12 illustrate that vectors comprising mutant integrases can deliver a payload (thymidine kinase as described herein) for cell killing in the presence of a prodrug (GCV). Figure 6 illustrates that expression of the payload (thymidine kinase, vTK) was not altered by the mutant integrase. Figures 7 and 8 illustrate that the vector containing the mutant integrase did not integrate into the host genome for at least 21 days after the host cell was contacted with the vector.

[0025]

[0054] In some embodiments, the mutant integrase comprises at least one, at least two, at least three, or more amino acid mutations. In some embodiments, the mutant integrase comprises one, two, three, or more amino acid mutations. In some embodiments, the mutant integrase comprises one amino acid mutation. In some embodiments, the mutant integrase comprises two amino acid mutations. In some embodiments, the mutant integrase comprises three amino acid mutations. In some embodiments, the mutant integrase comprises a Mg of the catalytic core domain of the mutant integrase. 2+In some embodiments, the mutant integrase comprises at least one, at least two, at least three, or more amino acid mutations in the binding motif. 2+ In some embodiments, the mutant integrase comprises one, two, three or more amino acid mutations in the binding motif. 2+ In some embodiments, the mutant integrase comprises a single amino acid mutation in the binding motif. 2+ In some embodiments, the mutant integrase comprises two amino acid mutations in the binding motif. 2+ The binding motif contains three amino acid mutations.

[0026]

[0055] In some embodiments, the mutant integrase comprises at least one amino acid mutation, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to an amino acid having a hydrophobic side chain. In some embodiments, the amino acid having a hydrophobic side chain is selected from the group consisting of valine (V), alanine (A), leucine (L), isoleucine (I), and phenylalanine (F). In some embodiments, the mutant integrase comprises at least one amino acid mutation, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to a phenylalanine (F). In some embodiments, the mutant integrase comprises at least one amino acid mutation, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to an alanine (A). In some embodiments, the mutant integrase comprises at least one amino acid mutation, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to an amino acid having a positively charged side chain. In some embodiments, the mutant integrase comprises at least one amino acid mutation, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to an amino acid having a positively charged side chain, the positively charged side chain being histidine (H). In some embodiments, the mutant integrase comprises at least one amino acid mutation, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to an amino acid having a polarly charged side chain. In some embodiments, the mutant integrase comprises at least one amino acid mutation, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to an amino acid having a polar charged side chain, the positively charged side chain being serine (S).In some embodiments, the mutant integrase comprises at least one amino acid mutation, the at least one amino acid mutation comprising changing an aspartic acid (D) or a glutamic acid (E) of the wild-type integrase to a cysteine ​​(C).

[0027]

[0056] In some embodiments, the mutant integrase comprises a Mg fragment of the catalytic core domain of the mutant integrase. 2+ The binding motif comprises at least one amino acid mutation, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to an amino acid having a hydrophobic side chain. In some embodiments, the amino acid having a hydrophobic side chain is selected from the group consisting of valine (V), alanine (A), leucine (L), isoleucine (I), and phenylalanine (F). In some embodiments, the mutant integrase comprises a Mg amino acid of the catalytic core domain of the mutant integrase. 2+ The binding motif comprises at least one amino acid mutation, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to a phenylalanine (F). In some embodiments, the mutant integrase comprises a Mg residue in the catalytic core domain of the mutant integrase. 2+ The binding motif comprises at least one amino acid mutation, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to an alanine (A). In some embodiments, the mutant integrase comprises a Mg residue in the catalytic core domain of the mutant integrase. 2+ In some embodiments, the mutant integrase comprises at least one amino acid mutation in the binding motif, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to an amino acid having a positively charged side chain. 2+In some embodiments, the mutant integrase comprises at least one amino acid mutation in the binding motif, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to an amino acid having a positively charged side chain, the positively charged side chain being histidine (H). 2+ In some embodiments, the mutant integrase comprises at least one amino acid mutation in the binding motif, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to an amino acid having a polar charged side chain. 2+ In some embodiments, the mutant integrase comprises at least one amino acid mutation in the binding motif, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to an amino acid having a polar charged side chain, the positively charged side chain being serine (S). 2+ The binding motif comprises at least one amino acid mutation, the at least one amino acid mutation comprising changing an aspartic acid (D) or glutamic acid (E) of the wild-type integrase to a cysteine ​​(C).

[0028]

[0057] In some embodiments, the mutant integrase comprises a single mutation. In some embodiments, the mutant integrase comprises a single mutation at position 125 of the wild-type integrase. In some embodiments, the mutant integrase comprises a single mutation comprising a substitution of aspartic acid (D) to alanine (A) at position 125 of the wild-type integrase, i.e., a D125A mutation. In some embodiments, the mutant integrase comprising a single mutation comprising a substitution of a D125A mutation comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to SEQ ID NO:2. In some embodiments, the mutant integrase comprising a single mutation comprising a substitution of a D125A mutation comprises a polypeptide sequence that is SEQ ID NO:2.

[0029]

[0058] In some embodiments, the mutant integrase comprises a single mutation at position 184 of the wild-type integrase. In some embodiments, the mutant integrase comprises a single mutation comprising an aspartic acid (D) to alanine (A) substitution at position 184 of the wild-type integrase, i.e., a D184A mutation. In some embodiments, the mutant integrase comprising a single mutation comprising a substitution of a D184A mutation comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to SEQ ID NO:3. In some embodiments, the mutant integrase comprising a single mutation comprising a substitution of a D184A mutation comprises a polypeptide sequence that is SEQ ID NO:3.

[0030]

[0059] In some embodiments, the mutant integrase comprises a single mutation at position 220 of the wild-type integrase. In some embodiments, the mutant integrase comprises a single mutation comprising a substitution of alanine (A) for glutamic acid (E) at position 220 of the wild-type integrase, i.e., an E220A mutation. In some embodiments, the mutant integrase comprising a single mutation comprising a substitution of an E220A mutation comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to SEQ ID NO:4. In some embodiments, the mutant integrase comprising a single mutation comprising a substitution of an E220A mutation comprises a polypeptide sequence that is SEQ ID NO:4.

[0031]

[0060] In some embodiments, the mutant integrase comprises two mutations. In some embodiments, the mutant integrase comprises two mutations, the first mutation comprises a substitution of aspartic acid (D) to alanine (A) at position 125 of wild-type integrase, i.e., D125A mutation; the second mutation comprises a substitution of aspartic acid (D) to alanine (A) at position 184 of wild-type integrase, i.e., D184A mutation. In some embodiments, the mutant integrase comprises two mutations, including a substitution of a D125A mutation and a substitution of a D184A mutation, comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to SEQ ID NO:5. In some embodiments, the mutant integrase comprises two mutations, including a substitution of a D125A mutation and a substitution of a D184A mutation, comprises a polypeptide sequence that is SEQ ID NO:5.

[0032]

[0061] In some embodiments, the mutant integrase comprises two mutations, the first mutation comprises an aspartic acid (D) to alanine (A) substitution at position 125 of the wild-type integrase, i.e., a D125A mutation; the second mutation comprises a glutamic acid (E) to alanine (A) substitution at position 220 of the wild-type integrase, i.e., an E220A mutation. In some embodiments, the mutant integrase comprising two mutations, comprising a D125A mutation substitution and an E220A mutation substitution, comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to SEQ ID NO:6. In some embodiments, the mutant integrase comprising two mutations, comprising a D125A mutation substitution and an E220A mutation substitution, comprises a polypeptide sequence that is SEQ ID NO:6.

[0033]

[0062] In some embodiments, the mutant integrase comprises two mutations, the first mutation comprises an aspartic acid (D) to alanine (A) substitution at position 184 of the wild-type integrase, i.e., a D184A mutation; the second mutation comprises a glutamic acid (E) to alanine (A) substitution at position 220 of the wild-type integrase, i.e., an E220A mutation. In some embodiments, the mutant integrase comprises two mutations, comprising a D184A mutation substitution and an E220A mutation substitution, comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to SEQ ID NO:7. In some embodiments, the mutant integrase comprises two mutations, comprising a D184A mutation substitution and an E220A mutation substitution, comprises a polypeptide sequence that is SEQ ID NO:7.

[0034]

[0063] In some embodiments, the mutant integrase comprises three mutations. In some embodiments, the mutant integrase comprises three mutations, the first mutation comprises a substitution of aspartic acid (D) at position 125 of wild-type integrase with alanine (A), i.e., D125A mutation; the second mutation comprises a substitution of aspartic acid (D) at position 184 of wild-type integrase with alanine (A), i.e., D184A mutation; and the third mutation comprises a substitution of glutamic acid (E) at position 220 of wild-type integrase with alanine (A), i.e., E220A mutation. In some embodiments, the mutant integrase comprising three mutations comprises a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more identical to SEQ ID NO:8. In some embodiments, the mutant integrase comprising three mutations comprises a polypeptide sequence that is SEQ ID NO:8.

[0035]

[0064] In some embodiments, the vector containing at least one amino acid mutation renders the integrase non-functional. In some embodiments, the mutant integrase is no longer able to introduce the vector (e.g., a recombinant retroviral vector) into the genome of a host cell containing the vector. In some embodiments, the vector containing the mutant integrase encodes at least one therapeutic agent or at least one antigen. In some embodiments, the at least one therapeutic agent comprises a cytokine. In some embodiments, the cytokine comprises an interleukin or an interferon. In some embodiments, the vector encodes at least one interleukin subunit. In some embodiments, the vector encodes at least two interleukin subunits, the at least two interleukin subunits being the same or different. In some embodiments, the vector encodes one interleukin subunit. In some embodiments, the vector encodes two interleukin subunits. In some embodiments, the vector encodes two different interleukin subunits. In some embodiments, the vector encodes two or more different interleukin subunits. In some embodiments, the vector comprises at least one initiation codon for expressing an interleukin, a subunit of an interleukin, or a combination thereof. In some embodiments, the vector comprises at least two initiation codons for expressing two interleukins, two subunits of an interleukin, or a combination thereof, hi some embodiments, the vector comprises two codons for expressing each of the interleukin subunits.Non-limiting examples of interleukins include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, or IL-41. In some embodiments, the interleukin comprises IL-7. In some embodiments, the interleukin comprises IL-12.

[0036]

[0065] In some embodiments, the vector comprising the mutant integrase encodes a therapeutic agent comprising an interferon, hi some embodiments, the interferon comprises IFNα, IFNβ, IFNγ, or a combination thereof.

[0037]

[0066] In some embodiments, the vector comprises at least one promoter for expressing at least one polynucleotide.For example, the vector comprises the CMV promoter for expressing at least one polynucleotide encoding interleukin (e.g., P40 subunit and P35 subunit) as described herein.In some embodiments, the promoter comprises muscle-specific promoter, such as HSA (human skeletal α-actin promoter), muscle creatine kinase (MCK) gene-based promoter, such as CK6 or MHCK7 promoter; desmin gene promoter (DES); constitutive human promoter EF-1α (elongation factor 1α). Other examples of promoters include retroviral LTRs, SV40 promoters, Rous sarcoma virus (RSV) promoters, histone promoters, polIII promoters, β-actin promoters, inducible promoters such as MMTV promoters, metallothionein promoters, heat shock promoters, adenovirus promoters, albumin promoters, ApoAI promoters, B19 parvovirus promoters, human globin promoters, viral thymidine kinase promoters such as herpes simplex virus thymidine kinase promoters, retroviral LTRs, human growth hormone promoters, and MxIFN inducible promoters. In some embodiments, the promoter is a tissue-specific promoter.In some embodiments, the tissue specific promoter is selected from the group including tyrosinase-related promoters (TRP-1 and TRP-2), DF3 enhancer (for breast cells), SLPI promoter (secretory leukoprotease inhibitor, expressed in many types of carcinomas), TRS (tissue specific regulatory sequence), alpha-fetoprotein promoter (specific for normal and transformed hepatocytes, respectively), carcinoembryonic antigen promoter (for use in transformed cells of the gastrointestinal tract, lung, breast and other tissues), tyrosine hydroxylase promoter (for melanocytes), choline acetyltransferase or neuron specific enolase promoter for use in neuroblastoma, gliofibroblastoma regulatory sequence, tyrosine hydroxylase promoter, c-erb B-2 promoter, PGK promoter, PEPCK promoter, whey acidic promoter (breast tissue), and casein promoter (breast tissue) and adipocyte P2 promoter. In some embodiments, the promoter is a virus-specific promoter (e.g., a retroviral promoter, as well as other promoters such as the HIV promoter), hepatitis, herpes (e.g., EBV). In some embodiments, the promoter is the native HSV-TK promoter. In some embodiments, the promoter is a bacterial, fungal, or parasite (e.g., malaria) specific promoter that is utilized to target specific cells or tissues infected with a virus, bacteria, fungus, or parasite. In some aspects, the vector comprises a nucleic acid sequence that encodes a tag, such as a His tag or a Flag tag, for purification, imaging, or expression control purposes.

