Compositions and methods for therapeutic delivery

Abstract

This article describes compositions and methods for therapeutic delivery using engineered viral vectors. The engineered viral vectors provided herein can improve therapeutic delivery, increase targeting efficiency, and reduce off-target or harmful effects.

Description

[0001] Cross-referencing This application claims the benefit of U.S. Provisional Application No. 63 / 599,385, filed November 15, 2023, and U.S. Provisional Application No. 63 / 643,887, filed May 7, 2024, each of which is incorporated herein by reference in its entirety. Background Technology

[0002] The use of viral particles or viral vectors for delivering therapeutic agents has become a fundamental part of modern medicine. However, the use of such viral particles or viral vectors can lead to off-target or harmful effects. Therefore, there remains a need for engineered viral particles or engineered viral vectors for delivering therapeutic agents that offer increased targeting efficiency and reduced off-target and harmful effects.

[0003] Incorporation All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually cited and incorporated herein by reference. In the event of any conflict between a publication, patent, or patent application cited and the disclosure contained herein, this specification is intended to substitute for and / or give precedence to any such conflicting material. Summary of the Invention

[0004] In one aspect, this disclosure provides an engineered viral vector comprising a modified envelope protein, wherein the modified envelope protein comprises: a) at least one targeting moiety, and b) at least one modification of a wild-type protein sequence (SEQ ID NO: 1); wherein, relative to a viral vector without the modified envelope protein, the modified envelope protein increases the transduction efficiency or specificity of the engineered viral vector for target cells. In some embodiments, the at least one modification is in the E2 domain.

[0005] In some embodiments, the engineered viral vector comprises an engineered retroviral vector. In some embodiments, the engineered retroviral vector comprises an engineered gamma retroviral vector. In some embodiments, the engineered gamma retroviral vector comprises an engineered murine leukemia virus (MLV) vector. In some embodiments, the modified envelope protein comprises a recombinant viral envelope protein derived from a DNA virus. In some embodiments, the modified envelope protein comprises a recombinant viral envelope protein derived from an RNA virus. In some embodiments, the RNA virus comprises an alphavirus. In some embodiments, the alphavirus comprises Sindbis virus. In some embodiments, the modified envelope protein derived from the Sindbis virus comprises an E3 domain, an E2 domain, a 6K domain, an E1 domain, or a combination thereof. In some embodiments, the modified envelope protein further comprises a protease cleavage site located between the E3 and E2 domains.

[0006] In some embodiments, the protease cleavage site includes a furin protease cleavage site.

[0007] In some embodiments, the at least one targeting portion is located within the E2 domain. In some embodiments, the at least one targeting portion includes a conjugation portion. In some embodiments, the conjugation portion comprises an IgG binding domain of a bacterial protein. In some embodiments, the bacterial protein is bacterial protein A. In some embodiments, the IgG binding domain of the bacterial protein is a ZZ protein domain.

[0008] In some embodiments, the engineered viral vector is further conjugated with an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof includes IgG-scFv, a single variable domain (V) on the heavy chain, and so on. H H), nanobodies, BiTE, biantibodies, DART, TandAb, sc biantibodies, sc biantibodies-CH3, triplebodies, mini-antibodies, minibodies, TriBi miniantibodies, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc biantibodies-Fc, biantibodies-Fc, tandem scFv-Fc, intracellular antibodies, their binding fragments, their chemically modified derivatives, heavy chains of variable fragments (V H ), light chains of variable segments (V L( ) or a combination thereof. In some embodiments, the antibody or its antigen-binding fragment binds to the target ligand of the target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell includes human cancer cells. In some embodiments, the cancer cell includes leukemia cells, myeloid cells, promyeloid cells, myeloid mononuclear cells, mononuclear cells, erythroleukemia cells, chronic myeloid (granulocytic) leukemia cells, chronic lymphocytic leukemia cells, lymphoma cells such as Hodgkin's and non-Hodgkin's, fibrosarcoma cells, myoma cells, liposarcoma cells, chondrosarcoma cells, osteosarcoma cells, angiosarcoma cells, endothelial sarcoma cells, Ewing's tumor cells, colon cancer cells, pancreatic cancer cells, breast cancer cells, ovarian cancer cells, prostate cancer cells, squamous cell carcinoma cells, basal cell carcinoma cells, adenocarcinoma cells, renal cell carcinoma cells, liver cancer cells, Wilms' tumor cells, cervical cancer cells, uterine cancer cells, testicular tumor cells, lung cancer cells, small cell lung cancer cells, bladder cancer cells. Carcinoma cells, epithelial cancer cells, glioma cells, astrocytoma cells, oligodendroglioma cells, melanoma cells, neuroblastoma cells, retinoblastoma cells, dysplastic and hyperplastic cells, prostatitis cells, benign prostatic hyperplasia (BPH) cells, prostatic paraganglioma cells, prostatic adenocarcinoma cells, prostatic intraepithelial tumor cells, prostatic-rectal fistula cells, atypical prostatic stromal lesion cells, bladder cancer cells, lung cancer cells, head and neck cancer cells, non-small cell lung cancer cells, renal cancer cells, glioblastoma cells, Merkel cell carcinoma cells, angiosarcoma cells, cutaneous T-cell lymphoma cells, cutaneous B-cell lymphoma cells, dermatofibrosarcoma protuberans cells, sebaceous gland cancer cells, or cutaneous neuroendocrine cancer cells.

[0009] In some embodiments, the target ligand includes cell surface markers. In some embodiments, the cell surface markers include cancer cell markers. In some embodiments, the cancer cell markers include CD47, CEA, HER2, EGFR, PSMA, TfR, nectin-4, muc-1, endoglin, EphA2, EphB2, folate receptor, GRP78, IGF-1R, cMET / HGFR, Tn antigen, EpCAM, ανβ3 integrin, CD44, mesothelin, PSCA, uPAR, CAIX, CD13, FAP-α, matriptase, MT-1 MMP, MT6-MMP, KDR, KIT, CD276, CD-83, or combinations thereof. In some embodiments, the target ligands include CD33, CD30, HER2, CD22, CD79b, nectin-4, Trop-2, BCMA, CD19, EGFR, folate receptor α, CD3, CD38, CD20, BCMA, CEA, PSMA, OX40, 4-1BB, CD16A, PD-1, PD-L1, CTLA-4, LAG-3, TIM3, VEGF, CD47, ICOS, MET, LGR5, IGF-1R, HER3, ANG2, DLL4, FAP, DR5, fibronectin ED-B, tendinin C, EpCAM, TNFα, digoxin, GPIIb / IIIa, rattlesnake venom, scorpion venom, CA6, PLVAP, 5T4, EDB, EGFRvIII, 6B11, CXCR4, MSLN, FN1, GPC2, GPC3, VCAN1, COL11A1, or MMR.

[0010] In some embodiments, the target ligand of the target cell includes an immune checkpoint protein or an immune checkpoint receptor. In some embodiments, the target ligand of the target cell is an immune checkpoint protein. In some embodiments, the immune checkpoint protein includes PD-L1, CTLA-4, B7RP1, HVEM, CD137L, OX40L, CD40, CD70, GAL9, MHCII, CD47, VISTA, or GITR. In some embodiments, the target ligand of the target cell is an immune checkpoint receptor. In some embodiments, the immune checkpoint receptor includes PD-1, ICOS (CD278), BTLA, CD137 (4-IBB), OX40 (CD134), CD40L, CD27, TIM3, CD20, or LAG3.

[0011] In some embodiments, the target cells are immune cells. In some embodiments, the immune cells include T cells, B cells, macrophages, natural killer (NK) cells, or dendritic cells. In some embodiments, the target ligands of the target cells include cell surface antigens.

[0012] In some embodiments, the at least one targeting portion includes a binding portion. In some embodiments, the binding portion includes a single-chain variable fragment (scFv), a single variable domain (V) on a biantibody or heavy chain. H H). In some embodiments, the binding portion includes the scFv. In some embodiments, the scFv is encoded by a nucleotide sequence in the E2 domain, wherein the nucleotide sequence encodes at least one base-linker, the heavy chain (V) of the variable fragment. H ), intermediate joints or light chains of the variable segments (V L In some implementations, the V encoding the scFv H or the V L The nucleic acid sequence described herein is derived from the same antibody. In some embodiments, the V encoding the scFv H or the V L The nucleic acid sequences described are derived from different antibodies.

[0013] In some embodiments, the nucleotide sequence further includes at least one restriction enzyme site. In some embodiments, the at least one restriction enzyme site is located upstream, downstream, or a combination thereof within the E2 domain of the nucleotide sequence. In some embodiments, the at least one restriction enzyme site includes a BstEII site.

[0014] In some embodiments, the at least one base linker comprises SEQ ID NO: 2-6. In some embodiments, the intermediate linker comprises at least 5 amino acids. In some embodiments, the intermediate linker comprises up to 25 amino acids. In some embodiments, the intermediate linker comprises between 5 and 25 amino acids. In some embodiments, the intermediate linker comprises a linker of 14 amino acids or a linker of 18 amino acids. In some embodiments, the intermediate linker comprises SEQ ID NO: 7-8. In some embodiments, the V L It's the opposite.

[0015] In some embodiments, the binding portion comprises the biantibody. In some embodiments, the biantibody comprises a dimer of a single-chain variable fragment (scFv). In some embodiments, the biantibody is encoded by a nucleotide sequence in the E2 domain, wherein the nucleotide sequence encodes at least one basal linker, at least one heavy chain (V) of the variable fragment. H ), at least one intermediate joint or at least one light chain (V) of the variable segment L In some embodiments, the at least one intermediate linker comprises at least 5 amino acids. In some embodiments, the at least one intermediate linker comprises up to 25 amino acids. In some embodiments, the at least one intermediate linker comprises between 5 and 25 amino acids. In some embodiments, the at least one intermediate linker comprises a linker of 18 amino acids. In some embodiments, the at least one intermediate linker comprises SEQ ID NO: 7-8. In some embodiments, the dimer of the scFv is derived from the same antibody. In some embodiments, the dimer of the scFv is derived from different antibodies. In some embodiments, the at least one V H Derived from the same antibody. In some embodiments, the at least one V H Derived from different antibodies. In some embodiments, the at least one light chain (V) of the variable fragment L The variable fragment is derived from the same antibody. In some embodiments, at least one light chain (V) of the variable fragment L () originates from different antibodies.

[0016] In some embodiments, the binding moiety further comprises at least one modification relative to the sequence of the wild-type binding moiety. In some embodiments, the at least one modification comprises an amino acid substitution that removes a protease cleavage site from the binding moiety. In some embodiments, the protease cleavage site is a furin protease cleavage site. In some embodiments, the at least one modification comprises a lysine-to-histidine substitution. In some embodiments, the binding moiety comprising the VHH comprises the formula: linker 1-VHH-linker 2. In some embodiments, linker 1 is selected from (GGGGS)nAAGHVG, AAGHVG(GGGGS)n, (GGGGS)nAAGHVG(GGGGS)n, (GGGGS)nGVHGAA, GVHGAA(GGGGS)n, and (GGGGS)nGVHGAA(GGGGS)n. In some embodiments, the connector 2 is selected from (GGGGS)nAAGHVG, AAGHVG(GGGGS)n, (GGGGS)nAAGHVG(GGGGS)n, (GGGGS)nGVHGAA, GVHGAA(GGGGS)n and (GGGGS)nGVHGAA(GGGGS)n.

[0017] In some embodiments, the binding portion comprises a nucleotide sequence encoding its antigen-binding fragment, wherein the antigen-binding fragment binds to a target ligand on the target cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell includes human cancer cells. In some embodiments, the cancer cell includes breast cancer cells, colon cancer cells, bladder cancer cells, lung cancer cells, head and neck cancer cells, squamous cell carcinoma cells, pancreatic cancer cells, prostate cancer cells, melanoma cells, non-small cell lung cancer cells, renal cancer cells, glioblastoma cells, Merkel cell carcinoma cells, angiosarcoma cells, cutaneous T-cell lymphoma cells, cutaneous B-cell lymphoma cells, dermatofibrosarcoma protuberans cells, sebaceous gland cancer cells, or cutaneous neuroendocrine cancer cells. In other embodiments, at least two target portions are combined together or separately with the engineered viral vector disclosed herein. In some cases, the at least two target portions include CD47, HER2, nectin-4, HLA, or EGFR. In some embodiments, the combination of the target portions has an increasing effect on the activity of the engineered viral vector disclosed herein (in some cases, a synergistic effect).

[0018] In some embodiments, the target ligand comprises a cell surface marker. In some embodiments, the cell surface marker comprises a cancer cell marker. In some embodiments, the cancer cell marker comprises CD47, CEA, HER2, EGFR, PSMA, TfR, nectin-4, muc-1, endothelin, EphA2, EphB2, folate receptor, GRP78, IGF-1R, cMET / HGFR, Tn antigen, EpCAM, ανβ3 integrin, CD44, mesothelin, PSCA, uPAR, CAIX, CD13, FAP-α, matriptase, MT-1 MMP, MT6-MMP, KDR, KIT, CD276, CD-83, or combinations thereof. In some embodiments, the target ligands include CD33, CD30, HER2, CD22, CD79b, nectin-4, Trop-2, BCMA, CD19, EGFR, folate receptor α, CD3, CD38, CD20, BCMA, CEA, PSMA, OX40, 4-1BB, CD16A, PD-1, PD-L1, CTLA-4, LAG-3, TIM3, VEGF, CD47, ICOS, MET, LGR5, IGF-1R, HER3, ANG2, DLL4, FAP, DR5, fibronectin ED-B, tendinin C, EpCAM, TNFα, digoxin, GPIIb / IIIa, rattlesnake venom, scorpion venom, CA6, PLVAP, 5T4, EDB, EGFRvIII, 6B11, CXCR4, MSLN, FN1, GPC2, GPC3, VCAN1, COL11A1, or MMR.

[0019] In some embodiments, the target ligand of the target cell includes an immune checkpoint protein or an immune checkpoint receptor. In some embodiments, the target ligand of the target cell is an immune checkpoint protein. In some embodiments, the immune checkpoint protein includes PD-L1 or CTLA-4. In some embodiments, the target ligand of the target cell is an immune checkpoint receptor. In some embodiments, the immune checkpoint receptor includes PD-1.

[0020] In some embodiments, the target cells are immune cells. In some embodiments, the immune cells include T cells, B cells, macrophages, natural killer (NK) cells, or dendritic cells. In some embodiments, the target ligands of the target cells include cell surface antigens.

[0021] In some embodiments, the engineered viral vector further comprises at least one amino acid modification in the E1 domain relative to the wild-type protein sequence (SEQ ID NO: 1). In some embodiments, the at least one amino acid modification in the E1 domain relative to the wild-type protein sequence (SEQ ID NO: 1) comprises at least one amino acid substitution. In some embodiments, the at least one amino acid substitution comprises an amino acid substitution at position 770, an amino acid substitution at position 771, or a combination thereof, wherein the amino acid position is based on the amino acid numbering in SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 770 comprises an A770S or A770Y substitution. In some embodiments, the amino acid substitution at position 771 comprises a K771G substitution. In some embodiments, the at least one amino acid substitution further comprises an amino acid substitution at position 226. In some embodiments, the amino acid substitution at position 226 comprises an E226G substitution.

[0022] In some embodiments, the at least one modification in the E2 domain relative to the wild-type protein sequence (SEQ ID NO: 1) comprises a substitution of one or more amino acids. In some embodiments, the substitution of one or more amino acids comprises an amino acid substitution at position 134, an amino acid substitution at position 135, an amino acid substitution at position 136, an amino acid substitution at position 137, an amino acid substitution at position 225, an amino acid substitution at position 226, or a combination thereof, wherein the amino acid positions are based on the amino acid numbers in SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 134 comprises an S134A substitution. In some embodiments, the amino acid substitution at position 135 comprises an L135A substitution. In some embodiments, the amino acid substitution at position 136 comprises a K136A substitution. In some embodiments, the amino acid substitution at position 137 comprises a Q137A substitution. In some embodiments, the amino acid substitution at position 225 comprises a K225A substitution. In some embodiments, the amino acid substitution at position 226 comprises an E226A substitution.