[0038]

[0067] In some embodiments, the vector (e.g., a recombinant retroviral vector described herein) comprises at least one modified untranslated region (UTR). In some embodiments, the at least one UTR is a 5'-UTR located at the 5' end of the nucleic acid sequence of the payload. In some embodiments, the at least one UTR is a 3'-UTR located at the 3' end of the nucleic acid sequence of the payload. In some embodiments, the at least one UTR comprises both a 5'-UTR and a 3'-UTR located at both the 5' and 3' ends of the nucleic acid sequence of the payload. In some embodiments, the at least one modified UTR increases expression of a payload compared to when the payload is flanked by wild-type UTRs.

[0039]

[0068] In some aspects, the vector is a viral vector, such as a retroviral vector. Viral vectors, particularly retroviral vectors, are becoming the most widely used method for inserting genes into mammalian cells, such as human cells. In some embodiments, other viral vectors are derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, adeno-associated virus, or sindbis virus. Non-limiting examples of viral vectors can include retroviral vectors, adenoviral vectors, adeno-associated virus vectors (AAV), pox vectors, parvovirus vectors, baculovirus vectors, measles virus vectors, or herpes simplex virus vectors (HSV). In some cases, recombinant retroviral vectors include gamma retroviral vectors, such as vectors derived from Moloney murine leukemia virus (MoMLV, MMLV, MuLV, or MLV) or murine stem cell virus (MSCV) genomes. In some cases, recombinant retroviral vectors also include lentiviral vectors, such as lentiviral vectors derived from human immunodeficiency virus (HIV) genomes. In some cases, the AAV vector comprises AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 serotype.In some cases, the viral vector is a chimeric viral vector that comprises viral parts from two or more viruses.In additional examples, the viral vector is a recombinant viral vector.

[0040]

[0069] In some embodiments, recombinant retroviral vector comprises at least one modification, and recombinant retroviral vector may code at least one amino acid mutation to increase the targeting efficiency of virus to cell type.For example, recombinant Sindbis retroviral vector may code E160G mutation in E2 protein of Sindbis virus, and in this case, E160G mutation increases the targeting of Sindbis virus to dendritic cells.In such a case, targeting of dendritic cells can increase the immune response and efficacy of vaccination by contacting dendritic cells with the antigen encoded by recombinant retroviral vector.

[0041]

[0070] In some aspects, a recombinant retroviral vector is described herein, comprising a first nucleic acid sequence encoding a mutant integrase described herein and a second nucleic acid sequence encoding at least one payload. In some embodiments, the mutant integrase has a Mg fragment length of the catalytic core domain when compared to the wild-type integrase. 2+ The at least one mutation in the binding motif may be included. For example, the at least one mutation may include a D125A mutation, a D184A mutation, or a combination of both D125 and D184A mutations. In some embodiments, the at least one payload includes an antigen. In some embodiments, the antigen induces an immune response in the cell. In some embodiments, the antigen includes a pathogen polypeptide. In some embodiments, the pathogen polypeptide includes a polypeptide from a pathogen, an alveolata pathogen, an amoeba pathogen, a fungal pathogen, a protozoan pathogen, a nematode pathogen, a flatworm pathogen, a viral pathogen, or a combination thereof. Table 1 provides a non-limiting list of examples of such pathogens as described above, which may serve as a basis for designing a nucleotide sequence encoding a pathogen-derived polypeptide for incorporation into a retroviral vector for transduction and subsequent expression as an antigen payload by the transduced cell.

[0042]

Table 1-1

[0043]

Table 1-2

[0044]

Table 1-3

[0045]

Table 1-4

[0046]

Table 1-5

[0047]

Table 1-6

[0048]

Table 1-7

[0049]

Table 1-8

[0050]

Table 1-9

[0051]

Table 1-10

[0052]

[0071] In some embodiments, the antigen comprises a viral polypeptide. In some embodiments, the viral polypeptide comprises a coronavirus polypeptide described herein. In some embodiments, the coronavirus polypeptide comprises a SARS-CoV-2 polypeptide. In some embodiments, the SARS-Cov-2 polypeptide comprises a spike protein or a fragment thereof. In some embodiments, the spike protein or a fragment thereof is a full-length spike protein. In some embodiments, the spike protein or a fragment thereof is a truncated spike protein. In some embodiments, the truncated spike protein comprises the N-terminal domain of the spike protein or the S2 domain of the spike protein. In some embodiments, the truncated spike protein comprises the N-terminal domain of the spike protein and the S2 domain of the spike protein. In some embodiments, the spike protein or a fragment thereof is a recombinant spike protein. In some embodiments, the spike protein or a fragment thereof comprises at least one modification. In some embodiments, the at least one modification comprises at least one amino acid mutation. In some embodiments, the at least one amino acid mutation eliminates a cleavage site, such as a furin cleavage or serine protease cleavage site in the spike protein. In some embodiments, the furin cleavage site comprises amino acid residues 682-685, amino acid residues 679-682, or amino acid residue 811 in SEQ ID NO:21 (full length spike protein) or SEQ ID NO:22 (omicron mutant spike protein).

[0053]

[0072] In some embodiments, the serine protease cleavage site comprises amino acid residues 986 and 987; or 983 and 984 in SEQ ID NO:21 (full length spike protein) or SEQ ID NO:22 (omicron mutant spike protein). In some embodiments, the at least one amino acid mutation comprises an amino acid substitution at amino acid residues 814, 889, 896, 939, 682-685, 679-682, 811, 986, 987, 983, 984, or combinations thereof in SEQ ID NO:21 (full length spike protein) or SEQ ID NO:22 (omicron mutant spike protein). In some embodiments, the at least one amino acid mutation comprises an amino acid substitution at amino acid residues 814, 889, 896, and 939 in SEQ ID NO:21 (full length spike protein) or SEQ ID NO:22 (omicron mutant spike protein). In some embodiments, the spike protein or fragment thereof comprises a signal peptide. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 13: MDAMKRGLCCVLLLCGAVFVSASQEIHARFRR. In some embodiments, the signal peptide is a secretory peptide comprising an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to IgE Fc receptor alpha. In some embodiments, the secretory peptide is IgE Fc receptor alpha.

[0054]

[0073] In some embodiments, the viral polypeptide comprises an influenza polypeptide. In some embodiments, the influenza polypeptide comprises an influenza A polypeptide, an influenza B polypeptide, an influenza C polypeptide, or an influenza D polypeptide. In some embodiments, the influenza polypeptide is an influenza A polypeptide. In some embodiments, the influenza A polypeptide comprises a neuraminidase (NA) or a fragment thereof or a hemagglutinin (HA) or a fragment thereof. In some embodiments, the influenza A polypeptide comprises a hemagglutinin (HA) or a fragment thereof. In some embodiments, the hemagglutinin (HA) or a fragment thereof is a full-length hemagglutinin (HA). In some embodiments, the hemagglutinin (HA) or a fragment thereof is a truncated hemagglutinin (HA). In some embodiments, the truncated hemagglutinin (HA) comprises a Stalk domain. In some embodiments, the hemagglutinin (HA) or a fragment thereof is a recombinant hemagglutinin (HA) comprising at least one modification. In some embodiments, the at least one modification comprises at least one amino acid mutation. In some embodiments, the at least one modification comprises hemagglutinin (HA) or a fragment thereof comprising the amino acid sequence of the extracellular domain of the M2 protein (M2e) of influenza A: SEQ ID NO: 14: MSLLTEVETPIRNEWGCRCNDSSD.

[0055]

[0074] In some embodiments, the recombinant retroviral vector encodes at least one envelope protein. In some embodiments, the envelope encoded by the recombinant retroviral vector is an amphotropic envelope. In some embodiments, the recombinant retroviral vector encodes at least one envelope protein for making an amphotropic envelope. In some embodiments, the at least one envelope protein comprises at least one alphavirus envelope protein. In some embodiments, the at least one Sindbis virus envelope protein comprises an E3 protein, an E2 protein, a 6K protein, an E1 protein, or a combination thereof. In some embodiments, the at least one Sindbis virus envelope protein comprises at least one mutation. In some embodiments, the at least one mutation increases the binding affinity between the at least one Sindbis virus envelope protein and human cells, such as dendritic cells. In some embodiments, the at least one mutation is E160G of the E2 protein.

[0056]

[0075] In some embodiments, the vector encodes a targeting moiety, such as an antibody, for targeting a cell surface marker (e.g., an antigen expressed on the cell surface of a cell associated with a disease or condition). In some embodiments, the cell surface marker is a tumor-associated antigen, such as Her2. In some embodiments, the targeting moiety comprises an antibody, a nanobody (e.g., a single chain variable fragment or scFv), or a combination thereof. In some embodiments, the targeting moiety is expressed on the surface of the viral envelope, where the vector described herein is packaged into the viral envelope. In some embodiments, the targeting moiety increases the targeting or delivery of the vector to cells or microenvironments associated with a disease or condition, such as cancer or a lesion.

[0057]

[0076] In some embodiments, the vector encodes an enzyme that is not an interleukin. In some aspects, the vector encodes an enzyme that can convert a nucleoside agent into a cytotoxic agent to kill cells associated with a disease or condition described herein. In some embodiments, the enzyme comprises a kinase that has a nucleic acid nucleotide as a substrate. In some embodiments, the kinase is a thymidine kinase, where the thymidine kinase is a salvage pathway enzyme that phosphorylates nucleoside analogs in addition to natural nucleoside substrates. In general, viral thymidine kinases can be therapeutically exploited by administering nucleoside analogs, such as ganciclovir or acyclovir, to cells expressing the viral thymidine kinase, where the viral thymidine kinase phosphorylates the nucleoside analogs to create a toxic product that can kill the cell. The viral thymidine kinases of the present disclosure can be prepared from a wide variety of viral thymidine kinases. In some embodiments, the viral thymidine kinase mutant is derived from a thymidine kinase from the Herpesviridae family, which includes both primate and non-primate herpesviruses, such as avian herpesviruses. Representative examples of suitable herpesviruses include, for example, Herpes Simplex Virus (HSV) type 1, Herpes Simplex Virus type 2, Varicella-Zoster Virus, Marmoset Herpesvirus, Feline Herpesvirus type 1, Pseudorabies Virus, Equine Herpesvirus type 1, Bovine Herpesvirus type 1, Turkey Herpesvirus, Marek's Disease Virus, Herpesvirus Saimiri, or Epstein-Barr Virus.

[0058]

[0077] In some embodiments, the thymidine kinase described herein may be a mutant thymidine kinase, where the mutant thymidine kinase comprises at least one amino acid mutation. In some embodiments, the mutant thymidine kinase is a mutant herpes simplex virus type 1 thymidine kinase (HSV1-TK) comprising at least one amino acid mutation compared to the wild-type amino acid sequence of HSV1-TK: MASYPGHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRPEQKMPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLR (SEQ ID NO: 11). In some embodiments, the mutant HSV1-TK comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the HSV1-TK amino acid sequence (e.g., SEQ ID NO: 11). In some embodiments, the mutant HSV-1-TK comprises a nuclear export sequence (NES). In some embodiments, the NES comprises the amino acid sequence of LQKKLEELELDG (SEQ ID NO: 12).

[0059]

[0078] Herpes viruses can be readily obtained from commercial sources, such as the American Type Culture Collection ("ATCC", Rockville, Md.). Herpes viruses can also be isolated from naturally occurring organisms (e.g., infected animals).

[0060]

[0079] In some embodiments, the mutant HSV1-TK comprises at least one amino acid mutation at amino acid residues 25, 26, 32, 33, 167, 168, or a combination thereof, compared to the wild-type amino acid sequence of HSV1-TK (SEQ ID NO: 11). In some embodiments, the mutation comprises substituting a wild-type amino acid with a polar, non-polar, basic, or acidic amino acid. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 167, 168, or both. In one example, the sequence is mutated at amino acid residue 167. In another example, the sequence is mutated at amino acid residue 168. In another example, the sequence is mutated at amino acid residues 167 and 168. Amino acid residue 167 may be mutated to histidine, lysine, cysteine, serine, and phenylalanine. Amino acid residue 168 may be mutated to histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25 and / or 26. The amino acid residues 25 and / or 26 may be mutated to an amino acid selected from the group consisting of glycine, serine, and glutamic acid. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 32 and / or 33. The amino acid residues 32 and / or 33 may be mutated to an amino acid selected from the group consisting of glycine, serine, cysteine, glutamic acid, and aspartic acid. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25, 26, 32, and / or 33. The amino acid residues 25, 26, 32, and / or 33 may be mutated to an amino acid selected from the group consisting of glycine, serine, cysteine, glutamic acid, and aspartic acid.

[0061]

[0080] In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25 and / or 26; and 167, where the mutation at amino acid residues 25 and / or 26 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 167 comprises histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25 and / or 26; and 168, where the mutation at amino acid residues 25 and / or 26 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 168 comprises histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25 and / or 26; and 167 and / or 168, wherein the mutation at amino acid residues 25 and / or 26 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residues 167 and / or 168 comprises histidine, lysine, cysteine, serine, or phenylalanine.