[0023] In some embodiments, the ligand is a hormone, neurotransmitter, cytokine, ion, or a subunit thereof. In some embodiments, the at least one targeting portion comprises a ligand of a cell surface receptor. In some embodiments, the ligand comprises a G protein-coupled receptor ligand or a small peptide ligand. In some embodiments, the ligand is encoded by a nucleotide sequence inserted into the E2 domain. In some embodiments, the G protein-coupled receptor ligand comprises endorphin or oxytocin. In some embodiments, the small peptide ligand comprises a ligand of a GPR78, EGFR, PD-1, or CD47 receptor. In some embodiments, the small peptide ligand is epidermal growth factor (EGF) or a subunit of EGF. In some embodiments, the subunit of EGF is an EGF core domain. In some embodiments, the EGF core domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24. In some embodiments, the EGF core domain comprises SEQ ID NO: 24. In some embodiments, the targeting portion comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 25 or 26. In some embodiments, the targeting portion comprises SEQ ID NO: 25 or 26. In some embodiments, the cell surface receptor is EGFR or an EGFR mutant. In some embodiments, the target cell is a cancer cell.

[0024] In some embodiments, the at least one targeting portion includes an immunogenic protein. In some embodiments, the immunogenic protein includes a viral protein. In some embodiments, the viral protein includes a viral envelope protein, a spike protein, or a combination thereof. In some embodiments, the viral protein is derived from a virus. In some embodiments, the virus includes a respiratory virus. In some embodiments, the respiratory virus includes SARS-CoV-2 or influenza virus. In some embodiments, the viral protein contains the N-terminal domain of SARS-CoV-2. In some embodiments, the viral protein contains the hemagglutinin (HA) of the influenza virus.

[0025] In some embodiments, the engineered viral vector further includes a payload vector. In some embodiments, the payload vector encodes at least one therapeutic agent.

[0026] In some embodiments, the at least one therapeutic agent comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises DNA, RNA, or a combination thereof. In some embodiments, the RNA comprises mRNA. In some embodiments, the at least one therapeutic agent comprises at least one therapeutic polypeptide. In some embodiments, the at least one therapeutic polypeptide comprises a suicide protein. In some embodiments, the suicide protein comprises thymidine kinase, cytosine deaminase, IL-2, nitroreductase (NR), carboxylesterase, β-glucuronidase, cytochrome p450, β-galactosidase, diphtheria toxin A chain (DT-A), carboxypeptidase G2 (CPG2), purine nucleoside phosphorylase (PNP), or deoxycytidine kinase (dCK). In some embodiments, the thymidine kinase is derived from herpes simplex virus (HSV-TK) or vesicular stomatitis virus (VSV-TK).

[0027] In some embodiments, the engineered viral vector is generated through transient transfection of a cell line. In some embodiments, the engineered viral vector is an integrative engineered viral vector. In some embodiments, the engineered viral vector is a non-integrative engineered viral vector.

[0028] In another aspect, this disclosure provides a cell comprising the engineered viral vector described herein. In another aspect, this disclosure provides a system comprising the engineered viral vector described herein.

[0029] In another aspect, this disclosure provides a pharmaceutical composition comprising the engineered viral vector, cell, or system described herein. In some embodiments, the pharmaceutical composition comprises at least one additional active ingredient. In some embodiments, the pharmaceutical composition comprises at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition stimulates an immune response in a subject.

[0030] In one aspect, this disclosure provides a method of delivering a therapeutic agent to target cells in a subject, the method comprising administering the pharmaceutical composition described herein to the subject via oral, bronchoalveolar lavage, sublingual, intratumoral, parenteral, intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, subarachnoid, intraocular, intrasternal, ocular, endothelial, local, intranasal, intrapulmonary, rectal, intraarterial, intrasheathal, inhalation, intralesional, intradermal, epidural, intracapsular, subcapsular, intracardiac, tracheal, subepidermal, or intraspinal administration, for example by injection or infusion.

[0031] In another aspect, this disclosure provides a method for inducing cell-killing activity in cancer cells of a subject, the method comprising administering the engineered viral vector described herein to the subject, thereby inducing cell-killing activity in the cancer cells.

[0032] In one aspect, this disclosure provides a method for inducing an immune response in a subject, the method comprising administering the engineered viral vector described herein to the subject, thereby inducing an immune response triggered by target cells in the subject. Attached Figure Description

[0033] This patent application contains at least one color drawing. The Patent Office will, upon request and upon payment of the necessary fees, provide a copy of this patent or patent application with the color drawing.

[0034] The features and advantages of the invention will be understood by referring to the following detailed description of illustrative embodiments utilizing the principles of the invention, along with the accompanying drawings, in which: Figure 1A-Figure 1B A list of amino acid sequences of the Sindbis (SB) envelope mutation and E1 domain, as described in this article, is presented. Figure 1A A series of SB envelope mutations are shown, where the GVO2.2 plasmid is generated based on the 2.2 plasmid, and this GVO2.2 plasmid is used as a base construct for further modifications. Gray vertical arrows indicate amino acid mutations, and boxes F indicate furin cleavage sites. ZZ represents an IgG binding domain derived from the B domain of bacterial protein A, which can bind to the Fc portion of any IgG. Tm indicates the targeting portion. Figure 1B The amino acid sequence of the E1 domain of the SB envelope in plasmids GVO2.2 or GVO2.2F is shown, which includes two mutations: A226Y and K227G.

[0035] Figure 2 This demonstrates the transduction of 293T cells with an engineered viral vector conjugated with an anti-HLA monoclonal antibody (mAb) in a dose-dependent manner. In this example, a firefly luciferase (Luc) reporter plasmid was used as the payload plasmid.

[0036] Figures 3A-3B The transduction of A375 human melanoma cells or Pit-2 knockout (Pit-2-KO) A375 cells with engineered viral vectors conjugated with anti-HLA mAb or bitropic (retroviral envelope (RVE)) viral vectors (which are Pit-2 dependent) is shown. Figure 3A The results of luciferase assays in wild-type A375 cells or Pit-2-KO A375 cells transduced with a GVO2.2 pseudotype vector conjugated to anti-HLA mAb are shown in an antibody concentration-dependent manner. Luciferase was used as a reporter molecule. Figure 3BThis indicates that because the anti-HLA mAb does not bind to the conventional RVE viral vector, the bitropic viral vector cannot transduce Pit-2-KOA375 cells. The RVE viral vector can only transduce wild-type A375 cells expressing Pit-2.

[0037] Figures 4A-4D The transduction results of various antibody-conjugated engineered viral vectors are shown. Different human cancer cell lines were tested. Figure 4A The results of luciferase assays in SKBR3 human breast cancer cells transduced with GVO 2.2 pseudotyped viral vectors conjugated to anti-HLA or anti-CD47 mAb are shown. The engineered viral vectors conjugated to anti-CD47 mAb showed limited transduction, saturating at an antibody concentration of 0.1 µg / mL, indicating distinct patterns of antigen-antibody interactions. Luciferase was used as a reporter molecule. Figure 4B The results of luciferase assays in SKBR3 cells transduced with GVO2.2 pseudovirus vectors conjugated to anti-HLA or anti-HER2 mAb are shown in a dose-dependent manner. Figure 4C The results of luciferase assays in HCT15 human colorectal cancer cells transduced with GVO 2.2 pseudotyped viral vectors conjugated to anti-HLA mAb or anti-EGFR mAb are shown. Transduction of HCT15 cells with viral vectors conjugated to anti-HLA mAb is not shown here because HCT15 cells do not express HLA class I antigens on their cell surface. Figure 4D The results of luciferase assays in HepG2 human hepatocellular carcinoma cells transduced with a GVO 2.2 pseudotype viral vector conjugated to anti-HLA or anti-EGFR mAb are shown, exhibiting results similar to those in HCT15 cells.

[0038] Figures 5A-5B The engineered viral vectors generated from the GVO2.2-scFv plasmid with various base sequences and configurations (#1-#14) are shown for transduction of U87MG human glioblastoma. In this experiment, the SB plasmid (or GVO2.2-scFv-C-1 plasmid) embedding human CD47-scFv was used to generate the engineered viral vectors. Figure 5A The engineered viral vector generated using the GVO2.2-scFv-C-1 plasmid is shown for transduction of U87MG cells. Different conformations of the adapters (#1-#14) were modified in each GVO2.2-scFv-C-1 plasmid. The conformations of adapters #1 to #7 correspond to the sequences in Table 1. The conformations of adapters #8 to #14 are the same as those of adapters #1 to #7, but with inverted V-shaped structures. L . Figure 5B A schematic diagram of the scFv configuration is shown.

[0039] Figure 6The results of luciferase assays using engineered viral vectors generated from the GVO2.2-scFv-C-1 plasmid are shown, compared to bitropic (RVE) viral vectors (whose transduction levels are relatively comparable across different cancer cell lines).

[0040] Figure 7 The diagram shows scFv (left inset) or dual antibody (right inset) incorporated into the E2 domain of the SB envelope protein as a targeting part.

[0041] Figure 8 Western blots of the anti-E2 domain of SB envelope antibodies on cell lysates of cells transfected with various vector particles (engineered viral vectors) or plasmids are shown. The predicted size of the engineered viral vector generated with the GVO2.2-scFv-C-1 plasmid is 81 kDa; the engineered viral vector generated with the GVO2.2-scFv-H-1 plasmid is 81.6 kDa; and the engineered viral vector generated with the GVO2.2-DB-H-1 plasmid is 110 kDa. Assuming the small fragments seen in lanes 3 and 4 are due to furin cleavage, the small fragment size for the engineered viral vector generated with the GVO2.2-scFv-H-1 plasmid is 15.6 kDa and the remainder is 65.8 kDa, and for the engineered viral vector generated with the GVO2.2-DB-H-1 plasmid, the small fragments are 15.6 kDa, 28.5 kDa, and the remainder is 65.9 kDa.

[0042] Figure 9 This study demonstrates the luciferase assay for transduction of SKOV3 human ovarian cancer cells using engineered viral vectors carrying HER2-targeting scFv (scFv-H-1 plasmid or scFv-H-2 plasmid) or HER2-targeting dual antibodies (DB-H-1 plasmid) in the targeting portion of the E2 domain. Modifications involving nucleotides encoding AAG(K) to CAC(H) in the HER2 scFv or HER2 DB targeting portion are termed K2H (K to H; lysine to histidine) modifications. In this experiment, the vectors with K2H modifications were scFv(K2H)-H-1, scFv-(K2H)-H-2, and DB(K2H)-H-1 plasmids. The data indicate that K2H modification increased the transduction of SKOV3 cells using engineered viral vectors (targeting HER2 on the cell surface) generated with scFv-H-1 and DB-H-1 plasmids (but not using H-2 due to low activity).

[0043] Figure 10The transduction of SKBR3 cells using engineered viral vectors generated with different mutations in the E1 domain is shown. As illustrated, the addition of the furin protease site (from GVO2.2 plasmid to GVO2.2F plasmid) increased transduction. Therefore, the amino acid mutation in YG also indicates a high transduction level similar to that of wild-type AK. All engineered viral vectors carried a luciferase reporter gene.

[0044] Figure 11 The luciferase activity of SKBR3 cells transduced with engineered viral vectors generated using HER2-targeting plasmids incorporating scFv or DB was demonstrated. Data showed that for engineered viral vectors generated using scFv and DB plasmids, the K2H mutation and the addition of the furin protease site increased transduction.

[0045] Figure 12 The luciferase activity of two distinct breast cancer cell lines (SKBR3 and MDA-MB-468) transduced using an engineered viral vector generated from the GVO2.2F-DB(K2H)-H-1 plasmid was demonstrated. SKBR3 cells overexpress HER2, while MDA-MB-468 cells are HER2-negative on their cell surface. The transduction of these two distinct breast cancer cell lines represents the specificity of binding target molecules to each cell line using an engineered viral vector incorporating HER2-DB.

[0046] Figures 13A-13B An example of another target (nectin-4) is shown. scFv-N-1 is a single-chain antibody derived from an anti-nectin-4 antibody and targets nectin-4 on the surface of cancer cells. Figure 13A This study demonstrates nectin-4 expression-specific transduction in three different cell lines using an engineered viral vector generated from the GVO2.2F-scFv-N-1 plasmid. Two different adapter sets, #5 or #6, were examined in this experiment. Figure 13B The cell surface staining of nectin-4 was shown in three cancer cell lines: A375 human melanoma, PC-3 human prostate cancer, and MDA-MB-468 human breast cancer cells, which have different nectin-4 expression levels.

[0047] Figures 14A-14B The relative potency assay of ganciclovir (GCV) mediated HSV1-TK in SKBR3 cells transduced with engineered viral vectors is shown. Figure 14AThe engineered viral vector generated using the GVO2.2F-scFv(K2H)-H-1 plasmid was able to transduce SKBR3 cells and successfully express HSV1-TK, killing transduced cells in a titer-dependent manner in the presence of GCV at levels similar to or higher than those of the bitropic enveloped viral vector (RDRS20A). Figure 14B The engineered viral vector generated using the GVO2.2F-DB(K2H)-H-1 plasmid showed the same results.

[0048] Figures 15A-15B Transduction of BT474 cells using a single agent (GVO-2.2-scFv-C-1, GVO-2.2-scFv-H-1, or GVO2.2-DB(K2H)-H-1 vector) or a combination of two GVO pseudotype vectors is demonstrated. Luciferase is used as a reporter molecule. Figure 15A The transduction results of GVO2.2-scFv-C-1, GVO2.2-scFv-H-1, and combinations of GVO2.2-scFv-C-1 and GVO2.2-scFv-H-1 in BT474 cells are shown. Figure 15B This study demonstrates the transduction results of GVO2.2-scFv-C-1, GVO2.2-DB(K2H)-H-1, and combinations of GVO2.2-scFv-C-1 and GVO2.2-DB(K2H)-H-1 in BT474 cells.

[0049] Figures 16A-16C This demonstrates the in vivo studies conducted using the RVE control vector and GVO2.2F-DB(K2H)-H-1 in an SKOV3 human ovarian cancer model in immunodeficient mice. Figure 16A A schematic diagram of a research design in a human ovarian cancer model in immunodeficient mice is shown. Figure 16B The study demonstrates the effective targeting and transduction of the GVO2.2F-DB(K2H)-H-1 vector in tumor cells compared to the RVE control vector, with p-values ​​less than 0.05. Transduction is expressed as the percentage of GFP+ / huβ2M+ tumor cells. Statistical data were calculated using one-way ANOVA. Figure 16C The percentage of tumor cells in the GFP-positive population in the GVO2.2F-DB(K2H)-H-1 group and the RVE control vector group is shown.

[0050] Figure 17A The percentage of GFP-positive cells in SKBR3 cells transduced with GVO2.2F-EGF adapter 5 or GVO2.2F-EGF adapter 6 is shown. NTC cells are untransduced control cells. Figure 17BThe percentage of GFP-positive cells in SKBR3 cells transduced with GVO2.2F-EGF adapter 5 or GVO2.2F-EGF adapter 6 is shown, as measured by flow cytometry.

[0051] Figure 18 The percentage of GFP-positive cells in HT29-Luc cells transduced with the RVE control vector, GVO2.2F-scFv-E-1, or GVO2.2F-EGF adapter 5 is shown. NTC cells are untransduced control cells.

[0052] Figure 19 The percentage of GFP-positive cells in U87MG wild-type (U87MGwt) or U87MG-EGFRvIII cells transduced with RVE control vector, GVO2.2F-scFv-E-1, or GVO2.2F-EGF adapter 5 is shown. NTCs are untransduced control cells.

[0053] Figure 20 The percentage of GFP-positive cells in wild-type 293T cells and EGFR-expressing 293T cells transduced with the RVE control vector, GVO2.2F-scFv-E-1, or GVO2.2F-EGF adapter 5 is shown at two different multiples of infection (MOI). NTCs are untransduced control cells.

[0054] Figure 21 The transduction levels (expressed as luciferase activity) of RKO or HT1080 cells transduced with the original GVO2.2F-VHH-PD-L1 vector (VHH-PD-L1 orig) or the optimized GVO2.2F-VHH-PD-L1 vector (VHH-PD-L1 delG) are shown.

[0055] Figure 22 The transduction level (expressed by luciferase activity) of RKO cells transduced with GVO2.2F-VHH-PD-L1 containing a linker, either v1-v6 or the GVO2.2F-VHH-PD-L1 optimized vector (VHH-PD-L1 delG), is shown.

[0056] Figure 23 The transduction level (expressed by luciferase activity) of SKBR3 cells transduced with GVO2.2F-VHH-PD-L1 containing the adapter is shown. The adapter is either v1-v6 or the GVO2.2F-VHH-PD-L1 optimized vector (VHH-PD-L1 delG). Detailed Implementation

[0057] Therapeutic delivery to target cells (such as gene therapy) is one of the fastest-growing areas of medical research and holds the promise of curing life-threatening diseases such as cancer, severe viral infections such as HIV, and genetic disorders in the future. Various strategies are being investigated to leverage the potential of this approach for efficient, widespread, and low-cost clinical application. Clinical therapeutic delivery protocols sometimes rely on stable therapeutic delivery methods for long-term expression of therapeutic genes, and these methods often involve engineered viruses (e.g., retroviruses) or other viral vectors as delivery tools. The ability to deliver therapeutic agents directly to specific types of target cells can be important not only for achieving therapeutic efficacy but also for limiting any potentially harmful or off-target effects associated with the therapeutic. Methods used for therapeutic delivery can also be tailored to deliver therapeutic agents efficiently and safely.

[0058] In some cases, methods such as transcriptional targeting and transduction targeting can be used for targeted therapy delivery. Transcriptional targeting manipulates transgene expression by modifying the promoter of the transgene (such as a tissue-specific promoter). Conversely, in transduction targeting, modified viral vectors can attach to the transgene with better specificity and transduce it into target cells. In viral pseudotypening, viral particles or viral vectors can be generated using viral envelope proteins from another virus to restrict or expand the host cell range.

[0059] As described herein, engineered viral vectors, generated by pseudotyped viruses or viral vectors with envelope proteins derived from different viruses, can improve transduction efficiency and specificity. Further modification of such envelope proteins can improve the therapeutic delivery and specificity of viruses or viral vectors to target cells of interest. Furthermore, delivery of engineered viral vectors to specific targets enables more efficient and precise therapeutic delivery. For example, tissues or cells refractory to conventional vectors can be targeted; cells in the tumor microenvironment that hide tumor cells can be specifically targeted before attacking tumor cells; and normal cells near the injection site at the tumor lesion can be protected by avoiding off-target effects. Viral particles or viral vectors with heterologous marker genes can be used for diagnostic applications and as research tools for transducing specific cell types. Further modification of the viral envelope proteins provided herein can also be used to target cell-cell interactions by expressing the targeted envelope in cells such as lymphocytes, and then allowing the cells to contact the targeted cells in vivo, in vitro, or ex vivo. In another aspect, this disclosure provides methods for delivering therapeutic agents to target cells in a subject using the engineered viral vectors described herein. The methods disclosed herein can also be used to stimulate an immune response to a specific antigen in a subject by delivering an engineered viral vector comprising a payload vector encoding an immunogenic protein to a target cell capable of modulating an immune response in the subject (e.g., antigen-presenting cells, such as dendritic cells).

[0060] Viral tropism describes the ability of a particular virus or viral vector's envelope protein to bind and fuse with the membrane of a specific target cell. As described herein, pseudotyped viruses or viral vectors with envelope proteins derived from different viruses can be used for targeted transduction. Pseudotypening is a method for generating viral particles or viral vectors combined with exogenous viral envelope proteins or non-wild-type envelope proteins. In this method, plasmids or vectors or nucleic acid constructs encoding non-surface proteins (e.g., transfer plasmids or packaging plasmids (e.g., gagpol plasmids)) can be used together with exogenous viral envelope plasmids or non-wild-type viral envelope plasmids for viral vector generation. As a result, pseudotyped viral particles or pseudotyped viral vectors are generated. Because pseudotyped viral particles or pseudotyped viral vectors have exogenous viral envelope proteins or non-wild-type envelope proteins instead of wild-type envelope proteins, this method can be used to alter viral tropism for specific cell types, thereby providing improvements in targeted cell infection for therapeutic delivery.

[0061] Enveloped viruses are characterized by a phospholipid bilayer membrane acquired when the virus buds from a host cell. Each enveloped virus contains its envelope glycoprotein (enveloping) gene in its genome. The envelope gene encodes a glycoprotein, or "spike protein," located on the surface of the virus. This glycoprotein or spike protein plays a crucial role in viral transmission by facilitating the attachment of the viral particle to the surface of the target cell via recognition of specific receptors. The binding targets of viral envelope glycoproteins are typically identified. For example, the envelope protein of 4070A ditropic murine leukemia virus (MLV) targets type III sodium-dependent phosphate transporter (Pit-2), while the envelope of gibberish leukemia virus (GALV) targets type III sodium-dependent phosphate transporter (Pit-1). The ditropic 4070A envelope protein of MLV consists of surface units (SU) and transmembrane domains (TM), which are processed from a single polypeptide encoded by the envelope gene. Although these domains are separated during maturation, they remain single proteins forming homotrimers. Following SU dissociation, the viral envelope undergoes binding, conformational changes, and fusion. Therefore, any modifications leading to targeting can affect the structure or conformation of the entire envelope protein. Once the viral particle binds to the cell surface, a series of conformational changes occur that promote proximity between the two membranes, followed by fusion of the cell and viral membranes via endocytosis or other pathways. Ultimately, this results in the release of the viral genome into the host cell.

[0062] Engineered viral vectors In one aspect, this document provides engineered viral vectors. In some embodiments, the engineered viral vector comprises a modified envelope protein. In some embodiments, the modified envelope protein comprises at least one targeting moiety. In some embodiments, the modified envelope protein comprises at least one modification in the E2 domain relative to the wild-type protein sequence. In some embodiments, the wild-type protein sequence has the sequence of SEQ ID NO: 1. In some embodiments, the modified envelope protein increases the transduction efficiency of the engineered viral vector to target cells relative to a viral vector without modified envelope protein. In some embodiments, the engineered viral vector comprises an engineered retroviral vector. In some embodiments, the engineered retroviral vector comprises an engineered gamma retroviral vector. In some embodiments, the engineered gamma retroviral vector comprises an engineered MLV vector. In some embodiments, the engineered viral vector generated by pseudotyped an enveloped virus with an envelope protein derived from different viruses comprises an MLV vector having a Sindbis envelope protein.

[0063] In some embodiments, the modified envelope protein includes a recombinant viral envelope protein derived from a DNA virus. In some embodiments, the modified envelope protein includes a recombinant viral envelope protein derived from an RNA virus. In some embodiments, the RNA virus includes an alphavirus. In some embodiments, the alphavirus includes Sindbis virus.

[0064] In one aspect, the Sindbis virus envelope protein can be engineered for antibody-mediated targeting, for example, by incorporating the IgG-binding domain (ZZ domain) of bacterial protein A into the E2 domain of the SB envelope gene. This modification allows IgG antibodies to conjugate to specific proteins, driving more specific binding to the target. Furthermore, modifications to the amino acid sequence of the Sindbis envelope gene can be performed to generate more effective and specific targeting for the fusion process while maintaining high viral titer quality. The modified Sindbis envelope can include different modifications, such as point mutations, insertions, or deletions, in the amino acid sequence at the E3, E2, 6K, E1 domains, or combinations thereof of wild-type Sindbis. The E3, E2, 6K, or E1 domains can have one or more mutations. Furthermore, the methods described herein cover combinations of mutations in the E3, E2, 6K, or E1 domains. Examples of wild-type sequences of the Sindbis envelope protein include, but are not limited to, SEQ ID NO: 1.

[0065] As described herein, in some embodiments, the modified envelope protein derived from Sindbis virus comprises an E3 domain, an E2 domain, a 6K domain, an E1 domain, or a combination thereof. In some embodiments, the modified envelope protein also comprises a protease cleavage site located between the E3 and E2 domains. Examples of protease cleavage sites include, but are not limited to, furin cleavage sites. Frin recognizes a specific amino acid sequence at the furin cleavage site, thereby proteolytically cleaving the target sequence. Frin is known to play an important role in viral envelope processing because it is involved in the maturation of functional viral envelope proteins. In some embodiments, the protease cleavage site includes a furin cleavage site. In various embodiments, the protease cleavage site located between the E3 and E2 domains (e.g., a furin cleavage site) is removed from the modified envelope protein.

[0066] In some embodiments, the modified envelope protein further comprises at least one targeting moiety. As described herein, a targeting moiety refers to a portion of a viral envelope protein that is modified to improve the targeting efficiency or specificity of an engineered viral vector for therapeutic delivery. In some embodiments, at least one targeting moiety is located within the E2 domain of the Sindbis envelope protein. In some embodiments, at least one targeting moiety includes a conjugation portion. In various embodiments, at least one targeting moiety includes a binding portion. In some embodiments, at least one targeting moiety includes a ligand for a cell surface receptor. In various embodiments, at least one targeting moiety includes an immunogenic protein.

[0067] In some embodiments, the targeting portion may target or bind to cell surface markers of target cells. In some embodiments, the targeting portion may target or bind to markers expressed by target cells. In some embodiments, the targeting portion may target or bind to markers associated with the microenvironment. In some embodiments, the targeting portion may target or bind to markers associated with a disease or condition. In some cases, the targeting portion includes a targeting peptide that mediates the selective localization of the engineered viral vector to cells in a specific cell type, tissue, or state (e.g., cancer).

[0068] In some embodiments, the targeting portion is configured to target cell surface markers. In some embodiments, the cell surface markers include cancer cell markers. In some embodiments, cancer cell markers include CD47, CEA, HER2, EGFR, PSMA, TfR, nectin-4, muc-1, endothelin, EphA2, EphB2, folate receptor, GRP78, IGF-1R, cMET / HGFR, Tn antigen, EpCAM, ανβ3 integrin, CD44, mesothelin, PSCA, uPAR, CAIX, CD13, FAP-α, matriptase, MT-1 MMP, MT6-MMP, KDR, KIT, CD276, CD-83, or combinations thereof. In some implementations, the targeting portion is configured to target CD33, CD30, HER2, CD22, CD79b, nectin-4, Trop-2, BCMA, CD19, EGFR, folate receptor α, CD3, CD38, CD20, BCMA, CEA, PSMA, OX40, 4-1BB, CD16A, PD-1, PD-L1, CTLA-4, LAG-3, TIM3, VEGF, CD47, and ICOS. The target moiety includes MET, LGR5, IGF-1R, HER3, ANG2, DLL4, FAP, DR5, fibronectin ED-B, tendinin C, EpCAM, TNFα, digoxin, GPIIb / IIIa, rattlesnake venom, scorpion venom, CA6, PLVAP, 5T4, EDB, EGFRvIII, 6B11, CXCR4, MSLN, FN1, GPC2, GPC3, VCAN1, COL11A1, or MMR. In some embodiments, the target moiety is configured to target CD47, HER2, nectin-4, HLA, or EGFR. In other embodiments, at least two target moieties are combined together or separately with the engineered viral vector disclosed herein. In some cases, at least two target moieties include CD47, HER2, nectin-4, HLA, or EGFR. In some embodiments, the combination of target moieties has an increasing effect on the activity of the engineered viral vector disclosed herein (in some cases, a synergistic effect).

[0069] In some embodiments, cell surface markers include pancreatic β-cell markers. In some embodiments, pancreatic β-cell markers include CD9 or ST8SA1.

[0070] In some implementations, the targeting portion is inserted into the E3 domain, E2 domain, 6K domain, or a combination thereof of the SB envelope protein. For example, the targeting portion (tm) can be inserted into the E2 domain of the SB envelope protein, such as... Figure 1AAs shown in the diagram. In some embodiments, the targeting portion can be inserted between the E3 domain, E2 domain, 6K domain, or a combination thereof of the SB envelope protein. In some embodiments, the E2 domain of the SB envelope gene is engineered to incorporate the IgG binding domain (ZZ protein domain) of bacterial protein A. In some embodiments, the targeting portion is inserted at the same location as the ZZ protein domain located within the E2 domain of the SB envelope protein. For example, as... Figure 1A As shown, the ZZ protein domain is replaced with the targeting portion.

[0071] In one aspect, the compositions and methods described herein can be used to improve the targeting efficiency, specificity, efficacy, and safety of viral therapeutic delivery by generating engineered viral vectors through exogenous or non-wild-type enveloped pseudotypes of enveloped viruses such as retroviruses (e.g., murine leukemia virus) obtained from other enveloped viruses such as RNA viruses (e.g., alphaviruses) to other enveloped viruses. Examples of enveloped viruses include, but are not limited to, DNA viruses (e.g., herpesviruses, poxviruses, hepatotropic DNA viruses), RNA viruses (e.g., flaviviruses, alphaviruses, encapsulated viruses, coronaviruses, hepatitis D viruses, orthomyxoviruses, paramyxoviruses, rhabdoviruses, Bunyaviruses, filoviruses), and retroviruses (e.g., lentiviruses, murine leukemia virus).

[0072] In some respects, the engineered viral vectors provided herein are viral vectors derived from retroviruses, lentiviruses, poxviruses, or herpesviruses. Examples of viral vectors may include, but are not limited to, retroviral vectors, poxvirus vectors, baculovirus vectors, measlesvirus vectors, or herpesvirus vectors. In some cases, recombinant retroviral vectors include gamma retroviral vectors, such as vectors derived from the genome of Moloney murine leukemia virus (MoMLV, MMLV, MuLV, or MLV) or murine stem cell virus (MSCV). In some embodiments, the engineered viral vectors include engineered gamma retroviral vectors. Examples of pseudotypeable gamma retroviral vectors include, but are not limited to, gamma retroviral vectors encoding the following viruses: chicken syncytial virus, feline leukemia virus, Finkel-Biskis-Jinkins murine sarcoma virus, Gardner-Arnstein feline sarcoma virus, gibbon leukemia virus, guinea pig C-type tumor virus, Hardy-Zuckerman feline sarcoma virus, Harvey murine sarcoma virus, Kirsten murine sarcoma virus, koala retrovirus, Moloney murine sarcoma virus, murine leukemia virus, porcine C-type tumor virus, reticuloendotheliosis virus, Snyder-Theilen feline sarcoma virus, Trager duck spleen necrosis virus, viper retrovirus, or velour monkey sarcoma virus. In some embodiments, engineered viral vectors include engineered gamma retroviral vectors. In some embodiments, engineered gamma retroviral vectors are engineered murine leukemia virus (MLV) vectors. In some cases, engineered retroviral vectors include lentiviral vectors, such as those derived from the human immunodeficiency virus (HIV) genome. In some cases, engineered viral vectors are chimeric viral vectors, which contain viral portions from two or more viruses. In other cases, engineered viral vectors are recombinant viral vectors.