[0062]

[0081] In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 32 and / or 33; and 167, where the mutation at amino acid residues 32 and / or 33 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 167 comprises histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 32 and / or 33; and 168, where the mutation at amino acid residues 32 and / or 33 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 168 comprises histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 32 and / or 33; and 167 and / or 168, where the mutation at amino acid residues 32 and / or 33 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residues 167 and / or 168 comprises histidine, lysine, cysteine, serine, or phenylalanine.

[0063]

[0082] In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25, 26, 32, and 33; and 167, where the mutations at amino acid residues 25, 26, 32, and 33 include glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 167 includes histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25, 26, 32, and 33; and 168, where the mutations at amino acid residues 25, 26, 32, and 33 include glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 168 includes histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25, 26, 32, and 33; and 167 and / or 168, where the mutations at amino acid residues 25, 26, 32, and 33 include glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutations at amino acid residues 167 and / or 168 include histidine, lysine, cysteine, serine, or phenylalanine.

[0064]

[0083] In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25 and 26 or 32 and 33; and 167, where the mutations at amino acid residues 25 and 26 or 32 and 33 include glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 167 includes histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25 and 26 or 32 and 33; and 168, where the mutations at amino acid residues 25 and 26 or 32 and 33 include glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 168 includes histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at amino acid residues 25 and 26 or 32 and 33; and 167 and / or 168, where the mutations at amino acid residues 25 and 26 or 32 and 33 include glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutations at amino acid residues 167 and / or 168 include histidine, lysine, cysteine, serine, or phenylalanine.

[0065]

[0084] In some embodiments, the mutant HSV1-TK is mutated at any one or more of amino acid residues 25, 26, 32, and / or 33; and 167, wherein the mutation at any one or more of amino acid residues 25, 26, 32, and / or 33 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 167 comprises histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at any one or more of amino acid residues 25, 26, 32, and / or 33; and 168, wherein the mutation at any one or more of amino acid residues 25, 26, 32, and / or 33 comprises glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residue 168 comprises histidine, lysine, cysteine, serine, or phenylalanine. In some embodiments, the mutant HSV1-TK is mutated at any one or more of amino acid residues 25, 26, 32 and / or 33; and 167 and / or 168, wherein the mutation at any one or more of amino acid residues 25, 26, 32 and / or 33 comprises a glycine, serine, cysteine, glutamic acid, or aspartic acid; and the mutation at amino acid residues 167 and / or 168 comprises a histidine, lysine, cysteine, serine, or phenylalanine.

[0066]

[0085] In some embodiments, the vector, in addition to encoding mutant HSV1-TK, may also encode PiT-2, PiT-1, mCat-1 (mouse cationic receptor-1; target of ecotropic Moloney MLV), or other receptors used by gammaretroviruses.

[0067]

[0086] In some embodiments, the mutant HSV1-TK comprises an increased enzymatic activity that converts a nucleoside agent to a cytotoxic agent compared to wild-type HSV1-TK. In some embodiments, the mutant HSV1-TK increases the enzymatic activity that converts a nucleoside agent to a cytotoxic agent by at least 0.1-fold, 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, or more compared to the enzymatic activity of wild-type HSV1-TK that converts the same nucleoside agent (e.g., a prodrug) to a cytotoxic agent.

[0068]

[0087] In some embodiments, the mutant HSV1-TK increases the bystander effect of cells capable of the bystander phenomenon to kill cells related to disease or condition. "Bystander effect" as used herein refers to the phenomenon that HSV1-TK positive cells (e.g., cells contacted with the vector described herein) exert a lethal effect on adjacent HSV1-TK negative cells after HSV1-TK expression is induced in the HSV1-TK positive cells. In some embodiments, the mutant HSV1-TK increases the bystander effect by at least 0.1-fold, 0.2-fold, 0.3-fold, 0.4-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, or higher, compared to the bystander effect induced by wild-type HSV1-TK positive cells.

[0069] Vaccination

[0088] In some embodiments, described herein are vectors (e.g., recombinant viral vectors) that include the mutant integrase described herein. In some embodiments, the mutant integrase prevents the vector from being inserted into the genome of a host cell that includes the vector. In some embodiments, the vector includes a mutant reverse transcriptase, which prevents the vector from being converted into DNA for insertion into the genome of the cell. In some embodiments, the vector includes both a mutant integrase and a mutant reverse transcriptase. Such vectors that include a mutant integrase and / or a mutant reverse transcriptase may be particularly useful for vaccination of a subject in need thereof, where an antigen encoded by the vector can be expressed in the subject for an extended period of time. The extended expression of the antigen can induce a sufficient immune response against the antigen, and thus the subject can be vaccinated against a disease or condition. In some embodiments, the expression of the antigen can be inducible, where the antigen is expressed (thereby vaccinating the subject) only when needed. For example, the expression of the antigen can be induced when immunity to the antigen is waning, resulting in a booster vaccination.

[0070]

[0089] In some cases, the antigen may comprise a polypeptide sequence of a viral protein. In some embodiments, the antigen may be a viral protein of a coronavirus. In some embodiments, the coronavirus may be a severe acute respiratory syndrome-related virus (SARS-CoV). In some embodiments, the SARS-CoV is SARS-CoV-2. In some embodiments, the epitope may be a viral protein, portion thereof, or combination thereof selected from ORF1a, ORF1ab, spike protein (S protein), 3a, 3b, envelope protein (E protein), matrix protein (M protein), p6, 7a, 7b, 8b, 9b, nucleocapsid protein (N protein), ORF14, Nsp1 (leader protein), Nsp2, Nsp3, Nsp4, Nsp5 (3C-like proteinase), Nsp6, Nsp7, Nsp8, Nsp9, Nsp10 (growth factor-like protein), Nsp12 (RNA-dependent RNA polymerase, or RdRp), Nsp13 (RNA 5'-triphosphatase), Nsp14 (3'→5' exonuclease), Nsp15 (endoRNAse), and Nsp16 (2'-O-ribose methyltransferase).

[0071]

[0090] In some embodiments, the antigen may comprise a polypeptide sequence of a viral protein of an influenza virus. In some embodiments, the influenza virus is selected from the genus consisting of influenza virus A, influenza virus B, influenza virus C, and influenza virus D. In further embodiments, the influenza A virus has a subtype H1N1, H2N2, H3N2, H5N1, H7N7, H1N2, H9N2, H7N2, H7N3, H10N7, H7N9, or H6N1. In further embodiments, the influenza virus comprises an influenza B virus of B / Yamagata / 16 / 88-like lineage or B / Victoria / 2 / 87-like lineage. In further embodiments, the influenza virus comprises an influenza A virus of A / California / 04 / 2009-like lineage or A / Brisbane / 59 / 2007-like lineage. In some embodiments, the influenza may be any strain of influenza virus or any serotype within a strain of influenza virus. In some cases, the influenza virus comprises any combination of viral surface glycoproteins hemagglutinin (H or HA) and neuraminidase (N or NA). In some embodiments, the antigen comprises a modified influenza protein comprising a hemagglutinin (HA) Stalk (conserved region) domain. In some embodiments, the HA Stalk comprises at least one modification. Non-limiting examples of HA Stalk modifications can include head removal; glycine linker loop; cross-link in Cys; transmembrane removal; loop fusion peptide; position of GCN4; or cross-link in Cys. In some embodiments, the antigen comprises the extracellular domain of influenza A M2 protein (M2e), having the amino acid sequence MSLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO: 14).

[0072]

[0091] In some embodiments, the antigen may comprise a polypeptide sequence of a pathogen protein, such as another viral protein, a bacterial protein, a parasite protein, a fungal protein, or a combination thereof. In some embodiments, the antigen may comprise a tumor antigen, such as Her2, where the subject is then immunized against the cancer.

[0073] Treatment Method

[0092] In some embodiments, methods of using the vectors described herein are disclosed herein. In some embodiments, the methods include treating a disease or condition in a subject in need thereof by administering to the subject a vector or a pharmaceutical composition comprising the vector described herein. In some embodiments, the methods include contacting a cell with the vector, followed by administering the cell to the subject. In some embodiments, the cell contacted with the vector is an autologous cell. For example, the cell may first be isolated from the subject, and optionally cultured or expanded before contacting with the vector. In some embodiments, the expression of an interleukin (e.g., IL-12, IL-7, or interferon P40 or P35) or HSV1-TK encoded by the vector can be verified in the cell before administering the cell to the subject.

[0074]

[0093] In some embodiments, the method includes administering two or more vectors to a subject, where a first of the two or more vectors encodes an interleukin (e.g., IL-12, IL-7, or interferon P40 or P35) as described herein, and a second of the two or more vectors encodes a thymidine kinase (e.g., mutated HSV1-TK) as described herein. In some embodiments, the method includes first contacting a cell with two or more vectors, followed by administering the cell to a subject. In embodiments, the interleukin (e.g., IL-12, IL-7, or interferon P40 or P35) and the thymidine kinase (e.g., mutated HSV1-TK) are encoded by the same vector. In some embodiments, the administration is by any suitable mode of administration, systemic administration (e.g., intravenous, inhalation, etc.). In some embodiments, the subject is a human. In some embodiments, the disease or condition is a cancer or a pathology. In some embodiments, the disease or condition is a metabolic disease.

[0075]

[0094] In some embodiments, the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition delivers an interleukin to a cell or microenvironment associated with a disease or condition. In some embodiments, the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition that delivers an interleukin reduces toxicity in a subject (e.g., as determined by reduced cell death or reduced expression of hot tumor genes in cells not associated with a disease or condition) compared to administering the interleukin directly to the subject. In some embodiments, the toxicity of delivering an interleukin by the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition described herein is reduced by at least 0.1-fold, 0.2-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, 50.0-fold, or more compared to the toxicity induced by administering the interleukin directly to a subject.

[0076]

[0095] In some embodiments, the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition delivers IL-12 to cells or microenvironments associated with a disease or condition. In some embodiments, the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition that delivers IL-12 (either as P40 and P35 subunits or recombinant IL-12) reduces toxicity in a subject (e.g., as determined by reduced cell death or reduced expression of hot oncogenes in cells not associated with a disease or condition) compared to the direct administration of IL-12 to the subject. In some embodiments, the toxicity of delivering IL-12 by the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition described herein is reduced by at least 0.1-fold, 0.2-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, 50.0-fold, or more compared to the toxicity induced by direct administration of IL-12 to a subject.

[0077]

[0096] In some embodiments, the IL-12 encoded by the vector is expressed or secreted by the cell. In some embodiments, the IL-12 expressed or secreted by the cell can stimulate innate immune signaling or response in the subject. In some embodiments, the method includes stimulating the production of an endogenous cytokine (e.g., IFN-γ) with the expressed or secreted interleukin (e.g., P40 or P35 of IL-12) to treat a disease or condition. Hot oncogene expression can refer to the expression of a gene product, such as a cytokine described herein, that initiates an endogenous immune response. Thus, hot oncogene expression can cause the death of a cell (e.g., cancer or tumor cell) associated with a disease or condition by an endogenous immune response.

[0078]

[0097] In some embodiments, the vector, cell comprising the vector, recombinant virus encoded by the vector, or pharmaceutical composition that delivers an interleukin has increased efficacy for treating a disease or condition in a subject compared to direct administration of the interleukin to the subject (e.g., as determined by increased cell death of tumor cells or increased expression of a hot tumor gene). In some embodiments, the efficacy for treating a disease or condition by delivering an interleukin by a vector, cell comprising the vector, recombinant virus encoded by the vector, or pharmaceutical composition described herein is reduced by at least 0.1-fold, 0.2-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, 50.0-fold, or more compared to the efficacy of treating the disease or condition by directly administering the interleukin to a subject.

[0079]

[0098] In some embodiments, a vector, a cell comprising a vector, a recombinant virus encoded by a vector, or a pharmaceutical composition that delivers IL-12 (either as the P40 and P35 subunits or recombinant IL-12) increases efficacy for treating a disease or condition in a subject (e.g., as determined by increased cell death of tumor cells or increased expression of hot oncogenes) compared to direct administration of IL-12 to the subject. In some embodiments, the efficacy for treating a disease or condition by delivering IL-12 by a vector, a cell comprising a vector, a recombinant virus encoded by a vector, or a pharmaceutical composition described herein is reduced by at least 0.1-fold, 0.2-fold, 0.5-fold, 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, 50.0-fold, or more compared to the efficacy of treating a disease or condition by direct administration of IL-12 to a subject.

[0080]

[0099] In some embodiments, the vector, cell containing the vector, recombinant virus encoded by the vector, or pharmaceutical composition is administered at least once during the period (e.g., every 2 days, twice a week, once a week, every week, 3 times a month, twice a month, once a month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, once a year). In some embodiments, the composition is administered twice or more during the period (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 times).

[0081]

[0100] In some aspects, described herein are methods of treating or preventing a disease or condition in a subject by vaccinating the subject, comprising administering to the subject a recombinant retroviral vector described herein, a recombinant virus encoded by a retroviral vector described herein, a cell transduced by a retroviral vector described herein, or a pharmaceutical composition described herein. In some embodiments, at least one payload encoded by the retroviral vector comprises an antigen that induces an immune response in the subject, thereby treating or preventing a disease or condition in the subject by vaccinating the subject. In some embodiments, the immune response comprises induction of neutralizing antibodies that target the antigen, thereby generating immunity to the antigen in the subject. In some embodiments, the immune response comprises induction of immunoglobulin antibodies that target the antigen, thereby generating immunity to the antigen in the subject. In some embodiments, the immunoglobulin antibodies comprise IgG antibodies, IgM antibodies, IgA antibodies, IgD antibodies, IgE antibodies, or combinations thereof. In some embodiments, the immunoglobulin antibodies comprise IgG antibodies.

[0082]

[0101] In some embodiments, at least one payload is expressed and secreted in a subject for a duration of at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, or longer. In some embodiments, at least one payload is expressed or secreted in a subject for a duration of at least 12 hours, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, or longer. In some embodiments, the duration of an immune response induced by at least one payload expressed or secreted for at least 12 hours is increased by at least 10%, at least 20%, at least 50%, at least 100%, at least 5-fold, at least 10-fold, or more, compared to the duration of an immune response induced by a comparable payload expressed or secreted for less than 12 hours. In some embodiments, the duration of an immune response induced by at least one payload expressed or secreted for at least one day is increased by at least 10%, at least 20%, at least 50%, at least 100%, at least 5-fold, at least 10-fold, or more compared to the duration of an immune response induced by a comparable payload expressed or secreted for less than one day. In some embodiments, the duration of an immune response induced by at least one payload expressed or secreted for at least two days is increased by at least 10%, at least 20%, at least 50%, at least 100%, at least 5-fold, at least 10-fold, or more compared to the duration of an immune response induced by a comparable payload expressed or secreted for less than two days.In some embodiments, the duration of an immune response induced by at least one payload expressed or secreted for at least 3 days is increased by at least 10%, at least 20%, at least 50%, at least 100%, at least 5-fold, at least 10-fold, or more compared to the duration of an immune response induced by a comparable payload expressed or secreted for less than 3 days. In some embodiments, the duration of an immune response induced by at least one payload expressed or secreted for at least 4 days is increased by at least 10%, at least 20%, at least 50%, at least 100%, at least 5-fold, at least 10-fold, or more compared to the duration of an immune response induced by a comparable payload expressed or secreted for less than 4 days. In some embodiments, the duration of an immune response induced by at least one payload expressed or secreted for at least 5 days is increased by at least 10%, at least 20%, at least 50%, at least 100%, at least 5-fold, at least 10-fold, or more compared to the duration of an immune response induced by a comparable payload expressed or secreted for less than 5 days. In some embodiments, the duration of an immune response induced by at least one payload expressed or secreted for at least 5 days is increased by at least 10%, at least 20%, at least 50%, at least 100%, at least 5-fold, at least 10-fold, or more compared to the duration of an immune response induced by a comparable payload expressed or secreted for less than 6 days. In some embodiments, the duration of an immune response induced by at least one payload expressed or secreted for at least 7 days is increased by at least 10%, at least 20%, at least 50%, at least 100%, at least 5-fold, at least 10-fold, or more compared to the duration of an immune response induced by a comparable payload expressed or secreted for less than 7 days.In some embodiments, the duration of the immune response induced by at least one payload expressed or secreted for at least 8 days is increased by at least 10%, at least 20%, at least 50%, at least 100%, at least 5-fold, at least 10-fold, or more compared to the duration of the immune response induced by a comparable payload expressed or secreted for less than 8 days. In some embodiments, the duration of the immune response induced by at least one payload expressed or secreted for at least 8 days is increased by at least 10%, at least 20%, at least 50%, at least 100%, at least 5-fold, at least 10-fold, or more compared to the duration of the immune response induced by a comparable payload expressed or secreted for less than 9 days. In some embodiments, the immune response persists in the subject for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 12 months, at least 2 years, at least 3 years, or more.

[0083]

[0102] In some embodiments, the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition is administered in a therapeutically effective amount by various forms and routes, such as, for example, oral or topical administration. In some embodiments, the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition may be administered by bronchial lavage, sublingual, intratumoral, parenteral, intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, subarachnoid, intraocular, intrasternal, intraocular, endothelial, topical, intranasal, intrapulmonary, intrarectal, intraarterial, intrathecal, inhalation, intralesional, intradermal, epidural, intracapsular, subcapsular, intracardiac, transtracheal, subcuticular, or intraspinal administration, for example, by injection or infusion. In some embodiments, the composition may be administered by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa administration). In some embodiments, the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition is delivered via multiple routes of administration.

[0084]

[0103] In some embodiments, the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition is administered by intravenous infusion. In some embodiments, the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition comprising the vector is administered by slow continuous infusion over a long period of time, for example, over a period of time longer than 24 hours. In some aspects, the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition may be administered in a localized manner, for example, via injection of the drug directly into an organ, and optionally in a depot or sustained release formulation or implant.

[0085]

[0104] In some embodiments, the method includes monitoring expression of an interleukin, such as IL-12, in the subject after the subject has been treated. In some aspects, the method includes monitoring expression levels of IL-12, and when IL-12 expression in the subject reaches a predetermined threshold, an interleukin inhibitor can be administered to the subject.

[0086]

[0105] In some embodiments, the vector, the cell containing the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition provided herein may be administered with at least one additional therapeutic agent, such as an antiviral therapeutic agent, a chemotherapeutic agent, an antibiotic, a cell therapeutic agent, a cytokine therapeutic agent, or an anti-inflammatory agent. In some embodiments, the at least one additional therapeutic agent comprises a nucleoside agent (e.g., a prodrug). Non-limiting examples of prodrugs include, for thymidine kinase, FHBG (9-[4-fluoro-3-(hydroxymethyl)butyl]guanine), FHPG (9-([3-fluoro-1-hydroxy-2-propoxy]methyl)guanine), FGCV (fluoroganciclovir), FPCV (fluoropenciclovir), FIAU (1-(2'-deoxy-2'-fluoro-1-β-D-arabinofuranosyl)-5-iodouracil), FEAU (fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil), FMAU (fluoro-5-methyl-1-beta-D-arabinofuranosyluracil), FHOMP (6-((1-fluoro-3-hydroxypropan-2-yloxy)methyl)-5-methylpyrimidine-2,4(1H,3H)-dione), ganciclovir, valganciclovir, acyclovir, Mention may be made of valacivlovir, penciclovir, radiolabeled pyrimidines with a 4-hydroxy-3-(hydroxymethyl)butyl side chain at N-1 (HHG-5-FEP), or 5-(2-)hydroxyethyl)- and 5-(3-hydroxypropyl)-substituted pyrimidine derivatives with 2,3-dihydroxypropyl, acyclovir, ganciclovir and penciclovir-like side chains attached; for oxidoreductases, ifosfamide; for VZV-TK, 6-methoxypurine arabinoside; for cytosine deaminase, 5-fluorocytosine; for beta-glucuronidase, doxorubicin; for nitroreductase, CB1954; and for carboxypeptidase A, N-(cyanoacetyl)-L-phenylalanine, or N-(3-chloropropionyl)-L-phenylalanine.In some embodiments, the nucleoside agent comprises ganciclovir, valganciclovir, acyclovir, valacyclovir, or penciclovir.

[0087]

[0106] In some embodiments, the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition provided herein can be administered before, during, or after the appearance of a disease or condition. In some embodiments, the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition can be used as a prophylactic and can be administered continuously to a subject. In some embodiments, the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition can be administered to a subject before the onset of symptoms associated with a disease or condition.

[0088]

[0107] The actual dosage level of the agent of the present disclosure (e.g., a vector, a cell containing a vector, a recombinant virus encoded by a vector, or a pharmaceutical composition) can be varied to obtain an amount of agent that achieves the desired therapeutic response for a particular subject, composition, and mode of administration without causing toxicity to the subject (e.g., a subject for immunization or a subject for treatment). The selected dosage level may depend on various pharmacokinetic factors, such as the activity of the particular composition of the present invention employed, the route of administration, the time of administration, the frequency of excretion, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0089]

[0108] Dosage regimens can be adjusted to provide the optimum desired response (e.g., therapeutic and / or prophylactic response). For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. Dosage unit form, as used herein, refers to physically discrete units suitable as a combined dosage for a subject (e.g., a subject for immunization or a subject for treatment); each unit contains a predetermined amount of active agent calculated to produce a desired therapeutic effect depending on the required pharmaceutical carrier. The details regarding dosage unit forms of the present disclosure can be determined by and directly depend on (a) the unique characteristics of the active agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art in compounding such active agents for the treatment of susceptibility in an individual. Dosage can be determined by reference to the plasma or local concentration of the circular polyribonucleotide or antibody or antigen-binding fragment thereof. Dosage can be determined by reference to the plasma or local concentration of the linear polyribonucleotide or antibody or antigen-binding fragment thereof.

[0090]

[0109] In some embodiments, the vectors, cells containing the vectors, recombinant viruses encoded by the vectors, or pharmaceutical compositions described herein may be in unit dosage form suitable for single administration of precise dosages. In unit dosage form, the formulation may be divided into unit doses containing appropriate amounts of the composition. In unit dosage form, the formulation may be divided into unit doses containing appropriate amounts of one or more linear polyribonucleotides, antibodies or antigen-binding fragments thereof, and / or therapeutic agents. The unit dosage form may be in the form of a package containing discrete amounts of the formulation. Non-limiting examples are packaged injectables, vials, and ampoules. The aqueous suspension compositions disclosed herein may be packaged in single-dose non-reclosable containers. Multi-dose reclosable containers may be used, for example, in combination with or without preservatives. The formulations for injection disclosed herein may be in unit dosage form, for example, in ampoules containing preservatives or in multi-dose containers.

[0091]

[0110] The dosage of the vector, the cell containing the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition is preferably within a circulating concentration range that includes the ED50 with little or no toxicity. The dosage can vary within this range depending on the dosage form employed and the route of administration utilized. The therapeutically effective dose can be initially estimated from cell culture assays. The dose can be provided in an animal model that achieves a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal infection or half-maximal inhibition) when determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by RT-qPCR or ddPCR methods.

[0092]

[0111] The effective amount or therapeutically effective amount of the vector disclosed herein, the cell containing the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition to be administered to a subject in need of treatment can be determined in various ways.As an example, the amount can be based on the titer or efficacy of the virus in an animal model.Alternatively, the dosage regimen used in clinical trials can be used as a general guideline.

[0093]

[0112] In some embodiments, the daily dose may be administered in a single dose or in several doses at various times throughout the day. In some embodiments, higher dosages may be required and may be reduced over time once an optimal initial response is obtained. In some embodiments, treatment may be continuous for days, weeks, or years, or may be spaced apart with intervening rest periods. In some embodiments, dosage is modified according to other treatments that the individual may have undergone. However, the method of treatment is in no way limited to a particular concentration or range of retroviral particles, which may vary for each individual being treated and each derivative used. Individualization of dosage may be required to achieve maximum effect for a given individual. In some embodiments, the dosage administered to the individual being treated varies depending on the individual's age, severity or stage of the disease, and response to treatment courses. In some embodiments, clinical parameters for determining dosage include, but are not limited to, tumor size, changes in levels of tumor markers used in clinical trials for a particular malignancy. In some embodiments, the treating physician determines the therapeutically effective amount to be used for a given individual. In some embodiments, the therapies disclosed herein are administered as frequently as needed, for as long as deemed necessary by the treating physician.

[0094]

[0113] In some embodiments, multiple courses of therapeutic agents (e.g., a first and a second course of therapeutic agents) are administered to a subject in need of treatment. In some embodiments, the first and / or second course of therapeutic agents are administered intravenously. In other embodiments, the first and / or second course of therapeutic agents are administered via intra-arterial infusion, including but not limited to infusion via the hepatic artery, cerebral artery, coronary artery, pulmonary artery, iliac artery, celiac artery, gastric artery, splenic artery, renal artery, gonadal artery, subclavian artery, vertebral artery, axillary artery, brachial artery, radial artery, ulnar artery, carotid artery, femoral artery, inferior mesenteric artery, and / or superior mesenteric artery. Intra-arterial infusion may be achieved using an endovascular procedure, a percutaneous procedure, or a surgical approach involving an incision. In some embodiments, the first and second courses of therapeutic agents may be administered sequentially. In yet other embodiments, the first and second courses of therapeutic agents may be administered simultaneously. In yet other embodiments, the optional third course of therapeutic agent may be administered sequentially or simultaneously with the courses of the first and second therapeutic agents.

[0095]

[0114] In some embodiments, the vector, cell containing the vector, recombinant virus encoded by the vector, or pharmaceutical composition disclosed herein may be administered with a course of therapeutic agent administered sequentially or simultaneously in high doses based on accumulation.For example, in some embodiments, the course of therapeutic agent may be administered systemically, e.g., intravenously, to a patient in need thereof based on accumulation.The course of the first therapeutic agent may be administered systemically.Alternatively, the course of the first therapeutic agent may be administered in a localized manner, e.g., intra-arterially, to a patient in need thereof via intra-arterial infusion based on accumulation.