[0073] In some implementation schemes, A viruses include Aura virus, Barmah Forest virus, Bebaru virus, Caaingua virus, Cabassou virus, Chikungunya virus, Easternequine encephalitis virus, Eilat virus, Everglades virus, Fort Morgan virus, Getah virus, Highlands J virus, Madariaga virus, Mayaro virus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumu virus, O'nyong'nyong virus, Pixuna virus, and Rio Negro virus. Negro virus, Ross River virus, Salmon pancreas disease virus, Semliki Forest virus, Sindbis virus (SB), Southern elephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus, or Whataroa virus.

[0074] Sindbis virus (SB) is an alphavirus with a high infectivity rate and the ability to infect non-dividing cells, thus presenting itself as a potential tool for in vivo therapeutic delivery. The SB envelope gene encodes four components: E3, E2, 6K, and E1, located from the N-terminus to the C-terminus, respectively. These four subunits have individual functions. They are proteatically separated and form homotrimers of these components, similar to the envelope of an MLV. In mature SB virus particles, the positive-sense RNA virus genome complexes with capsid proteins to form an icosahedral nucleocapsid, which is surrounded by a lipid bilayer containing two intact membrane glycoproteins, E1 and E2, thus forming a heterodimer and functioning as a unit. E1 and E2 are independently anchored in the membrane. E2 binds to the host cell receptor. When E1 is exposed, it can mediate membrane fusion. Although E3 separates after the E2 domain matures, the E2 domain acts as a binding molecule and regulates fusion via E1. Therefore, in some embodiments, modifications to E2 do not affect the structure and function of E1, such as in vector fusion.

[0075] As described herein, engineered viral vectors can be generated by transiently transfecting cell lines with one or more plasmids. In some embodiments, the one or more plasmids include an envelope plasmid, a gagpol plasmid, and a payload plasmid. In some embodiments, the one or more plasmids include an envelope plasmid, a payload plasmid, a gagpol plasmid, and one or more packaging plasmids. The envelope plasmid (or envelope vector or envelope construct) encodes a modified or unmodified viral envelope protein, which may be derived from the same or different viral species. In some cases, pseudotyped engineered viral vectors as described herein can use envelope plasmids encoding viral envelope proteins from different viral species, such as envelope plasmids encoding SB envelope proteins. This can alter infectivity and provide altered tropism. Furthermore, the nucleic acid sequence encoding the viral envelope protein of the envelope plasmid can be modified to alter transduction efficiency and specificity. The gagpol plasmid encodes a polypeptide originating from structural components (e.g., core protein, capsid, matrix protein, nucleoprotein, and nucleocapsid) and enzymes (e.g., proteases, reverse transcriptases, integrases, and polymerases). In some embodiments, the polypeptide encoded by the gagpol gene is isolated into one or more gagpol plasmids. In some cases, the one or more gagpol plasmids include a first gagpol plasmid and a second gagpol plasmid. In some embodiments, the first gagpol plasmid contains the gagpol gene and reverse transcriptase. In some embodiments, the first gagpol plasmid contains the gagpol gene but does not contain reverse transcriptase. In some embodiments, the gagpol gene encodes a polypeptide lacking integrase function. In some embodiments, the second gagpol plasmid contains reverse transcriptase. The packaging plasmid encodes all the proteins necessary for the transcription and packaging of the RNA copy of the payload construct into recombinant pseudoviral particles. In some embodiments, the packaging plasmid contains Rev, a transactivator protein that improves nuclear localization signals and is involved in the export of unspliced ​​or incompletely spliced ​​mRNA to the cytoplasm. Finally, the payload plasmid (or payload body or payload construct) with packaging signals carries at least one or more genes, transgenes, or therapeutic agents of interest that can be delivered to target cells via engineered viral vectors. One or more plasmids are then transfected into packaging cells to generate engineered viral particles or viral vectors. After assembly, the engineered viral vectors are released from the packaging cells, and these engineered viral vectors may contain payloads for therapeutic delivery, such as genes of interest, transgenes, or therapeutic agents. Other methods can be used to generate the engineered viral vectors described herein. In some embodiments, the engineered viral vectors described herein are reproducible engineered viral vectors. In some embodiments, the engineered viral vectors are oncolytic engineered viral vectors. In some embodiments, the engineered viral vectors are replication-defective engineered viral vectors.In some implementations, the engineered viral vector is an integrated engineered viral vector. In other implementations, the engineered viral vector is a non-integrated engineered viral vector.

[0076] In some cases, engineered viral vectors can be generated using one or more modified plasmids. In some embodiments, the one or more modified plasmids comprise one or more modified gagpol plasmids. In some embodiments, the one or more modified gagpol plasmids contain a modified viral gagpol gene or a mutated viral gagpol gene compared to the wild-type gagpol gene.

[0077] In some embodiments, the viral gagpol gene is the wild-type gagpol gene. In some embodiments, the engineered viral vector generated from the wild-type gagpol gene is an integrative engineered viral vector. In some cases, the viral gagpol gene is modified or mutated, and this can affect the integration ability of the engineered viral vector. In some embodiments, the viral gagpol gene is a mutated gagpol gene. For example, the integrase of the gagpol gene is modified by mutating the nucleic acid sequence encoding the integrase, thereby affecting the integration ability of the engineered viral vector. In some embodiments, the integrase is a wild-type integrase. In some embodiments, the mutated gagpol gene lacks integrase function. In some embodiments, the integrase is a modified integrase. In some embodiments, the modified integrase lacks integrase function. In some embodiments, the modified integrase contains mutations affecting integration ability, such as deletions or insertions. In some embodiments, the engineered viral vector generated from the mutated gagpol gene is a non-integrative engineered viral vector. In some embodiments, the engineered viral vector generated from the modified integrase is a non-integrative engineered viral vector.

[0078] In some embodiments, the engineered viral vector includes an engineered murine leukemia virus (MLV) vector. In some cases, the engineered MLV vector is generated from one or more plasmids within the packaging cell, which are modified SB protein plasmids, one or more viral gagpol plasmids, and a payload plasmid. In some embodiments, the one or more viral gagpol plasmids contain at least one structural protein and at least one polymerase. In some embodiments, the one or more viral gagpol plasmids contain a viral gagpol gene. In some embodiments, the viral gagpol gene is the murine leukemia virus (MLV) gagpol gene.

[0079] Joining part In various aspects of the engineered viral vectors provided herein, in some embodiments, the engineered viral vector includes a conjugation portion. As described herein, a conjugation portion refers to a type of targeting portion that is modified on the envelope protein after the engineered viral vector is generated to conjugate with an antibody or an antigen-binding fragment thereof. In some embodiments, an engineered viral vector having a conjugation portion on the envelope protein may be incubated with an antibody or an antigen-binding fragment thereof prior to being used for therapeutic delivery to target cells.

[0080] In some embodiments, the conjugation of the conjugated portion is an affinity-binding conjugation. In some embodiments, the conjugated portion includes an IgG-binding domain, an IgA-binding domain, an IgM-binding domain, an IgD-binding domain, an IgE-binding domain, or a combination thereof. In some embodiments, the conjugated portion includes an IgG-binding domain. In some embodiments, the IgG-binding domain is derived from a bacterial protein. In some embodiments, the bacterial protein is bacterial protein A. In some embodiments, bacterial protein A is derived from Staphylococcus aureus. In some embodiments, the IgG-binding domain of the bacterial protein is a ZZ protein domain. In some embodiments, the ZZ protein domain includes an Fc region binding domain. In some embodiments, the ZZ protein domain is a non-viral domain. For example, the ZZ protein domain can be obtained from mammalian, plant, bacterial, or insect sources. In some embodiments, the ZZ protein domain can be inserted into the E3 domain, E2 domain, 6K domain, or a combination thereof of the SB envelope protein. In some embodiments, the ZZ protein domain can be inserted into the E2 domain of the SB envelope protein, as shown in Figure 1. In some embodiments, the ZZ protein domain can be inserted between the E3 domain, E2 domain, 6K domain, or a combination thereof of the SB envelope protein. For example, the ZZ protein domain can be inserted between the E3 and E2 domains of the SB envelope protein. In some embodiments, the ZZ protein domain is conjugated to an antibody or an antigen-binding fragment thereof.

[0081] In some embodiments, the engineered viral vector is further conjugated with an antibody or its antigen-binding fragment. In some embodiments, the antibody or its antigen-binding fragment includes IgG-scFv, a single variable domain (V) on the heavy chain, and so on. HH), nanobodies, BiTE, biantibodies, DART, TandAb, sc biantibodies, sc biantibodies-CH3, triantibodies, microantibodies, microantibodies, TriBi microantibodies, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc biantibodies-Fc, biantibodies-Fc, tandem scFv-Fc, intracellular antibodies, their binding fragments, their chemically modified derivatives, heavy chains of variable fragments (V H ), light chains of variable segments (V L (or more) combinations.

[0082] In some embodiments, the antibody or its antigen-binding fragment binds to a target ligand on a target cell. In some embodiments, the target cell is a healthy cell. In some embodiments, the target cell is a non-healthy cell. In some embodiments, the target cell is a normal cell. In some embodiments, the target cell is an abnormal cell. In some embodiments, the target cell is a wild-type cell. In some embodiments, the target cell is a mutated cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the cancer cell includes human cancer cells. In some implementations, cancer cells include leukemia cells, myeloid cells, promyeloid cells, myeloid mononuclear cells, mononuclear cells, erythroleukemia cells, chronic myeloid (granulocytic) leukemia cells, chronic lymphocytic leukemia cells, lymphoma cells such as Hodgkin's and non-Hodgkin's lymphoma cells, fibrosarcoma cells, myoma cells, liposarcoma cells, chondrosarcoma cells, osteosarcoma cells, angiosarcoma cells, endothelial sarcoma cells, Ewing's tumor cells, colon cancer cells, pancreatic cancer cells, breast cancer cells, ovarian cancer cells, prostate cancer cells, squamous cell carcinoma cells, basal cell carcinoma cells, adenocarcinoma cells, renal cell carcinoma cells, liver cancer cells, and Wilms' tumor cells. Tumor cells, cervical cancer cells, uterine cancer cells, testicular tumor cells, lung cancer cells, small cell lung cancer cells, bladder cancer cells, epithelial cancer cells, glioma cells, astrocytoma cells, oligodendroglioma cells, melanoma cells, neuroblastoma cells, retinoblastoma cells, dysplastic and hyperplastic cells, prostatitis cells, benign prostatic hyperplasia (BPH) cells, prostatic paraganglioma cells, prostatic adenocarcinoma cells, prostatic intraepithelial neoplasia cells, prostatic-rectal fistula cells, atypical prostatic stromal lesion cells, bladder cancer cells, lung cancer cells, head and neck cancer cells, non-small cell lung cancer cells, renal cancer cells, or glioblastoma cells. In some implementations, cancer cells include breast cancer cells, colon cancer cells, bladder cancer cells, lung cancer cells, head and neck cancer cells, squamous cell carcinoma cells, pancreatic cancer cells, prostate cancer cells, melanoma cells, non-small cell lung cancer cells, kidney cancer cells, glioblastoma cells, Merkel cell carcinoma cells, angiosarcoma cells, cutaneous T-cell lymphoma cells, cutaneous B-cell lymphoma cells, cutaneous fibrosarcoma protuberans cells, sebaceous gland cancer cells, or cutaneous neuroendocrine cancer cells.

[0083] In some embodiments, the antibody or its antigen-binding fragment binds to a target ligand located on the cell surface of a target cell. In some embodiments, the target ligand includes a cell surface marker. In some embodiments, the cell surface marker includes a cancer cell marker. In some embodiments, the cancer cell marker includes CD47, CEA, HER2, EGFR, PSMA, TfR, nectin-4, muc-1, endothelin, EphA2, EphB2, folate receptor, GRP78, IGF-1R, cMET / HGFR, Tn antigen, EpCAM, ανβ3 integrin, CD44, mesothelin, PSCA, uPAR, CAIX, CD13, FAP-α, matriptase, MT-1 MMP, MT6-MMP, KDR, KIT, CD276, CD-83, or combinations thereof. In some implementations, target ligands include CD33, CD30, HER2, CD22, CD79b, nectin-4, Trop-2, BCMA, CD19, EGFR, folate receptor α, CD3, CD38, CD20, BCMA, CEA, PSMA, OX40, 4-1BB, CD16A, PD-1, PD-L1, CTLA-4, LAG-3, TIM3, VEGF, CD47, ICOS, and M. ET, LGR5, IGF-1R, HER3, ANG2, DLL4, FAP, DR5, fibronectin ED-B, tendinin C, EpCAM, TNFα, digoxin, GPIIb / IIIa, rattlesnake venom, scorpion venom, CA6, PLVAP, 5T4, EDB, EGFRvIII, 6B11, CXCR4, MSLN, FN1, GPC2, GPC3, VCAN1, COL11A1, or MMR. In some embodiments, the engineered viral vector is conjugated with an anti-CD47 antibody, an anti-HER2 antibody, an anti-nectin-4 antibody, an anti-HLA antibody, or an anti-EGFR antibody. In some embodiments, the engineered viral vector is conjugated with an anti-CD47 antibody generated from clone BRIC126. In some embodiments, the engineered viral vector is conjugated with the anti-HER2 antibody trastuzumab or an anti-HER2 antibody generated from clone 191924. In some embodiments, the nucleic acid sequence encodes the anti-HER2 antibody trastuzumab. In some embodiments, the nucleic acid sequence encodes the anti-nectin-4 antibody enfortumab vedotin. In some embodiments, the engineered viral vector is conjugated to the anti-EGFR antibody clone 528. In some embodiments, the engineered viral vector is conjugated to the anti-HLA antibody clone W6 / 32. In other embodiments, at least two antibodies are combined together or separately with the engineered viral vector disclosed herein.In some cases, at least two antibodies include CD47, HER2, nectin-4, HLA, or EGFR. In some embodiments, the combination of antibodies has an increasing effect on the activity of the engineered viral vector disclosed herein (in some cases, a synergistic effect).

[0084] In some embodiments, cell surface markers include pancreatic β-cell markers. In some embodiments, pancreatic β-cell markers include CD9 or ST8SA1.

[0085] Blocking the interaction between immune checkpoint proteins and immune checkpoint receptors, such as programmed death receptor 1 (PD-1) or its ligand (PD-L1), allows immune cells to kill cancer cells. In some embodiments, the target ligands of the target cells include immune checkpoint proteins or immune checkpoint receptors. In some embodiments, the target ligands of the target cells are immune checkpoint proteins. In some embodiments, immune checkpoint proteins include PD-L1, cytotoxic T-cell lymphocyte-associated protein 4 (CTLA-4), B7RP1, HVEM, CD137L, OX40L, CD40, CD70, GAL9, MHCII, CD47, VISTA, or GITR. In some embodiments, the target ligands of the target cells are immune checkpoint receptors. In some embodiments, immune checkpoint receptors include PD-1, ICOS (CD278), BTLA, CD137 (4-IBB), OX40 (CD134), CD40L, CD27, TIM3, CD20, or LAG3. In some embodiments, the target cells are immune cells. In some implementations, immune cells include T cells, B cells, macrophages, NK cells, or dendritic cells.

[0086] In some implementations, the target ligands of the target cells include cell surface antigens. Examples of cell surface antigens include, but are not limited to, cell surface proteins, such as human leukocyte antigen (HLA), cell surface glycoproteins, and transport proteins (e.g., type III sodium-dependent phosphate transporter (Pit-2)).