[0096]

[0115] In yet other embodiments, a subject in need thereof may receive a combination of systemic and intra-arterial infusion administration of high doses of the vector, cells comprising the vector, recombinant virus encoded by the vector, or pharmaceutical composition, either sequentially or simultaneously. For example, a patient in need thereof may first be administered a systemic dose of the vector, cells comprising the vector, recombinant virus encoded by the vector, or pharmaceutical composition on a depot basis, followed by an additional course of intra-arterial infusion, e.g., hepatic artery infusion, of the therapeutic agent as a depot-based delivery.

[0097]

[0116] A subject in need of treatment can also be administered a course of therapeutic agent that delivers a vector, a cell containing a vector, a recombinant virus encoded by a vector, or a pharmaceutical composition, either systemically or locally (e.g., intra-arterial infusion, e.g., hepatic artery infusion), over a period of time. In some embodiments, the period can be at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. Administration can also be performed in a chronic manner, i.e., for an indefinite period of time or indefinitely.

[0098]

[0117] The administration of the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition may also be performed in a regular manner, for example, at least once a day, at least twice a day, at least three times a day, at least four times a day, or at least five times a day. The regular administration of the delivery of the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition may depend on the mode of administration in addition to the time of delivery. For example, parenteral administration may be performed only once a day for an extended period of time, whereas the oral administration of the delivery of the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition may be performed more than once a day, in which case the administration of the delivery of the vector, the cell comprising the vector, the recombinant virus encoded by the vector, or the pharmaceutical composition is performed for a shorter period of time.

[0099]

[0118] In one embodiment, the subject is allowed to rest for 1-2 days between the first and second therapeutic courses. In some embodiments, the subject is allowed to rest for 2-4 days between the first and second therapeutic courses. In other embodiments, the subject is allowed to rest for at least 2 days between the first and second therapeutic courses. In still other embodiments, the subject is allowed to rest for at least 4 days between the first and second therapeutic courses. In still other embodiments, the subject is allowed to rest for at least 6 days between the first and second therapeutic courses. In some embodiments, the subject is allowed to rest for at least 1 week between the first and second therapeutic courses. In still other embodiments, the subject is allowed to rest for at least 2 weeks between the first and second therapeutic courses. In one embodiment, the subject is allowed to rest for at least 1 month between the first and second therapeutic courses. In some embodiments, the subject is allowed to rest for at least 1-7 days between the course of the second therapeutic agent and the optional course of the third therapeutic agent, hi yet other embodiments, the subject is allowed to rest for at least 1-2 weeks between the course of the second therapeutic agent and the optional course of the third therapeutic agent.

[0100]

[0119] In some embodiments, the vector, cell comprising the vector, recombinant virus encoded by the vector, or pharmaceutical composition is administered to increase the local concentration of interleukins (e.g., P40 or P35 of IL-12) and thymidine kinases (e.g., mutated HSV1-TK) in cells or microenvironments (e.g., cancer or lesions) associated with a disease or condition described herein. In some embodiments, the vector, cell comprising the vector, recombinant virus encoded by the vector, or pharmaceutical composition is administered via intra-arterial infusion, thereby increasing the local concentration of the therapeutic vector to a specific organ system. In yet other embodiments, the vector, cell comprising the vector, recombinant virus encoded by the vector, or pharmaceutical composition is administered intratumorally. In some embodiments, depending on the location of the target lesion, catheterization of the hepatic artery is followed by infusion into the pancreaticoduodenal artery, right hepatic artery, and middle hepatic artery, respectively, to locally target liver lesions. In some embodiments, local distribution of the polypeptide or delivery vector to other organ systems, such as the lungs, gastrointestinal, brain, reproductive tract, spleen, or other defined organ systems, is achieved via catheterization or other local delivery systems. In some embodiments, intra-arterial infusion is accomplished via any other available arterial source, including, but not limited to, infusion via the hepatic, cerebral, coronary, pulmonary, iliac, celiac, gastric, splenic, renal, gonadal, subclavian, vertebral, axillary, brachial, radial, ulnar, carotid, femoral, inferior mesenteric and / or superior mesenteric arteries. In some embodiments, intra-arterial infusion is accomplished using an endovascular procedure, a percutaneous procedure or a surgical approach involving an incision.

[0101] Pharmaceutical Compositions

[0120] Described herein are pharmaceutical compositions comprising therapeutic agents (e.g., vectors or cells comprising vectors described herein) or antigens for vaccinating subjects. In some embodiments, cells contacted with vectors described herein express interleukins (e.g., P40 or P35 of IL-12), thymidine kinases (e.g., mutated HSV1-TK), or antigens described herein in vivo or in vitro. In some embodiments, cells are obtained from a subject; expanded in an in vitro environment; and administered back to the subject to treat a disease or condition in the subject or vaccinate the subject. In some embodiments, at least one vector described herein or cells contacted with at least one vector can be formulated into a vaccine. In some embodiments, at least one vector described herein is formulated into an RNA vaccine. In some embodiments, at least one vector described herein is formulated into an mRNA vaccine, in which the antigen is encoded by the mRNA as the payload of the vector. In some embodiments, the pharmaceutical composition comprises a recombinant virus encoded by a vector described herein. For example, the pharmaceutical composition may include a modified Sindbis virus for targeting dendritic cells. In some embodiments, the vaccine includes at least two vectors described herein, and the at least two vectors may encode different payloads. In some embodiments, the vector includes a mutant integrase described herein, such that the vector can no longer insert into the genome of a cell. In some embodiments, the vector includes a mutant reverse transcriptase, such that the nucleic acid (e.g., RNA) of the vector can no longer be converted into DNA and subsequently inserted into the genome of a cell. In some embodiments, the vector includes both a mutant integrase and a mutant reverse transcriptase. In some aspects, the cells are obtained from a source that is not from the subject. In some aspects, the cells are obtained from a cell line. In some embodiments, the cells are formulated into a pharmaceutical composition.In some embodiments, a pharmaceutical composition comprises a nucleoside agent described herein.

[0102]

[0121] In some embodiments, the pharmaceutical composition comprises pharma- ceutical acceptable carrier, excipient, or diluent.In some embodiments, the pharmaceutical composition described herein comprises at least one additional active agent other than the cells described herein.In some embodiments, the at least one additional active agent is a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitor, an antihormonal agent, an antiangiogenic agent, or a checkpoint inhibitor.

[0103]

[0122] In some aspects, the pharmaceutical composition comprises an adjuvant for increasing the immune response for vaccination of a subject in need thereof. In some embodiments, the adjuvant may comprise an analgesic adjuvant. In some embodiments, the adjuvant may comprise an inorganic compound such as alum, aluminum hydroxide, aluminum phosphate, or hydroxyapatite. In some embodiments, the adjuvant may comprise mineral oil or paraffin oil. In some embodiments, the adjuvant may comprise bacterial products such as inactivated Bordetella pertussis, Mycobacterium bovis, tor oxoids, etc. In some embodiments, the adjuvant may comprise non-bacterial organic substances such as squalene. In some embodiments, the adjuvant may comprise the use of a delivery system such as detergent (Quil A). In some embodiments, the adjuvant may comprise a plant saponin, such as saponin from Quillaja, soybean, or Polygala senega. In some embodiments, the adjuvant may include Freund's complete or incomplete adjuvant, hi some embodiments, the adjuvant may include a food-based oil, such as peanut oil.

[0104]

[0123] In carrying out the methods of treatment or use provided herein, a therapeutically effective amount of the pharmaceutical composition described herein is administered to a mammal having a disease or condition, such as cancer or a lesion, to be treated.In some embodiments, the mammal is a human.The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used, and other factors.The therapeutic agent described herein, in some cases the composition, can be used alone or in combination with one or more therapeutic agents as components of a mixture.

[0105]

[0124] The pharmaceutical compositions described herein can be administered to a subject by suitable administration route, examples of which include, but are not limited to, bronchial lavage, sublingual, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration route.Compositions described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast dissolving formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.

[0106]

[0125] Pharmaceutical compositions containing a therapeutic agent can be manufactured in a conventional manner, e.g., by way of example only, conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, entrapping or compressing processes.

[0107]

[0126] Pharmaceutical compositions may contain at least exogenous therapeutic agents as active ingredients in free acid or free base form, or in pharmaceutical acceptable salt form.In addition, the methods and compositions described herein include the use of N-oxides (optionally), crystalline forms, amorphous phases, as well as active metabolites of these compounds with the same type of activity.In some embodiments, therapeutic agents are present in unsolvated form or in solvated form with pharmaceutical acceptable solvents, such as water, ethanol, etc.Solvated forms of therapeutic agents are also considered to be disclosed herein.

[0108]

[0127] In certain embodiments, the pharmaceutical compositions provided herein comprise one or more preservatives for inhibiting microbial activity.Suitable preservatives include mercury-containing substances, such as phenylmercuric borate and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0109]

[0128] In some embodiments, the pharmaceutical compositions described herein benefit from antioxidants, metal chelators, thiol-containing compounds and other general stabilizing agents. Examples of such stabilizers include, but are not limited to, (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, I about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfates and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[0110]

[0129] The pharmaceutical compositions described herein can be formulated into any suitable dosage form, including but not limited to aqueous oral dispersion, liquid, gel, syrup, elixir, slurry, suspension, solid oral dosage form, aerosol form, controlled release formulation, fast dissolving formulation, effervescent formulation, lyophilized formulation, tablet, powder, pill, dragee, capsule, delayed release formulation, sustained release formulation, pulsed release formulation, multiparticulate formulation, and mixed immediate release and controlled release formulation.In one embodiment, the therapeutic agent discussed herein, for example, therapeutic agent, is formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous or intravenous injection.In one embodiment, the formulation suitable for intramuscular, subcutaneous or intravenous injection includes physiologically acceptable sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and sterile powder for rehydration into sterile injectable solution or dispersion. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propylene glycol, polyethylene-glycol, glycerol, cremophor, etc.), suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preservatives, wetting agents, emulsifying agents, and dispensing agents. Prevention of microbial growth can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some cases, it is desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of substances that delay absorption, such as aluminum monostearate and gelatin.

[0111]

[0130] For intravenous injection or drip or infusion, the pharmaceutical compositions described herein are formulated in the form of an aqueous solution, preferably in the form of a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiologically buffered saline.For transmucosal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.For other parenteral injections, suitable formulations include aqueous or non-aqueous solutions, preferably with physiologically compatible buffers or excipients.

[0112]

[0131] Parenteral injections may include bolus injections or continuous infusions. Pharmaceutical compositions for injection may be in unit dosage form, for example in the form of ampoules or multi-dose containers with added preservatives. The pharmaceutical compositions described herein may be in a form suitable for parenteral injections, as sterile suspensions, solutions or emulsions in oily or aqueous vehicles, or may contain forming agents such as suspending, stabilizing and / or dispersing agents. In one embodiment, the active ingredient is in powder form for constitution with a suitable vehicle, for example sterile pyrogen-free water, before use.

[0113]

[0132] For administration by inhalation, the therapeutic agent is formulated for use as an aerosol, mist or powder. The pharmaceutical composition described herein is conveniently delivered in the form of an aerosol spray supply from a pressurized pack or nebulizer using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, such as, by way of example only, gelatin capsules and cartridges, may be formulated containing a powder mix of the therapeutic agent described herein and a suitable powder base, such as lactose or starch, for use in an inhaler or insufflator. The formulation containing the composition is prepared as a solution in saline employing benzyl alcohol or other suitable preservatives, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. Preferably, these compositions and formulations are prepared using suitable non-toxic pharma-ceutically acceptable ingredients. The selection of suitable carrier depends on the exact nature of the desired nasal dosage form, for example, solution, suspension, ointment or gel.Nasal dosage forms generally contain a large amount of water in addition to active ingredient.A small amount of other ingredients, such as pH adjusters, emulsifiers or dispersants, preservatives, surfactants, gelling agents, or buffering agents and other stabilizers and solubilizers, are optionally present.Preferably, nasal dosage forms should be isotonic with nasal secretions.

[0114]

[0133] In another embodiment, the dosage form includes microencapsulated formulation.In some embodiments, one or more other compatible materials are present in the microencapsulated material.Non-limiting examples of materials include pH adjuster, erosion promoter, antifoaming agent, antioxidant, flavoring agent, and carrier material, such as binder, suspending agent, disintegrant, filler, surfactant, solubilizer, stabilizer, lubricant, wetting agent, and diluent.

[0115]

[0134] The liquid formulation for oral administration is optionally an aqueous suspension selected from the group consisting of, but not limited to, pharma-ceutically acceptable aqueous oral dispersion, emulsion, solution, elixir, gel, and syrup.In addition to the therapeutic agent, the liquid formulation optionally comprises additives, such as (a) disintegrant; (b) dispersant; (c) wetting agent; (d) at least one preservative, (e) viscosity enhancing agent, (f) at least one sweetener, and (g) at least one flavoring agent.In some embodiments, the aqueous dispersion further comprises a crystal formation inhibitor.

[0116]

[0135] In some embodiments, the pharmaceutical compositions described herein may be self-emulsifying drug delivery systems (SEDDS). Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are created by forceful mechanical dispersion. In contrast to emulsions or microemulsions, SEDDS spontaneously form emulsions when added to excess water without any external mechanical dispersion or agitation. The advantage of SEDDS is that only gentle mixing is required to disperse the droplets throughout the solution. In addition, water or aqueous phase is optionally added immediately prior to administration, thereby ensuring the stability of unstable or hydrophobic active ingredients. Thus, SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provides an improvement in the bioavailability of hydrophobic active ingredients.

[0117]

[0136] Oral formulations are administered using various formulations known in the art.In addition, the oral dosage forms described herein may further comprise a biodegradable (hydrolyzable) polymer carrier, which also helps the dosage form to adhere to oral mucosa.For oral or sublingual administration, the composition may take the form of tablets, lozenges, or gels that are formulated in a conventional manner.

[0118]

[0137] For intravenous injection, the pharmaceutical composition is optionally formulated in the form of an aqueous solution, preferably in a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiologically buffered saline.For transmucosal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation.For other parenteral injections, suitable formulations include aqueous or non-aqueous solutions, preferably with physiologically compatible buffers or excipients.

[0119]

[0138] Parenteral injections optionally include bolus injections or continuous infusions. Preparations for injections are optionally in unit dosage form, for example in ampoules or multi-dose containers with added preservatives. In some embodiments, the compositions described herein are in a form suitable for parenteral injections as sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain forming agents such as suspending agents, stabilizing agents and / or dispersing agents. Compositions for parenteral administration include aqueous solutions of the agent that modulates the activity of the carotid body in water-soluble form. In addition, suspensions of the agent that modulates the activity of the carotid body, for example oily suspension injections, are optionally prepared as needed.

[0120]

[0139] Traditional formulation techniques include, for example, one or a combination of the following methods: (1) dry blending, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion. Other methods include, for example, spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., Wurster coating), tangential coating, top spraying, tableting, extrusion, and the like.

[0121]

[0140] In some embodiments, a pharmaceutical composition may be provided that includes particles of a therapeutic agent and at least one dispersing or suspending agent for oral administration to a subject. The formulation may be a powder and / or granule for suspension, which when mixed with water, provides a substantially uniform suspension.

[0122]

[0141] In some embodiments, the pharmaceutical composition may include an agent that facilitates the binding and / or entry of the vector into cells, either for in vivo or ex vivo application. For either co-administration with the vectors described herein or pre-formulation with such vectors before administration to a patient, subject, or cells obtained from a patient or subject, certain pharmaceutical compositions may include polycationic substances. Such polycationic substances include, but are not limited to, polybrene, protamine sulfate, or recombinant human fibronectin.

[0123]

[0142] In addition, the pharmaceutical composition optionally contains one or more pH adjusting or buffering agents, examples of which include acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane; and buffers such as citric acid / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition in an acceptable range.

[0124]

[0143] In addition, the pharmaceutical composition optionally contains one or more salts in an amount necessary to bring the osmolality of the pharmaceutical composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, with suitable salts including sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0125]

[0144] In one embodiment, the aqueous suspension and dispersion described herein remain homogeneous for at least 4 hours.In one embodiment, the aqueous suspension is resuspended into a homogeneous suspension by physical stirring lasting less than 1 minute.In yet another embodiment, stirring is not required to maintain a homogeneous aqueous dispersion.

[0126]

[0145] Aerosol formulations for nasal administration are generally aqueous solutions designed to be administered to the nasal passages in the form of drops or sprays. Nasal solutions may resemble nasal secretions in that they are generally isotonic and slightly buffered to maintain a pH of about 5.5 to about 6.5, although pH values ​​outside this range may also be used. Antimicrobial agents or preservatives may also be included in the formulation.

[0127]

[0146] Aerosol formulations for inhalation and inhalation can be designed to deliver the drug or drug combination to the subject's respiratory tree when administered via the nasal or oral respiratory route. Inhalation solutions may be administered, for example, by a nebulizer. Inhalation or insufflation containing micronized or liquid drugs may be delivered to the respiratory system as a pharmaceutical aerosol of a solution or suspension of the drug or drug combination in a propellant, for example, to aid in distribution. The propellant may be a liquefied gas, examples of which include halocarbons, e.g., fluorocarbons such as fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons, and hydrochlorocarbons, as well as hydrocarbons and hydrocarbon ethers. The aerosol formulation may also contain other ingredients, such as ethanol, isopropanol, propylene glycol, as well as surfactants or other ingredients such as oils and detergents. These ingredients may help stabilize the formulation and / or lubricate the valve components.

[0128] kit

[0147] In some embodiments, kits for using the vectors described herein are described. In some embodiments, the kits can be used to treat a disease or condition in a subject. In some embodiments, the kits can be used to vaccinate a subject. In some embodiments, the kits include a group of materials or components apart from the vector or cells containing the vector. In some embodiments, the kits include components for assaying the number of units of a biomolecule (e.g., a vector, a cell, a therapeutic agent including IL-12, a mutant HSV1-TK, an antigen such as a spike protein or an HA protein, or a combination thereof) synthesized and / or released or expressed by the cells described herein. In some embodiments, the kits include components for performing assays such as enzyme-linked immunosorbent assay (ELISA), single molecule array (Simoa), PCR, and qPCR. The exact nature of the components configured in the kit depends on its intended purpose. For example, the kits may be designed for the purpose of treating a disease or condition (e.g., cancer or lesion) disclosed herein in a subject. In some embodiments, the kits are specifically designed for the purpose of treating a mammalian subject. In some embodiments, the kits are specifically designed for the purpose of treating a human subject. In some embodiments, the kits are specifically designed for the purpose of vaccinating a mammalian subject. In some embodiments, the kits are specifically designed for the purpose of vaccinating a human subject.

[0129]

[0148] Instructions for use may be included in the kit. In some embodiments, the kit includes instructions for administering the vector, cell, or pharmaceutical composition described herein to a subject in need thereof. In some embodiments, the kit includes instructions for further engineering the vector or cell to express a biomolecule (e.g., IL-12, mutant HSV1-TK, or therapeutic agent including an antigen). In some embodiments, the kit includes instructions for thawing or otherwise restoring biological activity of cells that may have been preserved during storage or transportation. In some embodiments, the kit includes instructions for measuring the viability of preserved cells to confirm efficacy for its intended purpose (e.g., therapeutic efficacy when used to treat a subject).

[0130]

[0149] Optionally, the kit also contains other useful components, such as diluents, buffers, pharma- ceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, dressings, or other useful equipment. The assembled materials or components in the kit may be provided to the physician, stored in any convenient and suitable manner that preserves operability and usefulness. For example, the components may be in dissolved, dehydrated, or lyophilized form, and they may be provided at room temperature, refrigerated temperature, or frozen temperature. The components are typically contained in suitable packaging materials.

[0131]

[0150] The use of independent or consecutive terms, such as "will", "will not be", "shall be", "shall not be", "must", "do not have to be", "initialy", "initially", "next", "consequently", "before", "after", "finally", and "finally" are not meant to be limiting on the scope of the embodiments of the invention disclosed herein, but are meant to be exemplary.

[0132]

[0151] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Additionally, the terms "including," "including," "having," "having," "with," or derivatives thereof are intended to be inclusive in a manner similar to the term "comprising" to the extent such terms are used either in the detailed description and / or claims.

[0133]

[0152] The phrases "at least one," "one or more," and "and / or," as used herein, are open-ended expressions that are valid both conjunctively and disjunctively. For example, each of the expressions "at least one of A, B, and C," "at least one of A, B, or C," "one or more of A, B, and C," "one or more of A, B, or C," and "A, B, and / or C" means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together.

[0134]

[0153] "Or," as used herein, can refer to "and," "or," or "and / or," and can be used both exclusively and inclusively. For example, the term "A or B" can also refer to "A or B," "A but not B," "A but not B," and "A and B." In some cases, context may have a particular meaning.

[0135]

[0154] Any of the systems, methods, software, and platforms described herein are modular, and thus terms such as "first" and "second" do not necessarily indicate a priority, order of importance, or order of execution.

[0136]

[0155] The term "about," when used in reference to a number or numerical range, means that the stated number or numerical range is approximate within experimental variation (or within statistical experimental error), and that the number or numerical range may vary, for example, from 1% to 15% of the stated number or numerical range. In embodiments, the term "about" refers to ±10% of the stated number or value.

[0137]

[0156] The term "increased", "increasing" or "increase" is generally used herein to mean an increase in a statically significant amount. In some embodiments, the term "increased" or "increase" means an increase of at least 10% compared to a reference level, for example, an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of up to 100%, including 100%, or any increase between 10 and 100% compared to a reference level, standard, or control. Other examples of "increase" include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, or more compared to a reference level.

[0138]

[0157] The term "reduced", "reducing" or "reducing" is generally used herein to mean a statistically significant reduction. In some embodiments, "reduced" or "reducing" means a reduction of at least 10% compared to a reference level, for example, a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or a reduction of up to 100%, including 100% (for example, non-existent or undetectable compared to a reference level), or any reduction between 10 and 100%. In the context of a marker or symptom, these terms mean a statistically significant reduction of such a level. The reduction may be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably a reduction to a level that is accepted as being within the normal range for an individual without a given disease.

[0139]

[0158] "Nucleic acid" as used herein refers to a polynucleotide that contains at least two covalently linked nucleotide or nucleotide analog subunits. Nucleic acid is generally deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or an analog of DNA or RNA. Nucleic acid is generally single-stranded, double-stranded, or a mixture thereof. For the purposes herein, unless otherwise specified, or clear from the context, nucleic acid is double-stranded.

[0140]

[0159] "DNA," as used herein, is meant to include DNA molecules of all types and sizes, including, for example, cDNA, plasmids, and DNA containing modified nucleotides and nucleotide analogs.

[0141]

[0160] "Nucleotide," as used herein, includes nucleoside mono-, di-, and triphosphates. Nucleotides also include modified nucleotides, such as, but not limited to, phosphorothioate nucleotides and deazapurine nucleotides, as well as other nucleotide analogs.

[0142]

[0161] The term "polynucleotide" as used herein refers to a polymeric form of nucleotide of any length, examples of which include ribonucleotides and deoxyribonucleotides.Such terms also include single-stranded and double-stranded DNA, as well as single-stranded and double-stranded RNA.This term also includes modified polynucleotides, such as methylated or capped polynucleotides.

[0143]

[0162] The term "subject" as used herein refers to the animal, plant, insect and bird into which large DNA molecule is introduced.It also includes higher organisms such as mammals and birds, for example, humans, primates, rodents, cows, pigs, rabbits, goats, sheep, mice, rats, guinea pigs, cats, dogs, horses, chickens, etc.Subjects may or may not have a disease or condition.

[0144]

[0163] "Administering to a subject," as used herein, is a procedure in which one or more delivery agents and / or large nucleic acid molecules, together or separately, are introduced into or applied onto a subject such that target cells present in the subject ultimately come into contact with the agents and / or large nucleic acid molecules.

[0145]

[0164] "Delivery vector" or "delivery vehicle" or "therapeutic vector" or "therapeutic system" as used herein refers to both viral and non-viral particles that encapsulate and transport exogenous nucleic acid molecules to target cells or tissues. Viral vehicles include, but are not limited to, retroviruses, adenoviruses, lentiviruses, herpes viruses, and adeno-associated viruses. Non-viral vehicles include, but are not limited to, microparticles, nanoparticles, virosomes, and liposomes. "Targeting" as used herein refers to the use of ligands that associate with the delivery vehicle and target the vehicle to cells or tissues. Ligands include, but are not limited to, antibodies, receptors, and collagen binding domains.

[0146]

[0165] "Delivery" is used synonymously with "transduction" and as used herein refers to the process by which an exogenous nucleic acid molecule is transferred to a cell so that it is located inside the cell. Delivery of a nucleic acid is a process separate from expression of the nucleic acid.

[0147]

[0166] "Expression" as used herein refers to the process that nucleic acid is translated into polypeptide or transcribed into RNA that can be translated into polypeptide or protein.If nucleic acid is derived from genomic DNA, expression includes splicing of mRNA if appropriate eukaryotic host cell or organism is selected.When expressing heterologous nucleic acid in host cell, heterologous nucleic acid must first be delivered to cell, and then, once inside cell, it will end up in nucleus.In some embodiments, expression occurs independently of gene integration.

[0148]

[0167] "Course of therapeutic agent" as used herein refers to regular or timed administration of the vector disclosed herein within a given period of time. Such period may be at least 1 day, at least 2 days, at least 3 days, at least 5 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, or at least 6 months. Administration may also be in a chronic manner, i.e., for an indefinite period of time. Regular or timed administration includes administration once a day, twice a day, three times a day, or other set timed administration.