[0087] Combined part In some aspects of the engineered viral vectors provided herein, in some embodiments, modification is performed on the nucleic acid sequence of a viral envelope protein encoding an envelope plasmid (or envelope vector) to incorporate a binding moiety into the envelope protein of the engineered viral vector. As described herein, a binding moiety refers to a type of targeting portion modified on the envelope protein and derived from a nucleic acid sequence encoding an antibody or antigen-binding fragment. In some embodiments, once engineered viral vectors are generated, the envelope protein of these engineered viral vectors may express a binding moiety that can bind to a specific target (such as an antigen, ligand, or receptor) of a target cell, thereby increasing transduction and targeting efficiency. In some embodiments, the binding moiety is part of the envelope protein. In some embodiments, once engineered viral vectors are generated, the envelope protein of these engineered viral vectors may express a binding moiety that can bind a peptide, small molecule, compound, agent, enzyme, biomolecule, target protein, or combination thereof. In some embodiments, engineered viral vectors having a binding moiety on the envelope protein may be incubated with a peptide, small molecule, compound, agent, enzyme, biomolecule, target protein, or combination thereof prior to being used for therapeutic delivery to target cells.

[0088] In some embodiments, the binding moiety includes a conjugation site. In some embodiments, the conjugation site includes an antibody or antigen-binding fragment that can bind to a peptide, small molecule, compound, agent, enzyme, biomolecule, target protein, or a combination thereof. In some embodiments, the conjugation of the binding moiety includes nonspecific conjugation. In some embodiments, the conjugation of the binding moiety includes site-specific conjugation. In some embodiments, the conjugation of the binding moiety includes affinity conjugation, enzyme-linked conjugation, or chemical-linked conjugation.

[0089] In some embodiments, the binding portion includes an antibody or antigen-binding fragment encoded by a nucleic acid sequence inserted into a viral envelope plasmid (e.g., an SB envelope plasmid). In some embodiments, the binding portion is modified to bind to a target ligand expressed on the cell surface of a target cell. Examples of binding portions include, but are not limited to, single-stranded variable fragments (scFv), dual antibodies (DB), IgG-scFv, and single variable domains (V) on the heavy chain. HH), nanobodies, BiTE, DART, TandAb, sc biantibodies, sc biantibodies-CH3, triantibodies, microantibodies, microantibodies, TriBi microantibodies, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc biantibodies-Fc, biantibodies-Fc, tandem scFv-Fc, intracellular antibodies, their binding fragments, their chemically modified derivatives, heavy chains of variable fragments (V H ) or a light chain of variable fragments (V L In some implementations, the binding portion includes a single-chain variable fragment (scFv), a single variable domain (V) on a biantibody or heavy chain. H H), which is encoded by the nucleic acid sequence of the antibody.

[0090] In some embodiments, the binding portion comprises a nucleic acid sequence encoding its antigen-binding fragment. In some embodiments, its antigen-binding fragment binds to a target ligand on a target cell. In some embodiments, the target cell includes cancer cells. In some embodiments, the cancer cells include human cancer cells. In some embodiments, the cancer cells include leukemia cells, myeloid cells, promyeloid cells, myeloid mononuclear cells, mononuclear cells, erythroleukemia cells, chronic myeloid (granulocytic) leukemia cells, chronic lymphocytic leukemia cells, lymphoma cells such as Hodgkin's and non-Hodgkin's lymphoma cells, fibrosarcoma cells, myoma cells, liposarcoma cells, chondrosarcoma cells, osteosarcoma cells, angiosarcoma cells, endothelial sarcoma cells, Ewing's tumor cells, colon cancer cells, pancreatic cancer cells, breast cancer cells, ovarian cancer cells, prostate cancer cells, squamous cell carcinoma cells, basal cell carcinoma cells, adenocarcinoma cells, renal cell carcinoma cells, liver cancer cells, Wilms' tumor cells, etc. Tumor cells, cervical cancer cells, uterine cancer cells, testicular tumor cells, lung cancer cells, small cell lung cancer cells, bladder cancer cells, epithelial cancer cells, glioma cells, astrocytoma cells, oligodendroglioma cells, melanoma cells, neuroblastoma cells, retinoblastoma cells, dysplastic and hyperplastic cells, prostatitis cells, benign prostatic hyperplasia (BPH) cells, prostatic paraganglioma cells, prostatic adenocarcinoma cells, prostatic intraepithelial tumor cells, prostatic-rectal fistula cells, atypical prostatic stromal lesion cells, bladder cancer cells, lung cancer cells, head and neck cancer cells, non-small cell lung cancer cells, renal cancer cells, glioblastoma cells, Merkel cell carcinoma cells, angiosarcoma cells, cutaneous T-cell lymphoma cells, cutaneous B-cell lymphoma cells, cutaneous fibrosarcoma protuberans cells, sebaceous gland cancer cells, or cutaneous neuroendocrine cancer cells. In some embodiments, the target ligand includes cell surface markers. In some embodiments, the cell surface markers include cancer cell markers. In some implementations, cancer cell markers include CD47, CEA, HER2, EGFR, PSMA, TfR, nectin-4, muc-1, endothelin, EphA2, EphB2, folate receptor, GRP78, IGF-1R, cMET / HGFR, Tn antigen, EpCAM, ανβ3 integrin, CD44, mesothelin, PSCA, uPAR, CAIX, CD13, FAP-α, matriptase, MT-1 MMP, MT6-MMP, KDR, KIT, CD276, CD-83, or combinations thereof.Other cell markers include, but are not limited to, all known differentiation cluster (CD) markers (e.g., CD4, CD8, CD19, CD20, CD33, CD34, CD133), tumor antigens exposed on cell surfaces (e.g., mucin-1), cell surface molecules of the nervous system (e.g., neurotransmitter receptors, such as acetylcholine or GABA receptors), growth factor receptors (e.g., EGFR and VEGFR-2), olfactory receptors, and G protein-coupled receptors. In some embodiments, cell surface markers include pancreatic β-cell markers. In some embodiments, pancreatic β-cell markers include CD9 or ST8SA1.

[0091] In some implementations, target ligands include CD33, CD30, HER2, CD22, CD79b, nectin-4, Trop-2, BCMA, CD19, EGFR, folate receptor α, CD3, CD38, CD20, BCMA, CEA, PSMA, OX40, 4-1BB, CD16A, PD-1, PD-L1, CTLA-4, LAG-3, TIM3, VEGF, CD47, ICOS, and M. ET, LGR5, IGF-1R, HER3, ANG2, DLL4, FAP, DR5, fibronectin ED-B, tendinin C, EpCAM, TNFα, digoxin, GPIIb / IIIa, rattlesnake venom, scorpion venom, CA6, PLVAP, 5T4, EDB, EGFRvIII, 6B11, CXCR4, MSLN, FN1, GPC2, GPC3, VCAN1, COL11A1, or MMR. In some embodiments, the binding portion is encoded by a nucleic acid sequence derived from an anti-CD47 antibody, an anti-HER2 antibody, an anti-nectin-4 antibody, or an anti-EGFR antibody. In some embodiments, the nucleic acid sequence encodes the anti-CD47 antibody clone BRIC126. In some embodiments, the nucleic acid sequence encodes the anti-HER2 antibody trastuzumab or clone 191924. In some embodiments, the nucleic acid sequence encodes the anti-nectin-4 antibody vementumab. In some embodiments, the nucleic acid sequence encodes the anti-EGFR antibody clone 528. In some implementations, the nucleic acid sequence encodes the anti-HLA antibody clone W6 / 32.

[0092] In some embodiments, the target ligands of the target cells include immune checkpoint proteins or immune checkpoint receptors. In some embodiments, the target ligands of the target cells are immune checkpoint proteins. In some embodiments, immune checkpoint proteins include PD-L1, cytotoxic T-cell lymphocyte-associated protein 4 (CTLA-4), B7RP1, HVEM, CD137L, OX40L, CD40, CD70, GAL9, MHCII, CD47, VISTA, or GITR. In some embodiments, the target ligands of the target cells are immune checkpoint receptors. In some embodiments, immune checkpoint receptors include PD-1, ICOS (CD278), BTLA, CD137 (4-IBB), OX40 (CD134), CD40L, CD27, TIM3, CD20, or LAG3. In some embodiments, the target cells are immune cells. In some embodiments, immune cells include T cells, B cells, macrophages, NK cells, or dendritic cells.

[0093] In some implementations, the target ligands of the target cells include cell surface antigens. Examples of cell surface antigens include, but are not limited to, cell surface proteins (e.g., human leukocyte antigen (HLA)), cell surface glycoproteins, and transport proteins (e.g., type III sodium-dependent phosphate transporter (Pit-2)).

[0094] Single-chain variable fragments (scFv) In some embodiments, the binding portion includes an scFv. In some embodiments, the scFv is an artificial construct having a heavy and light chain of an immunoglobulin linked to a peptide linker. In some embodiments, the scFv is approximately 25 kDa compared to a 150 kDa immunoglobulin molecule. In some cases, the nucleic acid sequence encoding the scFv can be obtained by sequencing the amino acid sequences of the heavy and light chains of an antibody. The amino acid sequence information is then used to create the nucleic acid sequence encoding the scFv. In some embodiments, this nucleic acid sequence encoding the scFv is designed and generated with restriction enzyme sites flanking both ends, such that the nucleic acid sequence can be incorporated into an envelope plasmid (or envelope vector) having the same restriction enzyme sites. In some cases, the restriction enzyme sites at both ends of the nucleic acid sequence encoding the scFv are the same restriction enzyme sites. In some cases, the restriction enzyme sites at both ends of the nucleic acid sequence encoding the scFv are different restriction enzyme sites. In some embodiments, the nucleic acid sequence also includes at least one restriction enzyme site. In some embodiments, at least one restriction enzyme site is located upstream, downstream, or a combination thereof of the nucleic acid sequence within the E2 domain. In some implementations, at least one restriction enzyme site includes the BstEII site.

[0095] In some implementations, scFv is encoded by a nucleic acid sequence inserted into the E2 domain of the SB envelope plasmid, and this nucleic acid sequence encodes at least one adapter, a variable fragment heavy strand (V H ) or a light chain of variable fragments (V L In some cases, at least one adapter facilitates the folding of the structure of the modified envelope protein (e.g., a modified SB envelope protein), thereby increasing transduction efficiency. The length of at least one adapter can also affect transduction efficiency. In some embodiments, the binding portion and the envelope protein encoded by the modified envelope gene are linked by at least one peptide adapter. In various embodiments, the adapter has virtually any sequence that produces a generally flexible peptide. In some embodiments, the entire nucleic acid insert is flanked by nucleic acid sequences encoding cysteine ​​residues that can create a loop structure closed by disulfide bonds. In some embodiments, the entire nucleic acid insert is flanked by restriction enzyme sites.

[0096] In some implementations, scFv is encoded by a nucleic acid sequence inserted into the E2 domain of the SB envelope plasmid, wherein the nucleic acid sequence encodes at least one basal adapter, a variable fragment heavy strand (V H A light chain with at least one intermediate joint or variable segment (V) L In some embodiments, at least one nucleic acid sequence encoding at least one scFv is inserted into a nucleic acid sequence within the E2 domain of the SB envelope plasmid. In some embodiments, the scFv is encoded by a nucleic acid sequence within the E2 domain, wherein the nucleic acid sequence encodes a first basal adapter, V, from its 5' end to its 3' end. H Intermediate joint, V L And the second base connector. In some embodiments, the V encoding scFv H or V L The nucleic acid sequences are derived from the same antibody. In some implementations, the V encoding scFv H or V L The nucleic acid sequences are derived from different antibodies.

[0097] In some implementations, the scFv is encoded by a nucleic acid sequence derived from an antibody targeting the following: CD47, CEA, HER2, EGFR, PSMA, TfR, nectin-4, muc-1, endothelin, EphA2, EphB2, folate receptor, GRP78, IGF-1R, cMET / HGFR, Tn antigen, EpCAM, ανβ3 integrin, CD44, mesothelin, PSCA, uPAR, CAIX, CD13, FAP-α, matriptase, MT-1 MMP, MT6-MMP, KDR, KIT, CD276, CD-83, or combinations thereof. In some implementations, the scFv is encoded by a nucleic acid sequence derived from antibodies targeting the following: CD33, CD30, HER2, CD22, CD79b, nectin-4, Trop-2, BCMA, CD19, EGFR, folate receptor α, CD3, CD38, CD20, BCMA, CEA, PSMA, OX40, 4-1BB, CD16A, PD-1, PD-L1, CTLA-4, LAG-3, TIM3, and VEGF. The scFv contains the following antibodies: CD47, ICOS, MET, LGR5, IGF-1R, HER3, ANG2, DLL4, FAP, DR5, fibronectin ED-B, tendinin C, EpCAM, TNFα, digoxin, GPIIb / IIIa, rattlesnake venom, scorpion venom, CA6, PLVAP, 5T4, EDB, EGFRvIII, 6B11, CXCR4, MSLN, FN1, GPC2, GPC3, VCAN1, COL11A1, or MMR. In some embodiments, the scFv is encoded by a nucleic acid sequence derived from an anti-CD47 antibody, an anti-HER2 antibody, an anti-nectin-4 antibody, an anti-HLA antibody, or an anti-EGFR antibody. In some embodiments, the nucleic acid sequence encodes the anti-CD47 antibody clone BRIC126. In some embodiments, the nucleic acid sequence encodes the anti-HER2 antibody trastuzumab or clone 191924. In some embodiments, the nucleic acid sequence encodes the anti-nectin-4 antibody vemetramumab. In some embodiments, the nucleic acid sequence encodes anti-EGFR antibody clone 528. In some embodiments, the nucleic acid sequence encodes anti-HLA antibody clone W6 / 32.

[0098] In some cases, V H and V L The orientation of the fold can affect protein folding and stability. In some embodiments, from the N-terminus to the C-terminus of the envelope protein, V... H and V L The orientation is the same. For example, from the N-terminus to the C-terminus of the envelope protein, V... H and VL The two orientations are not opposite, or V H and V L The orientations of the two are opposite. In some implementations, from the N-terminus to the C-terminus of the envelope protein, V... H and V L Their orientations are different. V H and V L Examples of different orientations include, but are not limited to, those from the N-terminus to the C-terminus of envelope proteins, V H It's the opposite, but V L No, V L It's the opposite, but V H No. In some implementations, V H The orientation is reversed. In some implementations, V L The orientation is reversed.

[0099] In some embodiments, at least one connector includes a peptide connector. In some embodiments, at least one connector includes at least one base connector. In some embodiments, at least one connector includes at least two base connectors. In some embodiments, at least one connector includes at least one intermediate connector. In some embodiments, at least one connector includes at least two intermediate connectors. In some embodiments, at least one connector includes at least three intermediate connectors. In some embodiments, at least one connector includes at least four intermediate connectors. In some embodiments, at least one connector includes at least five intermediate connectors. In some embodiments, at least one connector includes at least six intermediate connectors.

[0100] In some embodiments, at least one joint includes at least one base joint and at least one intermediate joint. In some embodiments, at least one joint includes at least two base joints and at least one intermediate joint.

[0101] In some embodiments, at least one joint includes at least one intermediate joint and at least one base joint. In some embodiments, at least one joint includes at least two intermediate joints and at least one base joint. In some embodiments, at least one joint includes at least three intermediate joints and at least one base joint. In some embodiments, at least one joint includes at least four intermediate joints and at least one base joint. In some embodiments, at least one joint includes at least five intermediate joints and at least one base joint. In some embodiments, at least one joint includes at least six intermediate joints and at least one base joint.

[0102] In some embodiments, at least one connector includes at least one intermediate connector and at least two base connectors. In some embodiments, at least one connector includes at least two intermediate connectors and at least two base connectors. In some embodiments, at least one connector includes at least three intermediate connectors and at least two base connectors. In some embodiments, at least one connector includes at least four intermediate connectors and at least two base connectors. In some embodiments, at least one connector includes at least five intermediate connectors and at least two base connectors. In some embodiments, at least one connector includes at least six intermediate connectors and at least two base connectors.

[0103] In some embodiments, at least one adapter includes the same adapter. In some embodiments, at least one adapter includes different adapters. In some embodiments, the different adapters contain different peptide adapter sequences. In some embodiments, the different adapters include adapters of different lengths.