[0149]

[0168] The terms "co-administered," "administered in combination with," and their grammatical equivalents, as used herein, are meant to encompass administration of a selected therapeutic agent to a single patient and are intended to include treatment regimens in which the agents are administered by the same or different routes of administration or at the same or different times. In some embodiments, the therapeutic agents disclosed in the present application are expected to be co-administered with other agents. These terms encompass administration of two or more agents to an animal such that both agents and / or their metabolites are present in the animal at the same time. Examples include co-administration in separate compositions, administration at different times in separate compositions, and / or administration in a composition in which both agents are present. Thus, in some embodiments, the therapeutic agent and the other agent are administered in the form of a single composition. In some embodiments, the therapeutic agent and the other agent are admixed in a composition. In further embodiments, the therapeutic agent and the other agent are administered in separate doses at separate times.

[0150]

[0169] The term "mutant thymidine kinase," as used herein, refers not only to the specific proteins described herein (as well as the nucleic acid sequences encoding these proteins), but also to derivatives thereof, which may include various structural forms of the primary protein that retain biological activity.

[0151]

[0170] The term "mutated" or "replaced with another nucleotide" as used herein means that a nucleotide at a particular position is replaced with a nucleotide other than the one present at that position in the unmutated or previously mutated sequence. That is, in some cases, a particular modification may be made with a different nucleotide. In some embodiments, the replacement is made such that the relevant splice donor and / or acceptor site is no longer present in the gene.

[0152]

[0171] "Polar amino acid," as used herein, refers to the amino acid residues Asn (N), Cys (C), Gln (Q), Gly (G), Ser (S), Thr (T) or Tyr (Y).

[0153]

[0172] "Nonpolar amino acid," as used herein, refers to the amino acid residues Ala (A), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), Trp (W), or Val (V).

[0154]

[0173] "Basic amino acid," as used herein, refers to the amino acid residues Arg (R), His (H), or Lys (K).

[0174] "Acidic amino acid," as used herein, refers to the amino acid residues Asp (D) or Glu (E).

[0155]

[0175] "Adjuvant" as described herein refers to a substance that, in combination with an antigen, promotes an adaptive immune response to the antigen. "Immunostimulatory compound" refers to a substance that specifically interacts with the innate immune system to initiate a "danger signal" that ultimately triggers the development of adaptive components of the immune response (e.g., B cells, T cells). Immunostimulatory compounds include pathogen-associated molecular patterns (PAMPs), either naturally occurring or synthetic, such as dsRNA, lipopolysaccharide, and CpG DNA. Immunostimulatory compounds are agonists of various innate immune receptors, such as Toll-like receptors (TLRs), NOD-like receptors, RIG-1 or MDA-5 receptors, C-type lectin receptors, or STING pathways.

[0156]

[0176] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided within the specification. Although the present invention has been described with reference to the above specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present invention. Furthermore, it is to be understood that all aspects of the present invention are not limited to the specific depictions, arrangements, or relative proportions described herein, which depend upon various conditions and variables. It is to be understood that various alternatives to the embodiments of the present invention described herein can be employed in the practice of the present invention. It is therefore anticipated that the present invention shall cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are thereby covered. EXAMPLES

[0157]

[0177] The following illustrative examples are representative of embodiments of the stimuli, systems, and methods described herein and are not meant to be limiting in any way. Example 1. Generation of an integrase-deficient vector

[0178] A simplified version of the viral cycle of retroviruses: the enzyme reverse transcriptase (RT) generates the cDNA of the retroviral RNA genome, which then produces a double-stranded DNA that immediately binds to the viral enzyme integrase. Integrase is the main enzyme that transports the viral DNA and catalyzes its integration into the host genome. Integrase is part of the gagpol gene, which is translated into a polyprotein sequence that is processed by the viral protease to yield seven proteins. Integrase (INT) is a 408 aa protein at the C-terminus of Gag-Pol (Table 2).

[0158] [Table 2]

[0159]

[0179] Comparative studies with integrase-deficient retroviral vectors have determined the structure of their integrase and different domains. Figure 1 illustrates the D, D, and E amino acid triplet that forms the Mg2+-binding motif that is important for integrase function.

[0160]

[0180] In addition, comparative studies were utilized to determine amino acid residues that could be mutated in reverse transcriptase (RT) to generate reverse transcriptase-deficient retroviral vectors. Two approaches were employed: three amino acid mutations in the active site of the RT enzyme; and base mutations in the RT binding region on the payload vector described herein (artificial primer binding site (aPBS)). Generation of seven mutant integrase sequences

[0181] Base substitutions were introduced to change amino acids in the integrase catalytic core domain sequence of the gagpol gene of MLV. Single base mutations were introduced in the catalytic core domain by site-directed mutagenesis (using the Q5 Site-Directed Mutagenesis Kit, NEB) with primers designed to introduce single base mutations into the wild-type gagpol (wtGP) sequence, vector pGP340-VKS. The first mutant generated was D184A (Figure 2). A single base codon was changed from GAC to GCC. (Point mutation at base 4541 of the gagpol gene). The same procedure was used to generate single mutants D125A (GAC to GcG) and E220A (GAG to GcG) (primer design; Figure 3). Before proceeding to generate double mutants, the single mutations were confirmed by sequencing (Laragen, Inc., CA). Double mutants were generated via a second round of site mutagenesis performed with primers selected against previously obtained and sequenced single mutants. A triple mutant was generated by a third round of site-directed mutagenesis on the double mutant D184A / E220A. All vectors were analyzed by restriction enzyme digestion and mutations were confirmed by sequencing (Laragen, Inc., CA). Table 3 illustrates seven exemplary mutant gagpol sequences described herein. Table 4 illustrates the polypeptide sequences of the integrase fragments spanning amino acid position 106 to amino acid position 287 of the wild-type integrase (SEQ ID NO:1) and seven mutant integrases (SEQ ID NOs:2-8). The last two bases of the codon were changed from GAT to GCC (point mutations at and near base 4541 of the gagpol gene). Single point mutations were used in the mutations D184A and E220A, converting GAC to GCC and GAG to GCG, respectively.

[0161] [Table 3]

[0162] [Table 4-1]

[0163] [Table 4-2]

[0164] Comparison with particles produced by wild-type Gag-pol

[0182] Viral supernatants were produced with each integrase-deficient vector (using triple transfection of 293T cells with envelope and payload / reporter plasmids) and properties were compared with particles made with the wtGP vector. Similar titers were obtained (Figure 4), demonstrating that the integrase mutations do not interfere with other functions of Gag-pol required to generate viral particles. Infectivity and payload expression over time

[0183] The conventional target cell line A375 (human melanoma) was transduced with viral vector supernatant. Payload expression was analyzed visually (GFP payload), by FACS (for vTK), or by payload activity (luciferase reporter gene in the payload) or vTK activity (GCV-induced cell killing assay). The results demonstrated that the mutations did not interfere with viral envelope production. The level of payload protein expression demonstrated that the integrase mutations did not interfere with the level of payload expression (by cell killing reflecting the expression and activity of the payload vTK; in addition, examined by Western blotting of the vTK protein; Figures 5 and 6). The timing of payload protein expression demonstrated that mutations that rendered integrase dysfunctional were unable to integrate the payload gene into the host genome, resulting in a decrease in the level of payload protein expression over time (loss of GCV sensitivity in cell killing assay, Figure 5; attenuation of vTK protein band intensity in Western blotting, Figure 6). Lack of vTK gene integration was demonstrated over time by qPCR of relative integration, confirming that the mutations disabled the integrase (Figures 7 and 8). After careful evaluation of the characteristics recorded for each mutant integrase sequence generated, a single mutant (D184A) and a double mutant (D125A-D184A) were selected. Generation of manufacturing vector integrase-deficient cell lines

[0184] To facilitate the generation of packaging cell lines carrying each integrase-deficient vector, gagpol sequences carrying selected integrase mutations were cloned into gagpol vector constructs and sequenced. Envelope-expressing 293T packaging cell lines were transformed with integrase-deficient gagpol retroviral vectors and tested. Packaging cell line testing

[0185] The integrase-deficient packaging lines were compared with wtGP by transfecting all three packaging cell lines with the vectors described herein. The titers obtained were in a similar range (Table 5). To express vTK and observe nonintegration from integrase-deficient particles, the viral vector supernatant was used to transduce the conventional target cell line A375 at 2e7 vector genomes / mL. The expression of vTK is reflected by cell killing assays in which the cells were exposed to GCV at a given time after transduction. At day 3 after transduction, the cell killing percentages were 79%, 59% and 69% for wtGP, mutGP-D184A and mutGP-D125A / D184A, respectively. At day 7, the cell killing activity was approximately 80% less in cells transduced with integrase-deficient particles, reaching almost zero at day 23.

[0165] [Table 5]

[0166] Generation of cell lines for producing integrase-deficient vectors

[0186] A 293T packaging cell line expressing envelope and integrase-deficient gagpol was transformed with the payload vectors described herein to generate integrase-deficient vector manufacturing cell lines, followed by single cell cloning if the payload sequence does not carry a drug selection marker.

[0167]

[0187] The same integrase-deficient packaging cell line harboring the D184A vector was also used in preclinical studies by transfection with a retroviral vector expressing mouse GMCSF, followed by cell cloning. Integrase-deficient D184A vector strain

[0188] The first round of cloning resulted in the selection of four positive clones (BC6, BF6, H6, C8). Viral supernatants produced by these manufacturing cell lines were compared for titer and vTK expression over time in transduced A375 cells by cell killing assays exposing cells to GCV at different times post-transduction (Table 6; Figure 10).

[0168] [Table 6]

[0169] Clone BC6 was selected for a second round of cloning based on titer, vTK expression profile, cell growth and morphology. Second clones BC6-C3 and BC6-E12 were selected from 18 tested based on titer and cell lethality levels of transduced A375 cells (FIG. 11). Clone BC6-C3 was expanded and adapted to suspension. Integrase-deficient D184A vector strain carrying mouse GMCSF

[0189] The first round of cloning resulted in the selection of four infectious clones (Table 7). Among all, clone G7 was chosen as the best, with a titer of 1.25E+09 vector genomes / mL and 20 psi sequence copies integrated.

[0170] [Table 7]

[0171] Integrase-deficient vectors drive payload expression in various cancer cell lines tested

[0190] Various cancer cell lines were tested with integrase-deficient vectors. When cells were successfully transduced with integrase-deficient retroviral vectors, expression of the payload disappeared between D7 and D14 for most lines tested, as demonstrated by WB, GCV-cell killing assays, and qPCR of relative integration. Figures 12A-C illustrate the vTK / GCV cell killing activity of various cancer cell lines from the wtGP vector (Figure 12A) and the integrase-deficient mutant vector (Figure 12B). Figure 12C illustrates the relative integration of vector payload by qPCR in cells treated with the wtGP vector and the integrase-deficient mutant vector.

[0172] Example 2. Expression of SARS-CoV-2 antigens

[0191] Integrase-deficient retroviral vectors (IDRVs) can be generated with an integrase-deficient Gagpol and any payload gene of interest. Transient protein expression of such IDRV systems offers potential vaccine use. Three SARS-CoV-2 vaccine payloads were designed and generated. Tests were performed with three plasmids: the vaccine payload, IDRV1 (D184A), or IDRV2 (D125A / D184A) Gagpol, and transient transfection of amphotropic envelope or modified Sindbis envelope targeted to antigen-presenting dendritic cells. Figure 13 illustrates an exemplary schematic of a recombinant retroviral vector containing a nucleic acid sequence encoding the spike protein of SARS-CoV-2 or a fragment thereof. Top: Diagram of a retroviral vector encoding the SARS-CoV-2 full-length spike protein (Wuhan sequence with modifications). Center: Diagram of a retroviral vector encoding the SARS-CoV-2 spike protein fragment (Wuhan sequence with modifications) of the N-terminal domain (NTD) and the S2 domain. Bottom: Diagram of the retroviral vector encoding the SARS-CoV-2 full-length spike protein (omicron BA.2 sequence with modifications).

[0173]

[0192] The first vaccine was a retroviral vector containing a nucleic acid sequence encoding a full-length spike protein based on the Wuhan strain with modifications. The nucleic acid sequence was codon-optimized for expression in human cells. A secretory leader sequence (IgEpsylon Fc receptor alpha) was added to the N-terminus. The furin cleavage site at residues 682-685 was modified from RRAR to SRAG to stabilize the spike protein. The serine protease cleavage site was modified to proline at residues 986 and 987 to stabilize the pre-fusion form of the spike structure.

[0174]

[0193] The second vaccine was a retroviral vector containing a nucleic acid sequence encoding the N-terminal domain and S2 protein based on the SARS-CoV-2 Wuhan strain with modifications. The nucleic acid sequence was codon-optimized for expression in human cells. A secretory leader sequence (IgEpsylon Fc receptor alpha) was added to the N-terminus of S2. The furin cleavage site at residues 682-685 was modified from RRAR to SRAG to stabilize the spike peptide. The serine protease cleavage site was modified to proline at residues K986 and V987 to stabilize the pre-fusion conformation of the structure.