[0104] In some embodiments, at least one base linker comprises at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 16 amino acids, at least 17 amino acids, at least 18 amino acids, at least 19 amino acids, at least 20 amino acids, at least 21 amino acids, at least 22 amino acids, at least 23 amino acids, at least 24 amino acids, at least 25 amino acids, at least 26 amino acids, at least 27 amino acids, at least 28 amino acids, at least 29 amino acids, at least 30 amino acids, at least 31 amino acids, at least 32 amino acids, at least 33 amino acids, at least 34 amino acids, or at least 35 amino acids.

[0105] In some embodiments, at least one base linker contains up to 5 amino acids, up to 6 amino acids, up to 7 amino acids, up to 8 amino acids, up to 9 amino acids, up to 10 amino acids, up to 11 amino acids, up to 12 amino acids, up to 13 amino acids, up to 14 amino acids, up to 15 amino acids, up to 16 amino acids, up to 17 amino acids, up to 18 amino acids, up to 19 amino acids, up to 20 amino acids, up to 21 amino acids, up to 22 amino acids, up to 23 amino acids, up to 24 amino acids, up to 25 amino acids, up to 26 amino acids, up to 27 amino acids, up to 28 amino acids, up to 29 amino acids, up to 30 amino acids, up to 31 amino acids, up to 32 amino acids, up to 33 amino acids, up to 34 amino acids, or up to 35 amino acids.

[0106] In some embodiments, at least one base linker comprises 5 to 35 amino acids, 6 to 35 amino acids, 7 to 35 amino acids, 8 to 35 amino acids, 9 to 35 amino acids, 10 to 35 amino acids, 11 to 35 amino acids, 12 to 35 amino acids, 13 to 35 amino acids, 14 to 35 amino acids, 15 to 35 amino acids, 16 to 35 amino acids, 17 to 35 amino acids, 18 to 35 amino acids, 19 to 35 amino acids, 20 to 35 amino acids, 21 to 35 amino acids, 22 to 35 amino acids, 23 to 35 amino acids, 24 to 35 amino acids, 25 to 35 amino acids, 26 to 35 amino acids, 27 to 35 amino acids, 28 to 35 amino acids, 29 to 35 amino acids, 30 to 35 amino acids, 31 to 35 amino acids, 32 to 35 amino acids, 33 to 35 amino acids, or 34 to 35 amino acids. In some embodiments, at least one base connector contains between 5 and 25 amino acids, between 6 and 25 amino acids, between 7 and 25 amino acids, between 8 and 25 amino acids, between 9 and 25 amino acids, between 10 and 25 amino acids, between 11 and 25 amino acids, between 12 and 25 amino acids, between 13 and 25 amino acids, between 14 and 25 amino acids, between 15 and 25 amino acids, between 16 and 25 amino acids, between 17 and 25 amino acids, between 18 and 25 amino acids, between 19 and 25 amino acids, between 20 and 25 amino acids, between 21 and 25 amino acids, between 22 and 25 amino acids, between 23 and 25 amino acids, or between 24 and 25 amino acids.

[0107] In some embodiments, at least one base linker comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids.

[0108] Non-limiting examples of at least one adapter provided herein are shown in Table 1 (SEQ ID NO. 2-8). In some embodiments, at least one base adapter comprises GGGGSGGGGS (SEQ ID NO: 2); GSTGSGSKPGSGEGSTKG (SEQ ID NO: 3; Whitlow / 218 adapter, as described in Whitlow et al., Protein Eng, 1993, 6:989-95, which is incorporated herein by reference); ESKYGPPCPSCPAPEFLGGP (SEQ ID NO: 4; IgG4 hinge region between Fac and Fc); EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 5; hIgG1 hinge region) or AAGHVG (SEQ ID NO: 6).

[0109] In some embodiments, the intermediate linker comprises at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 16 amino acids, at least 17 amino acids, at least 18 amino acids, at least 19 amino acids, at least 20 amino acids, at least 21 amino acids, at least 22 amino acids, at least 23 amino acids, at least 24 amino acids, at least 25 amino acids, at least 26 amino acids, at least 27 amino acids, at least 28 amino acids, at least 29 amino acids, at least 30 amino acids, at least 31 amino acids, at least 32 amino acids, at least 33 amino acids, at least 34 amino acids, or at least 35 amino acids.

[0110] In some implementations, the intermediate linker contains up to 5 amino acids, up to 6 amino acids, up to 7 amino acids, up to 8 amino acids, up to 9 amino acids, up to 10 amino acids, up to 11 amino acids, up to 12 amino acids, up to 13 amino acids, up to 14 amino acids, up to 15 amino acids, up to 16 amino acids, up to 17 amino acids, up to 18 amino acids, up to 19 amino acids, up to 20 amino acids, up to 21 amino acids, up to 22 amino acids, up to 23 amino acids, up to 24 amino acids, up to 25 amino acids, up to 26 amino acids, up to 27 amino acids, up to 28 amino acids, up to 29 amino acids, up to 30 amino acids, up to 31 amino acids, up to 32 amino acids, up to 33 amino acids, up to 34 amino acids, or up to 35 amino acids.

[0111] In some embodiments, the intermediate linker comprises 5 to 35 amino acids, 6 to 35 amino acids, 7 to 35 amino acids, 8 to 35 amino acids, 9 to 35 amino acids, 10 to 35 amino acids, 11 to 35 amino acids, 12 to 35 amino acids, 13 to 35 amino acids, 14 to 35 amino acids, 15 to 35 amino acids, 16 to 35 amino acids, 17 to 35 amino acids, 18 to 35 amino acids, 19 to 35 amino acids, 20 to 35 amino acids, 21 to 35 amino acids, 22 to 35 amino acids, 23 to 35 amino acids, 24 to 35 amino acids, 25 to 35 amino acids, 26 to 35 amino acids, 27 to 35 amino acids, 28 to 35 amino acids, 29 to 35 amino acids, 30 to 35 amino acids, 31 to 35 amino acids, 32 to 35 amino acids, 33 to 35 amino acids, or 34 to 35 amino acids. In some embodiments, at least one base connector contains between 5 and 25 amino acids, between 6 and 25 amino acids, between 7 and 25 amino acids, between 8 and 25 amino acids, between 9 and 25 amino acids, between 10 and 25 amino acids, between 11 and 25 amino acids, between 12 and 25 amino acids, between 13 and 25 amino acids, between 14 and 25 amino acids, between 15 and 25 amino acids, between 16 and 25 amino acids, between 17 and 25 amino acids, between 18 and 25 amino acids, between 19 and 25 amino acids, between 20 and 25 amino acids, between 21 and 25 amino acids, between 22 and 25 amino acids, between 23 and 25 amino acids, or between 24 and 25 amino acids.

[0112] In some implementations, the intermediate linker comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids.

[0113] In some embodiments, the intermediate linker comprises a 14-amino acid linker or an 18-amino acid linker. In some embodiments, the intermediate linker comprises GGGGSGGGGSGGGG (SEQ ID NO: 7) or SSGGGGSGGGGGGGSSRSS (SEQ ID NO: 8).

[0114] In some embodiments, the base linker comprises the amino acid sequence of any one of SEQ ID NO: 2-6. In some embodiments, the intermediate linker comprises the amino acid sequence of any one of SEQ ID NO: 7-8. In some embodiments, the base linker comprises the amino acid sequence of SEQ ID NO: 2 and the intermediate linker comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the base linker comprises the amino acid sequence of SEQ ID NO: 2 and the intermediate linker comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the base linker comprises the amino acid sequence of SEQ ID NO: 3 and the intermediate linker comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the base linker comprises the amino acid sequence of SEQ ID NO: 3 and the intermediate linker comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the base linker comprises the amino acid sequence of SEQ ID NO: 4 and the intermediate linker comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the base linker comprises the amino acid sequence of SEQ ID NO: 4 and the intermediate linker comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the base linker comprises the amino acid sequence of SEQ ID NO: 5 and the intermediate linker comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the base linker comprises the amino acid sequence of SEQ ID NO: 5 and the intermediate linker comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the base linker comprises the amino acid sequence of SEQ ID NO: 6 and the intermediate linker comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the base linker comprises the amino acid sequence of SEQ ID NO: 6 and the intermediate linker comprises the amino acid sequence of SEQ ID NO: 8.

[0115] In some embodiments, the amino acid sequence of the SB envelope plasmid includes the amino acid sequence of any one of SEQ ID NO: 9-15.

[0116] Double antibody In some implementations, the binding portion includes a biantibody. The biantibody (DB) is a non-covalent dimer of scFv; therefore, the size of DB is approximately 55 kDa, which is about twice the size of scFv (approximately 25 kDa). Within DB, each V H The structural domain is connected to a V via a short connector. LThe domains are linked to form two antigen-binding sites, which provides a higher binding affinity for the antigen within its structure. Therefore, incorporating DB into the viral envelope protein can provide better binding to target cells, thereby performing more efficient vector transduction.

[0117] In some embodiments, the biantibody comprises a dimer of a single-chain variable fragment (scFv). In some embodiments, the biantibody is encoded by a nucleic acid sequence inserted into the E2 domain of the SB envelope plasmid, and this nucleic acid sequence encodes at least one adapter, at least one heavy chain (V) of the variable fragment. H ) or at least one light chain of a variable fragment (V L In some embodiments, at least one adapter includes at least one base adapter, at least one intermediate adapter, or a combination thereof. In some embodiments, the dual antibody is encoded by a nucleic acid sequence in the E2 domain, wherein the nucleic acid sequence encodes at least one base adapter and at least one V... H At least one intermediate joint or at least one V L In some embodiments, the dual antibody is encoded by a nucleic acid sequence inserted into the E2 domain of the SB envelope plasmid, wherein the nucleic acid sequence, from the 5' end to the 3' end, encodes a first basal adapter and a first V... H First intermediate joint, first V L Second intermediate joint, second V H Third intermediate joint, second V L And a second basal linker. In some cases, the nucleic acid sequence encoding DB can be obtained by sequencing the amino acid sequences of the heavy and light chains of the antibody. The nucleic acid sequence encoding DB is then assembled using the information from the amino acid sequences. This nucleic acid sequence encoding DB is then designed and generated with restriction enzyme sites flanking both ends, so that the nucleic acid sequence can be incorporated into an enveloped plasmid or enveloped vector having the same restriction enzyme sites. In some cases, the restriction enzyme sites at both ends of the nucleic acid sequence encoding DB are the same restriction enzyme sites. In some cases, the restriction enzyme sites at both ends of the nucleic acid sequence encoding DB are different restriction enzyme sites. In some embodiments, the nucleic acid sequence further includes at least one restriction enzyme site. In some embodiments, at least one restriction enzyme site is located upstream, downstream, or a combination thereof of the nucleic acid sequence in the E2 domain. In some embodiments, at least one restriction enzyme site includes a BstEII site.

[0118] In some cases, at least one V of the biantibody HThis constitutes a chimeric scFv. Such a chimeric scFv can be derived from the amino acid sequences of antibodies generated from different clones. In some embodiments, the dimer of the biantibody scFv comprises a chimeric scFv. In some embodiments, the dimer of the biantibody scFv is derived from antibodies generated from different clones. In some embodiments, the dimer of the biantibody scFv is derived from the same antibody. In some embodiments, the dimer of the biantibody scFv is derived from different antibodies. In some embodiments, at least one V of the biantibody... H Including chimeric V H In some implementations, at least one V of the biantibody H Antibodies derived from different clones. In some embodiments, at least one V of the biantibody. H Derived from the same antibody. In some implementations, at least one V of the biantibody... H Derived from different antibodies. In some implementations, at least one V of the dual antibody L Including chimeric V L In some implementations, at least one V of the biantibody L Antibodies derived from different clones. In some embodiments, at least one V of the biantibody. L Derived from the same antibody. In some implementations, at least one V of the biantibody... L They originate from different antibodies.

[0119] In some implementations, the dual antibody is encoded by a nucleic acid sequence derived from an antibody targeting the following: CD47, CEA, HER2, EGFR, PSMA, TfR, nectin-4, muc-1, endothelin, EphA2, EphB2, folate receptor, GRP78, IGF-1R, cMET / HGFR, Tn antigen, EpCAM, ανβ3 integrin, CD44, mesothelin, PSCA, uPAR, CAIX, CD13, FAP-α, matriptase, MT-1 MMP, MT6-MMP, KDR, KIT, CD276, CD-83, or combinations thereof. In some implementations, the dual antibody is encoded by a nucleic acid sequence derived from antibodies targeting the following: CD33, CD30, HER2, CD22, CD79b, nectin-4, Trop-2, BCMA, CD19, EGFR, folate receptor α, CD3, CD38, CD20, BCMA, CEA, PSMA, OX40, 4-1BB, CD16A, PD-1, PD-L1, CTLA-4, LAG-3, TIM3, VEGF, CD47, ICOS, MET, LGR5, IGF-1R, HER3, ANG2, DLL4, FAP, DR5, fibronectin ED-B, tendinin C, EpCAM, TNFα, digoxin, GPIIb / IIIa, rattlesnake venom, scorpion venom, CA6, PLVAP, 5T4, EDB, EGFRvIII, 6B11, CXCR4, MSLN, FN1, GPC2, GPC3, VCAN1, COL11A1, or MMR. In some embodiments, the biantibody is encoded by a nucleic acid sequence derived from an anti-CD47 antibody, an anti-HER2 antibody, an anti-nectin-4 antibody, an anti-HLA antibody, or an anti-EGFR antibody. In some embodiments, the nucleic acid sequence encodes the anti-CD47 antibody clone BRIC126. In some embodiments, the nucleic acid sequence encodes the anti-HER2 antibody trastuzumab or clone 191924. In some embodiments, the nucleic acid sequence encodes the anti-nectin-4 antibody vementumab. In some embodiments, the nucleic acid sequence encodes anti-EGFR antibody clone 528. In some embodiments, the nucleic acid sequence encodes anti-HLA antibody clone W6 / 32.

[0120] In some cases, at least one V H and at least one V L The orientation of the membrane can affect protein folding and stability. In some embodiments, at least one V-shaped fold from the N-terminus to the C-terminus of the envelope protein... H and at least one V L The orientations are the same. For example, from the N-terminus to the C-terminus of the envelope protein, at least one V...H and at least one V L The orientation is not reversed, or at least V H and at least one V L The orientations are all reversed. In some embodiments, at least one V-shaped segment extends from the N-terminus to the C-terminus of the envelope protein. H and at least one V L The orientations are different. At least one V H and at least one V L Examples of different orientations include, but are not limited to, from the N-terminus to the C-terminus of the envelope protein, the first V H It's the opposite, but the first V L No, at the same time, the second V H Second V L Not the reverse, or the first V H Not the reverse but the first V L It's the opposite, and at the same time, the second V H Second V L Not the reverse, or any combination thereof. In some implementations, at least one V H The orientation is reversed. In some implementations, at least one V L The orientation is reversed. In some implementations, at least one V H The orientation is opposite, but at least one V L Not the reverse. In some implementations, at least one V L The orientation is opposite, but at least one V H It's not the other way around.

[0121] In some cases, at least one linker helps fold the structure of the modified envelope protein, thereby increasing transduction efficiency. The length of at least one linker can also affect transduction efficiency. In some embodiments, the binding portion (e.g., DB) and the envelope protein encoded by the modified envelope gene are linked by at least one peptide linker. In various embodiments, the linker has virtually any sequence that produces a generally flexible peptide. In some embodiments, the entire nucleic acid insert is flanked by nucleic acid sequences encoding cysteine ​​residues that can create a loop structure closed by disulfide bonds. In some embodiments, the entire nucleic acid insert is flanked by restriction enzyme sites.