[0175]

[0194] The third vaccine was a retroviral vector containing a nucleic acid sequence encoding the spike protein of the Omicron BA.2 strain with modifications. The nucleic acid sequence was codon-optimized for humans. The furin cleavage site at residues 679-682 was modified from RRAR to SRAG to stabilize the spike peptide. An additional furin cleavage site was removed at K811A. The serine protease cleavage site was modified to proline at residues K983 and V984 to stabilize the pre-fusion shape of the spike structure. An additional four further residues were modified to proline at residues F814, A889, A896, and A939. A signal peptide was added to the N-terminus, which was the propeptide of tissue plasminogen activator (SEQ ID NO: 13: MDAMKRGLCCVLLLCGAVFVSASQEIHARFRR).

[0176]

[0195] In some embodiments, the vaccine composition can be packaged and expressed by Sindbis virus. In some cases, the Sindbis virus can be a modified Sindbis virus. Sindbis envelope. In Sindbis virus (SB), one of the alphavirus species, the envelope gene encodes four components E3, E2, 6K, and E1 (N-terminal to C-terminal). They separate by proteolysis and form homotrimers. The unique feature of the SB envelope is that these four subunits have their own functions. E3 dissociates when the E2 domain matures; the E2 domain serves as a binding molecule; fusion is regulated by E1, whereas the function of 6K is unknown. Binding and fusion of viral particles are done in two separate movements that allow modification of E2 without affecting the structure and function of E1. Here, residue 160 in E2 is mutated to target human dendritic cells by pseudotyping retroviral vectors containing the payload described herein. The modified Sindbis envelope containing the E160G mutation was confirmed by Sanger sequencing at Laragen (Culver City, CA). The E160G mutation was predicted to target the recombinant retroviral vector to antigen-presenting cells (e.g., dendritic cells) at the injection site (Figure 15).

[0177]

[0196] All transgenes were designed as described above and synthesized at Genscript (Piscataway, NJ). Sequences were verified by Sanger sequencing at Genscript. Plasmids expressing the transgenes were digested with appropriate restriction enzymes and inserted into payload retroviral and lentivectors described herein (e.g., Figures 14A-14C). All final plasmids were sequenced at Primordium Labs (Arcadia, CA).

[0178]

[0197] Retroviral vectors were generated by calcium / phosphate transient transfection method with wild-type gagpol or IDRV1(D184A) gagpol with amphotropic envelope and full-length spike payload or NTD-S2 payload. A375 cells were transduced with retroviral vectors at 1E+08 vector genomes / mL, and the culture supernatants were examined by Western blotting. The results (Figure 16A and Figure 16B) demonstrated stable expression of spike protein secreted with IDRV1, although the level of expression was lower than with wild-type gagpol. Similar results were shown with NTD-S2 payload. The experiment was repeated with IDRV2(D125AD184A gagpol) vector, and similar results were obtained, as shown in Figure 16C. Both payloads showed stable expression in the supernatant. Retroviral vectors were also generated by transient transfection method with omicron mutants of spike protein, and wild-type gagpol or IDRV2 gagpol. Intracellular spike protein from retroviral vector-transduced A375 cells was examined by Western blotting, and the results demonstrated high levels of full-length spike protein expression, as shown in Figure 17A.

[0179]

[0198] A full-length spike payload lentivector was used to generate a manufacturing cell line with a fully codon-optimized amphotropic envelope and wild-type gagpol, and the cell line was cloned. Table 8 shows the physical titer of the cell line clones, which was measured for the Psi sequence in the extracted vector RNA by RT-qPCR (reverse transcriptase quantitative PCR). The results demonstrated an acceptable titer of the manufacturing cell line at 9.6E+07 vector genomes / mL.

[0180] [Table 8]

[0181]

[0199] The recombinant retroviral vectors produced from the cell line clones were tested in A375 melanoma cells. After transduction, the cells and supernatants were examined for the expression of spike protein, S2 region, by Western blotting. The results demonstrated that full-length spike protein was produced in transduced cells and secreted from the cells, as shown in Figure 17B.

[0182]

[0200] A recombinant retroviral vector with a payload encoding the omicron mutant spike protein was obtained by transiently co-transfecting 293T with wtGP and amphotropic envelope. The titer was 4.81E+07 vector genomes / mL as determined by RT-qPCR. A manufacturing cell line expressing IDRV2 and amphotropic envelope was generated to produce omicron mutant payload retroviral vector particles. The titers from two exemplary clones measured by RT-qPCR were 1.63E+08 vector genomes / mL and 4.67E+07 vector genomes / mL, respectively. When test cells were transduced with the omicron mutant spike retroviral vector, spike protein was detected in both cell lysates and conditioned medium (cell culture supernatant) by Western blotting (Figure 18A). Immunocytochemical staining of the omicron mutant spike protein demonstrated the expression of spike protein and its presence in the perinuclear and plasma membrane regions of the test cells (Figure 18B). A375 cells transduced with omicron retroviral vectors were fractionated by simple centrifugation, and the plasma membrane fraction and non-membrane soluble proteins were subjected to Western blotting (FIG. 18C). The spike protein was differentially present in the plasma membrane fraction along with the plasma membrane marker cadherin.

[0183]

[0201] To determine the best transient transfection conditions, various amounts of various payloads, wild type and IDRV gagpol, as well as envelope, can be tested by the transient transfection method. Retroviral vectors expressing Sindbis E160G can be tested using dendritic cells (Figure 15). The vaccine payload of interest can be produced as a retroviral vector on a large scale, for example in CS10 flasks, and then purified on a column. Two types of animal experiments may be performed. The purified retroviral vectors can be tested for antibody production in standard BALB / c and C57BL / 6J mice by four different routes (intramuscular, sublingual, intranasal, and intradermal). Both IgM and IgG production can be tested. After the results from the first animal experiment, the purified retroviral vectors can be tested in animals challenged with the real virus. For example, hamsters that normally express ACE receptors can be challenged with COVID virus and treated with omicron mutant retroviral vectors.

[0184] Experimental Methods

[0202] All transgenes were designed as described above and synthesized in Genscript. Sequences were confirmed by Sanger sequencing in Genscript. Plasmids expressing the transgenes were digested with appropriate restriction enzymes and inserted into the payload retroviral vectors described herein (e.g., Figures 14A-14C). All final plasmids were sequenced at Primordium Labs (Arcadia, CA). Payload, wild-type gagpol or IDRV, and envelope were transiently transfected into 293T cells. Full-length spike manufacturing cell lines were generated using a packaging 293T cell line platform expressing full-length amphotropic envelope with modifications, fully cloned (clone G8), and stably expressing wild-type gagpol. Cell lines were further cloned individually. For western blotting, A375 cells were transduced with retroviral vectors at appropriate dilutions in 8 μg / mL polybrene for 3 days. Samples were run on 4-20% TGX (Bio-Rad) gels at 135 V for 75 min. After transfer to PVDF membranes (Thermo) and blocking with 3% BSA (bovine serum albumin, Sigma) in 1× TBST (Tris-buffered saline, 0.1% Tween 20, Santa Cruz) for 1 h at room temperature, the blots were incubated overnight at 4°C with anti-S2 antibody (MAB1080100) from R&D Systems at 0.5 mg / mL. The following day, after three washes with 1× TBST, the blots were incubated with alkaline phosphatase-conjugated anti-rabbit antibody at a dilution of 1:2000 for 1 h at room temperature and developed with NBT / BCIP substrate (Millipore) after another series of washes.

[0185] Example 3. Expression of influenza antigens

[0203] Figure 19 illustrates a retroviral vector described herein that includes a payload for encoding M2e with a hemagglutinin (HA) Stalk (conserved region) domain with modifications. The nucleic acid sequence was codon-optimized for humans. The extracellular domain of the M2 protein (M2e) of influenza A has the amino acid sequence of MSLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO: 14). The additional HA Stalk with modifications included head deletion; glycine linker loop; intra-Cys bridge; transmembrane deletion; loop fusion peptide; GCN4 position; and inter-Cys bridge. Figure 20 illustrates the presence of M2e and HA4900 (influenza) transgenes as payloads of retroviral vectors or lentivectors described herein.

[0186]

[0204] Recombinant retroviral vectors with payloads encoding M2e and HA4900 were produced by 293T transiently co-transfected with wtGP and amphotropic envelope, and vector titers were measured by RT-qPCR. Retroviral vectors encoding influenza antigens showed 1.56E+08 vector genomes / mL. Titers of influenza antigen vector particles produced from the clonal manufacturing cell lines, measured by RT-qPCR, were 1.94E+08 and 1.74E+07 vector genomes / mL for two exemplary clones. The manufacturing cell lines expressed IDRV2 and amphotropic envelope. Test cells were transduced with such retroviral vectors, and HA protein (hemagglutinin) was detected in cell lysates by Western blotting (Figure 21A). Immunocytochemical staining of HA protein demonstrated expression and localization of HA protein in the perinuclear region of the test cells (Figure 21B).

[0187]

[0205] Although the foregoing disclosure has been described in some detail for clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail may be made without departing from the true scope of the present disclosure. For example, all of the techniques and devices described above may be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually and separately indicated to be incorporated by reference for all purposes.

[0188] [Table 9-1]

[0189] [Table 9-2]

[0190] [Table 9-3]

Claims

1. 1. A recombinant retroviral vector for use in treating or preventing a disease or condition in a subject, comprising a first nucleic acid sequence encoding a mutant integrase and a second nucleic acid sequence encoding at least one payload, wherein the mutant integrase has a reduced Mg content in the catalytic core domain when compared to a wild-type integrase having the sequence of SEQ ID NO:

1. ２＋ A recombinant retroviral vector comprising at least one mutation selected from the group consisting of D125A, D184A, and E220A in the binding motif; and a recombinant murine leukemia virus (MLV).

2. A recombinant retroviral vector as described in claim 1, wherein the payload comprises a cytokine, thymidine kinase, or antigen.

3. A recombinant retroviral vector as described in claim 1, wherein at least one payload is expressed in a subject for at least three days.

4. A recombinant retroviral vector as described in claim 2, wherein the antigen induces an immune response in a subject.

5. A recombinant retroviral vector as described in claim 1, which has deficient retroviral integration activity compared to a recombinant retroviral vector containing wild-type integrase.

6. The recombinant retroviral vector of claim 2 , wherein the antigen comprises a pathogen polypeptide or a fragment thereof or a cancer polypeptide or a fragment thereof.

7. A recombinant retroviral vector as described in claim 1, encoding at least one envelope protein including at least one alphavirus envelope protein.

8. 8. The recombinant retroviral vector of claim 7, wherein the at least one alphavirus envelope protein comprises at least one Sindbis virus envelope protein comprising an E3 protein, an E2 protein, a 6K protein, an E1 protein, or a combination thereof.

9. 9. The recombinant retroviral vector of claim 8, wherein at least one Sindbis virus envelope protein comprises at least one mutation, and the at least one mutation may increase the binding affinity between the at least one Sindbis virus envelope protein and a human cell.

10. 10. The recombinant retroviral vector of claim 9, wherein at least one mutation is E160G in the E2 protein of the Sindbis virus envelope protein when compared to the wild-type sequence.

11. The recombinant retroviral vector of claim 4, wherein the immune response includes the induction of neutralizing antibodies that target the antigen, thereby generating immunity to the antigen in the subject.

12. 5. The recombinant retroviral vector of claim 4, wherein the immune response includes the induction of immunoglobulin antibodies that target the antigen, thereby generating immunity to the antigen in the subject, and the immunoglobulin antibodies may include IgG antibodies, IgM antibodies, IgA antibodies, IgD antibodies, IgE antibodies, or a combination thereof.

13. 12. The recombinant retroviral vector of any one of claims 1 to 11, wherein at least one payload is expressed in a subject for at least 1 day, at least 3 days, at least 5 days, or at least 9 days.

14. 12. The recombinant retroviral vector of any one of claims 1 to 11, wherein at least one payload is secreted in the subject for at least 1 day, at least 3 days, at least 5 days, or at least 9 days.

15. 15. The recombinant retroviral vector of claim 14, wherein the duration of an immune response induced by at least one payload expressed for at least 1 day, at least 3 days, at least 5 days, or at least 9 days is increased by at least 10%, at least 20%, at least 50%, at least 100%, at least 5-fold, at least 10-fold, or more, compared to the duration of an immune response induced by a comparable payload expressed in less than 1 day, less than 3 days, less than 5 days, or less than 9 days.

16. 16. The recombinant retroviral vector of claim 15, wherein the duration of an immune response induced by at least one payload secreted for at least 1 day, at least 3 days, at least 5 days, or at least 9 days is increased by at least 10%, at least 20%, at least 50%, at least 100%, at least 5-fold, at least 10-fold, or more, compared to the duration of an immune response induced by a comparable payload expressed in less than 1 day, less than 3 days, less than 5 days, or less than 9 days.

17. 12. The recombinant retroviral vector of any one of claims 1 to 11, wherein the immune response persists in the subject for at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 12 months, or longer.

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