[0122] In some embodiments, at least one connector includes a peptide connector. In some embodiments, at least one connector includes at least one base connector. In some embodiments, at least one connector includes at least two base connectors. In some embodiments, at least one connector includes at least one intermediate connector. In some embodiments, a connector includes at least two intermediate connectors. In some embodiments, at least one connector includes at least three intermediate connectors. In some embodiments, at least one connector includes at least four intermediate connectors. In some embodiments, at least one connector includes at least five intermediate connectors. In some embodiments, at least one connector includes at least six intermediate connectors.

[0123] In some embodiments, at least one connector includes at least one base connector and at least one intermediate connector. In some embodiments, at least one connector includes at least two base connectors and at least one intermediate connector. In some embodiments, at least one connector includes at least two base connectors and at least two intermediate connectors. In some embodiments, at least one connector includes at least two base connectors and at least two intermediate connectors. In some embodiments, at least one connector includes at least two base connectors and at least three intermediate connectors. In some embodiments, at least one connector includes at least two base connectors and at least four intermediate connectors. In some embodiments, at least one connector includes at least two base connectors and at least five intermediate connectors. In some embodiments, at least one connector includes at least two base connectors and at least six intermediate connectors.

[0124] In some embodiments, at least one joint includes at least one intermediate joint and at least one base joint. In some embodiments, at least one joint includes at least two intermediate joints and at least one base joint. In some embodiments, at least one joint includes at least three intermediate joints and at least one base joint. In some embodiments, at least one joint includes at least four intermediate joints and at least one base joint. In some embodiments, at least one joint includes at least five intermediate joints and at least one base joint. In some embodiments, at least one joint includes at least six intermediate joints and at least one base joint.

[0125] In some embodiments, at least one connector includes at least one intermediate connector and at least two base connectors. In some embodiments, at least one connector includes at least two intermediate connectors and at least two base connectors. In some embodiments, at least one connector includes at least three intermediate connectors and at least two base connectors. In some embodiments, at least one connector includes at least four intermediate connectors and at least two base connectors. In some embodiments, at least one connector includes at least five intermediate connectors and at least two base connectors. In some embodiments, at least one connector includes at least six intermediate connectors and at least two base connectors.

[0126] In some embodiments, at least one adapter includes the same adapter. In some embodiments, at least one adapter includes different adapters. In some embodiments, the different adapters contain different peptide adapter sequences. In some embodiments, the different adapters include adapters of different lengths.

[0127] In some embodiments, at least one base linker comprises at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 16 amino acids, at least 17 amino acids, at least 18 amino acids, at least 19 amino acids, at least 20 amino acids, at least 21 amino acids, at least 22 amino acids, at least 23 amino acids, at least 24 amino acids, at least 25 amino acids, at least 26 amino acids, at least 27 amino acids, at least 28 amino acids, at least 29 amino acids, at least 30 amino acids, at least 31 amino acids, at least 32 amino acids, at least 33 amino acids, at least 34 amino acids, or at least 35 amino acids.

[0128] In some embodiments, at least one base linker contains up to 5 amino acids, up to 6 amino acids, up to 7 amino acids, up to 8 amino acids, up to 9 amino acids, up to 10 amino acids, up to 11 amino acids, up to 12 amino acids, up to 13 amino acids, up to 14 amino acids, up to 15 amino acids, up to 16 amino acids, up to 17 amino acids, up to 18 amino acids, up to 19 amino acids, up to 20 amino acids, up to 21 amino acids, up to 22 amino acids, up to 23 amino acids, up to 24 amino acids, up to 25 amino acids, up to 26 amino acids, up to 27 amino acids, up to 28 amino acids, up to 29 amino acids, up to 30 amino acids, up to 31 amino acids, up to 32 amino acids, up to 33 amino acids, up to 34 amino acids, or up to 35 amino acids.

[0129] In some embodiments, at least one base linker comprises 5 to 35 amino acids, 6 to 35 amino acids, 7 to 35 amino acids, 8 to 35 amino acids, 9 to 35 amino acids, 10 to 35 amino acids, 11 to 35 amino acids, 12 to 35 amino acids, 13 to 35 amino acids, 14 to 35 amino acids, 15 to 35 amino acids, 16 to 35 amino acids, 17 to 35 amino acids, 18 to 35 amino acids, 19 to 35 amino acids, 20 to 35 amino acids, 21 to 35 amino acids, 22 to 35 amino acids, 23 to 35 amino acids, 24 to 35 amino acids, 25 to 35 amino acids, 26 to 35 amino acids, 27 to 35 amino acids, 28 to 35 amino acids, 29 to 35 amino acids, 30 to 35 amino acids, 31 to 35 amino acids, 32 to 35 amino acids, 33 to 35 amino acids, or 34 to 35 amino acids. In some embodiments, at least one base connector contains between 5 and 25 amino acids, between 6 and 25 amino acids, between 7 and 25 amino acids, between 8 and 25 amino acids, between 9 and 25 amino acids, between 10 and 25 amino acids, between 11 and 25 amino acids, between 12 and 25 amino acids, between 13 and 25 amino acids, between 14 and 25 amino acids, between 15 and 25 amino acids, between 16 and 25 amino acids, between 17 and 25 amino acids, between 18 and 25 amino acids, between 19 and 25 amino acids, between 20 and 25 amino acids, between 21 and 25 amino acids, between 22 and 25 amino acids, between 23 and 25 amino acids, or between 24 and 25 amino acids.

[0130] In some embodiments, at least one base linker comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids.

[0131] In some embodiments, the intermediate linker comprises at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 16 amino acids, at least 17 amino acids, at least 18 amino acids, at least 19 amino acids, at least 20 amino acids, at least 21 amino acids, at least 22 amino acids, at least 23 amino acids, at least 24 amino acids, at least 25 amino acids, at least 26 amino acids, at least 27 amino acids, at least 28 amino acids, at least 29 amino acids, at least 30 amino acids, at least 31 amino acids, at least 32 amino acids, at least 33 amino acids, at least 34 amino acids, or at least 35 amino acids.

[0132] In some implementations, the intermediate linker contains up to 5 amino acids, up to 6 amino acids, up to 7 amino acids, up to 8 amino acids, up to 9 amino acids, up to 10 amino acids, up to 11 amino acids, up to 12 amino acids, up to 13 amino acids, up to 14 amino acids, up to 15 amino acids, up to 16 amino acids, up to 17 amino acids, up to 18 amino acids, up to 19 amino acids, up to 20 amino acids, up to 21 amino acids, up to 22 amino acids, up to 23 amino acids, up to 24 amino acids, up to 25 amino acids, up to 26 amino acids, up to 27 amino acids, up to 28 amino acids, up to 29 amino acids, up to 30 amino acids, up to 31 amino acids, up to 32 amino acids, up to 33 amino acids, up to 34 amino acids, or up to 35 amino acids.

[0133] In some embodiments, the intermediate linker comprises 5 to 35 amino acids, 6 to 35 amino acids, 7 to 35 amino acids, 8 to 35 amino acids, 9 to 35 amino acids, 10 to 35 amino acids, 11 to 35 amino acids, 12 to 35 amino acids, 13 to 35 amino acids, 14 to 35 amino acids, 15 to 35 amino acids, 16 to 35 amino acids, 17 to 35 amino acids, 18 to 35 amino acids, 19 to 35 amino acids, 20 to 35 amino acids, 21 to 35 amino acids, 22 to 35 amino acids, 23 to 35 amino acids, 24 to 35 amino acids, 25 to 35 amino acids, 26 to 35 amino acids, 27 to 35 amino acids, 28 to 35 amino acids, 29 to 35 amino acids, 30 to 35 amino acids, 31 to 35 amino acids, 32 to 35 amino acids, 33 to 35 amino acids, or 34 to 35 amino acids. In some embodiments, at least one base connector contains between 5 and 25 amino acids, between 6 and 25 amino acids, between 7 and 25 amino acids, between 8 and 25 amino acids, between 9 and 25 amino acids, between 10 and 25 amino acids, between 11 and 25 amino acids, between 12 and 25 amino acids, between 13 and 25 amino acids, between 14 and 25 amino acids, between 15 and 25 amino acids, between 16 and 25 amino acids, between 17 and 25 amino acids, between 18 and 25 amino acids, between 19 and 25 amino acids, between 20 and 25 amino acids, between 21 and 25 amino acids, between 22 and 25 amino acids, between 23 and 25 amino acids, or between 24 and 25 amino acids.

[0134] In some implementations, the intermediate linker comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids.

[0135] In some embodiments, the first intermediate linker of the biantibody comprises an amino acid sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids. In some embodiments, the first intermediate linker of the biantibody comprises a 15-amino acid linker. In some embodiments, the first intermediate linker of the biantibody comprises the amino acid sequence SSSSGSSSSGSSSSG (SEQ ID NO: 16).

[0136] In some embodiments, the second intermediate linker of the biantibody comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids. In some embodiments, the second intermediate linker of the biantibody comprises an 18-amino acid linker. In some embodiments, the second intermediate linker of the biantibody comprises the amino acid sequence SSGGGGSGGGGGGSSRSS (SEQ ID NO: 8).

[0137] Single variable structural domain (V) on heavy chain H H） V H H is an antigen-binding fragment or variable domain of the heavy chain containing the antibody. In some embodiments, the binding portion includes V. H H. In some cases, V H H is an antibody obtained from the Camelidae family. Examples of Camelidae include, but are not limited to, camels, llamas, alpacas, llamas, and guanacos.

[0138] In some implementations, the encoding V H The nucleic acid sequence of H can be obtained by sequencing the amino acid sequence of the heavy chain of an antibody obtained from the Camelidae family. Then, the information from the amino acid sequence is used to create the code for V. HThe nucleic acid sequence of H. In some implementations, this encodes V. H The nucleic acid sequence of H was designed and generated with restriction enzyme sites flanking both ends, allowing the sequence to be incorporated into enveloped plasmids (or enveloped vectors) containing the same restriction enzyme sites. In some cases, in encoding V... H The restriction enzyme sites at both ends of the H nucleic acid sequence are the same restriction enzyme sites. In some cases, in the encoding V... H The restriction enzyme sites at both ends of the H nucleic acid sequence are different restriction enzyme sites. In some embodiments, the nucleic acid sequence further includes at least one restriction enzyme site. In some embodiments, at least one restriction enzyme site is located upstream, downstream, or a combination thereof of the nucleic acid sequence within the E2 domain. In some embodiments, at least one restriction enzyme site includes a BstEII site.

[0139] In some implementation schemes, V H H is encoded by a nucleic acid sequence in the E2 domain of the inserted SB envelope plasmid, and this nucleic acid sequence encodes at least one adapter or a single variable domain on the heavy chain. In some cases, at least one adapter helps fold the structure of the modified envelope protein (e.g., a modified SB envelope protein), thereby increasing transduction efficiency. The length of at least one adapter can also affect transduction efficiency. In some embodiments, the binding portion and the envelope protein encoded by the modified envelope gene are linked by at least one peptide adapter. In various embodiments, the adapter has virtually any sequence that produces a generally flexible peptide. In some embodiments, the entire nucleic acid insert is flanked by nucleic acid sequences encoding cysteine ​​residues that can create a loop structure closed by disulfide bonds. In some embodiments, the entire nucleic acid insert is flanked by restriction enzyme sites.

[0140] In some implementation schemes, V H H is encoded by a nucleic acid sequence inserted into the E2 domain of the SB envelope plasmid, wherein the nucleic acid sequence encodes at least one single variable domain on the basal linker or heavy strand. In some embodiments, at least one V will be encoded. H At least one nucleic acid sequence of H is inserted into the nucleic acid sequence within the E2 domain of the SB envelope plasmid. In some embodiments, V H H is encoded by a nucleic acid sequence in the E2 domain, which encodes the first basal adapter (adapter 1) from the 5' end to the 3' end of the nucleic acid sequence. H H and the second base connector (connector 2). In some embodiments, V H H includes the following formula: Connector 1-V HH-Connector 2. In some embodiments, connector 1 is selected from (GGGGS)nAAGHVG, AAGHVG(GGGGS)n, (GGGGS)nAAGHVG(GGGGS)n, (GGGGS)nGVHGAA, GVHGAA(GGGGS)n, and (GGGGS)nGVHGAA(GGGGS)n, where n equals 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, connector 2 is selected from (GGGGS)nAAGHVG, AAGHVG(GGGGS)n, (GGGGS)nAAGHVG(GGGGS)n, (GGGGS)nGVHGAA, GVHGAA(GGGGS)n, and (GGGGS)nGVHGAA(GGGGS)n, where n equals 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some implementation schemes, V H H includes any of the following formulas: GGGGS-AAGHVG-V H H-GVHGAA, AAGHVG-V H H-GVHGAA-GGGGS, GGGGS-AAGHVG-V H H-GVHGAA-GGGGS, GGGGS-GGGGS-AAGHVG-V H H-GVHGAA, AAGHVG-V H H-GVHGAA-GGGGS-GGGGS, GGGGS-GGGGS-AAGHVG-V H H-GVHGAA-GGGGS-GGGGS.

[0141] In some implementation schemes, V H H is encoded by a nucleic acid sequence derived from an antibody obtained from the Camelidae family that targets the following: CD47, CEA, HER2, EGFR, PSMA, TfR, nectin-4, muc-1, endothelin, EphA2, EphB2, folate receptor, GRP78, IGF-1R, cMET / HGFR, Tn antigen, EpCAM, ανβ3 integrin, CD44, mesothelin, PSCA, uPAR, CAIX, CD13, FAP-α, matriptase, MT-1 MMP, MT6-MMP, KDR, KIT, CD276, CD-83, or combinations thereof. In some embodiments, V HH is encoded by a nucleic acid sequence derived from antibodies obtained from camelids that target the following: CD33, CD30, HER2, CD22, CD79b, nectin-4, Trop-2, BCMA, CD19, EGFR, folate receptor α, CD3, CD38, CD20, BCMA, CEA, PSMA, OX40, 4-1BB, CD16A, PD-1, PD-L1, CTLA-4, LAG-3, TIM3, VEGF, CD 47. ICOS, MET, LGR5, IGF-1R, HER3, ANG2, DLL4, FAP, DR5, fibronectin ED-B, tendinin C, EpCAM, TNFα, digoxin, GPIIb / IIIa, rattlesnake venom, scorpion venom, CA6, PLVAP, 5T4, EDB, EGFRvIII, 6B11, CXCR4, MSLN, FN1, GPC2, GPC3, VCAN1, COL11A1, or MMR. In some embodiments, V H H is encoded by a nucleic acid sequence derived from an anti-CD47 antibody, anti-HER2 antibody, anti-nectin-4 antibody, anti-HLA antibody, or anti-EGFR antibody obtained from the Camelidae family.

[0142] In some embodiments, at least one adapter includes a peptide adapter. In some embodiments, at least one adapter includes at least one base adapter. In some embodiments, at least one adapter includes at least two base adapters.

[0143] In some embodiments, at least one adapter includes the same adapter. In some embodiments, at least one adapter includes different adapters. In some embodiments, the different adapters contain different peptide adapter sequences. In some embodiments, the different adapters include adapters of different lengths.

[0144] In some embodiments, at least one base linker comprises at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 16 amino acids, at least 17 amino acids, at least 18 amino acids, at least 19 amino acids, at least 20 amino acids, at least 21 amino acids, at least 22 amino acids, at least 23 amino acids, at least 24 amino acids, at least 25 amino acids, at least 26 amino acids, at least 27 amino acids, at least 28 amino acids, at least 29 amino acids, at least 30 amino acids, at least 31 amino acids, at least 32 amino acids, at least 33 amino acids, at least 34 amino acids, or at least 35 amino acids.

[0145] In some embodiments, at least one base linker contains up to 5 amino acids, up to 6 amino acids, up to 7 amino acids, up to 8 amino acids, up to 9 amino acids, up to 10 amino acids, up to 11 amino acids, up to 12 amino acids, up to 13 amino acids, up to 14 amino acids, up to 15 amino acids, up to 16 amino acids, up to 17 amino acids, up to 18 amino acids, up to 19 amino acids, up to 20 amino acids, up to 21 amino acids, up to 22 amino acids, up to 23 amino acids, up to 24 amino acids, up to 25 amino acids, up to 26 amino acids, up to 27 amino acids, up to 28 amino acids, up to 29 amino acids, up to 30 amino acids, up to 31 amino acids, up to 32 amino acids, up to 33 amino acids, up to 34 amino acids, or up to 35 amino acids.

[0146] In some embodiments, at least one base linker comprises 5 to 35 amino acids, 6 to 35 amino acids, 7 to 35 amino acids, 8 to 35 amino acids, 9 to 35 amino acids, 10 to 35 amino acids, 11 to 35 amino acids, 12 to 35 amino acids, 13 to 35 amino acids, 14 to 35 amino acids, 15 to 35 amino acids, 16 to 35 amino acids, 17 to 35 amino acids, 18 to 35 amino acids, 19 to 35 amino acids, 20 to 35 amino acids, 21 to 35 amino acids, 22 to 35 amino acids, 23 to 35 amino acids, 24 to 35 amino acids, 25 to 35 amino acids, 26 to 35 amino acids, 27 to 35 amino acids, 28 to 35 amino acids, 29 to 35 amino acids, 30 to 35 amino acids, 31 to 35 amino acids, 32 to 35 amino acids, 33 to 35 amino acids, or 34 to 35 amino acids. In some embodiments, at least one base connector contains between 5 and 25 amino acids, between 6 and 25 amino acids, between 7 and 25 amino acids, between 8 and 25 amino acids, between 9 and 25 amino acids, between 10 and 25 amino acids, between 11 and 25 amino acids, between 12 and 25 amino acids, between 13 and 25 amino acids, between 14 and 25 amino acids, between 15 and 25 amino acids, between 16 and 25 amino acids, between 17 and 25 amino acids, between 18 and 25 amino acids, between 19 and 25 amino acids, between 20 and 25 amino acids, between 21 and 25 amino acids, between 22 and 25 amino acids, between 23 and 25 amino acids, or between 24 and 25 amino acids.

[0147] In some embodiments, at least one base linker comprises 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids.

[0148] Non-limiting examples of at least one adapter provided herein are shown in Table 1 (SEQ ID NO. 2-8). In some embodiments, at least one base adapter comprises GGGGSGGGGS (SEQ ID NO: 2); GSTGSGSKPGSGEGSTKG (SEQ ID NO: 3; Whitlow / 218 adapter, as described in Whitlow et al., Protein Eng, 1993, 6:989-95, which is incorporated herein by reference); ESKYGPPCPSCPAPEFLGGP (SEQ ID NO: 4; IgG4 hinge region between Fac and Fc); EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 5; hIgG1 hinge region) or AAGHVG (SEQ ID NO: 6). In some embodiments, at least one base adapter comprises the amino acid sequence of any one of SEQ ID NO: 2-6.

[0149] The binding site of the protease cleavage site In some cases, the encoding binding portion (such as scFv, biantibody, or V) H The nucleic acid sequence of H) may contain at least one protease cleavage site. At least one protease cleavage site may interfere with the formation of a functional binding moiety on the envelope protein of an engineered viral vector. Examples of at least one protease cleavage site include, but are not limited to, furin protease cleavage sites.

[0150] Frin proteases are proteases that cleave specific peptide sequences and contribute to the activation of many protein processing and signaling molecules. Frin proteases are known to play a crucial role in viral envelope processing because they are involved in the maturation of functional viral envelope proteins. In some cases, protease cleavage sites located at binding sites can be predicted using online tools such as the ProP-1.0 website, which predicts arginine and lysine propeptide cleavage sites in eukaryotic protein sequences. Once a potential protease cleavage site (e.g., a furin protease cleavage site) is predicted, amino acid sequence modifications at that location can be performed to remove the protease cleavage site.

[0151] In some embodiments, the binding moiety further comprises at least one modification relative to the sequence of the wild-type binding moiety. In some embodiments, the at least one modification includes an amino acid substitution, insertion, or deletion, or a combination thereof, that removes a protease cleavage site from the binding moiety. In some embodiments, the protease cleavage site is a furin protease cleavage site. In some embodiments, at least one modification that removes a protease cleavage site (e.g., a furin protease cleavage site) includes an amino acid substitution. In some embodiments, at least one modification includes an amino acid substitution from lysine to histidine. In some embodiments, at least one modification includes an amino acid substitution from lysine to arginine.

[0152] ligands of cell surface receptors In various aspects of the engineered viral vectors provided herein, in some embodiments, the targeting portion of the envelope protein is modified to include a ligand capable of binding to a specific cell surface receptor of a target cell. As described herein, in some embodiments, at least one targeting portion includes a nucleic acid sequence encoding a ligand capable of binding to a specific cell surface receptor of a target cell, and this strategy can be used to initiate transduction of the engineered viral vector.

[0153] In some embodiments, the ligand is encoded by a nucleic acid sequence in the E2 domain of the inserted envelope plasmid. In some embodiments, the targeting portion comprises a nucleic acid sequence encoding its antigen-binding fragment. In some embodiments, its antigen-binding fragment comprises a receptor ligand. In some embodiments, the receptor ligand binds to a target receptor on a target cell. Examples of such receptor ligands include, but are not limited to, G protein-coupled receptor ligands (such as endorphins and oxytocin), ligand peptides of G protein-coupled receptor 78 (GPR78) (Mandelin et al., PNAS, 2015, 112:3776-81), epidermal growth factor receptor (EGFR) (Hossein-Nejad-Ariani et al., Sci Rep, 2019, 9:2723), and small peptide ligands of the programmed cell death protein 1 (PD-1) receptor, as well as the C-terminal domain of the platelet-reactive protein of CD47 (Gao et al., J Biol Chem, 1996, 271:21-4). In some embodiments, the ligand comprises a G protein-coupled receptor ligand, a small peptide ligand, or the C-terminal domain of a thromboretin. In some embodiments, the G protein-coupled receptor ligand comprises endorphin or oxytocin. In some embodiments, the small peptide ligand comprises a ligand of GPR78, EGFR, programmed cell death protein 1 (PD-1) receptor, or CD47 receptor. In some embodiments, the ligand of the PD-1 receptor comprises programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), or a combination thereof. In other embodiments, at least two receptor ligands are combined together or separately with the engineered viral vector disclosed herein. In some cases, at least two receptor ligands comprise GPR78, HER2, PD-1, or CD47 receptors. In some embodiments, the combination of receptor ligands has an increasing effect on the activity of the engineered viral vector disclosed herein (in some cases, a synergistic effect).

[0154] In some embodiments, the small peptide ligand is epidermal growth factor (EGF) or a subunit of EGF. In some embodiments, the EGF subunit is the EGF core domain. In some embodiments, the EGF core domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 24. In some embodiments, the EGF core domain comprises SEQ ID NO: 24. In some embodiments, the targeting portion comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 25 or 26. In some embodiments, the targeting portion comprises SEQ ID NO: 25 or 26. In some embodiments, the cell surface receptor is EGFR or an EGFR mutant. In some embodiments, the EGFR mutant comprises a deletion of EGFR. In some embodiments, the EGFR mutant comprises a deletion of domain I (L1) and domain II (CRI) of EGFR. In some embodiments, the EGFR mutant comprises a deletion of amino acids 6-273 of EGFR. In some embodiments, the EGFR mutant is a constitutively active mutant. In some embodiments, the EGFR mutant comprises amino acid substitutions. In some embodiments, the EGFR mutant comprises at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, or at least twenty substitutions. In some embodiments, the EGFR mutant comprises insertions. In some embodiments, the EGFR mutant comprises at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, or at least twenty insertions.

[0155] In some implementations, the target cells are cancer cells. In some implementations, cancer cells include human cancer cells. In some implementations, cancer cells include leukemia cells, myeloid cells, promyeloid cells, myeloid mononuclear cells, mononuclear cells, erythroleukemia cells, chronic myeloid (granulocytic) leukemia cells, chronic lymphocytic leukemia cells, lymphoma cells such as Hodgkin's and non-Hodgkin's lymphoma cells, fibrosarcoma cells, myoma cells, liposarcoma cells, chondrosarcoma cells, osteosarcoma cells, angiosarcoma cells, endothelial sarcoma cells, Ewing's tumor cells, colon cancer cells, pancreatic cancer cells, breast cancer cells, ovarian cancer cells, prostate cancer cells, squamous cell carcinoma cells, basal cell carcinoma cells, adenocarcinoma cells, renal cell carcinoma cells, liver cancer cells, Wilms' tumor cells, cervical cancer cells, uterine cancer cells, testicular tumor cells, lung cancer cells, small cell lung cancer cells, and bladder cancer cells. Carcinoma cells, epithelial cancer cells, glioma cells, astrocytoma cells, oligodendroglioma cells, melanoma cells, neuroblastoma cells, retinoblastoma cells, dysplastic and hyperplastic cells, prostatitis cells, benign prostatic hyperplasia (BPH) cells, prostatic paraganglioma cells, prostatic adenocarcinoma cells, prostatic intraepithelial tumor cells, prostatic-rectal fistula cells, atypical prostatic stromal lesion cells, bladder cancer cells, lung cancer cells, head and neck cancer cells, non-small cell lung cancer cells, renal cancer cells, glioblastoma cells, Merkel cell carcinoma cells, angiosarcoma cells, cutaneous T-cell lymphoma cells, cutaneous B-cell lymphoma cells, dermatofibrosarcoma protuberans cells, sebaceous gland cancer cells, or cutaneous neuroendocrine cancer cells.

[0156] Immunogenic proteins In various aspects of the engineered viral vectors provided herein, in some embodiments, at least one targeting portion includes an antigenic protein, such as an immunogenic protein or immunogen. In some cases, the immunogen is encoded by a heterologous nucleic acid sequence inserted into the envelope plasmid. As described herein, engineered viral vectors generated from modified SB envelope plasmids that also express immunogens on envelope proteins can stimulate an immune response in a subject. In some cases, the engineered viral vectors can be modified into vaccines that not only carry a payload (e.g., an immunogenic protein) but also express immunogenic proteins on envelope proteins. Engineered viral vectors can present immunogenic proteins over extended periods, allowing stimulation of intrinsic immune responses at different stages.

[0157] In some implementations, the antigenic protein is derived from self-antigens, allergens, tumor-associated antigens, pathogenic viruses, pathogenic bacteria, pathogenic protozoa, pathogenic worms, or any other pathogenic organism from which the infected object originates. For example, a nucleic acid sequence encoding the N-terminal domain (NTD) of the SARS-CoV-2 spike protein can be inserted into an SB envelope plasmid, and engineered viral vectors (e.g., MLV vectors) can be generated using conventional three-plasmid transfection. Such engineered viral vectors contain an MLV core and a modified SB envelope protein that also expresses the NTD of the SARS-CoV-2 spike protein. The MLV core of the engineered viral vector can carry any gene of interest, such as the full-length spike or specific region of the SARS-CoV-2 virus. Such engineered viral vectors can be used as vaccines to stimulate an immune response to the SARS-CoV-2 virus.

[0158] In some embodiments, the immunogenic protein includes a viral protein. In some embodiments, the viral protein includes a viral envelope protein, a spike protein, or a combination thereof. In some embodiments, the viral protein is derived from a virus. In some embodiments, the virus includes a respiratory virus. In some embodiments, the respiratory virus includes SARS-CoV-2 or influenza virus. In some embodiments, the viral protein contains the N-terminal domain of SARS-CoV-2. In some embodiments, the viral protein contains hemagglutinin (HA) of influenza virus.

[0159] In some implementations, the immunogenic protein includes a viral antigen. In some cases, the viral antigen is derived from known pathogens that cause disease, including but not limited to SARS-CoV-2, measles, mumps, rubella, poliomyelitis, hepatitis A, hepatitis B (e.g., GenBank accession number E02707) and hepatitis C (e.g., GenBank accession number E06890) and other hepatitis viruses, influenza, adenoviruses (e.g., types 4 and 7), rabies (e.g., GenBank accession number M34678), respiratory syncytial virus (RSV), yellow fever, Japanese encephalitis (e.g., GenBank accession number E07883), dengue virus (e.g., GenBank accession number M24444), Hantavirus, and human immunodeficiency virus (e.g., GenBank accession number U18552).

[0160] In some implementations, the immunogenic protein includes bacterial antigens or parasitic antigens. In some cases, the bacterial antigens or parasitic antigens include those derived from known pathogens that cause diseases, including but not limited to diphtheria, pertussis (e.g., GenBank accession number M35274), tetanus (e.g., GenBank accession number M64353), tuberculosis, and bacterial and fungal pneumonia (e.g., Haemophilus influenzae, Pneumocystis carinii). (e.g., carinii), cholera, typhoid fever, plague, shigella, salmonellosis (e.g., GenBank accession number L03833), Legionnaires' disease, Lyme disease (e.g., GenBank accession number U59487), malaria (e.g., GenBank accession number X53832), hookworm disease, onchocerciasis (e.g., GenBank accession number M27807), schistosomiasis (e.g., GenBank accession number L08198), trypanosomiasis, leishmaniasis, giardiasis (e.g., GenBank accession number M33641), amoebiasis, filariasis (e.g., GenBank accession number J03266), spirochetosis, and trichinosis.

[0161] Modifications in the E1 structural domain In various aspects of the engineered viral vectors provided herein, in some embodiments, the E1 domain of the SB envelope protein is modified. In some embodiments, the SB envelope protein further comprises at least one amino acid modification in the E1 domain relative to the wild-type protein sequence (SEQ ID NO: 1). In some embodiments, the at least one amino acid modification in the E1 domain relative to the wild-type protein sequence (SEQ ID NO: 1) comprises at least one amino acid substitution. In some embodiments, the at least one amino acid substitution comprises an amino acid substitution at position 226, an amino acid substitution at position 227, or a combination thereof, and such amino acid position is based on the amino acid numbering in the E1 domain. In some embodiments, the at least one amino acid substitution comprises an amino acid substitution at position 770, an amino acid substitution at position 771, or a combination thereof, and such amino acid position is based on the amino acid numbering in SEQ ID NO: 1.

[0162] In some embodiments, the amino acid substitution at position 226 includes an A226S or A226Y substitution. In some embodiments, the amino acid substitution at position 227 includes a K227G substitution. In some embodiments, at least one amino acid substitution includes an A226S substitution, an A226Y substitution, a K227G substitution, or a combination thereof. In some embodiments, at least one amino acid substitution includes an A226S substitution or a K227G substitution. In some embodiments, at least one amino acid substitution includes an A226Y substitution or a K227G substitution.

[0163] In some embodiments, at least one amino acid substitution further includes an amino acid substitution at position 160, and this amino acid position is based on the amino acid numbering on the E1 domain. In some embodiments, the amino acid substitution at position 160 includes an E160G substitution.

[0164] Additional modifications in the E2 domain In another aspect of the engineered viral vector provided herein, in some embodiments, at least one modification in the E2 domain relative to the wild-type protein sequence (SEQ ID NO: 1) comprises a substitution of one or more amino acids. In some embodiments, the substitution of one or more amino acids comprises amino acid substitutions or combinations thereof, wherein the amino acid positions are based on the amino acid numbers on the E2 domain. In some embodiments, the substitution of one or more amino acids comprises an amino acid substitution at position 134, an amino acid substitution at position 135, an amino acid substitution at position 136, an amino acid substitution at position 137, an amino acid substitution at position 225, an amino acid substitution at position 226, or a combination thereof, wherein the amino acid positions are based on the amino acid numbers of SEQ ID NO: 1.

[0165] In some embodiments, the amino acid substitution at position 134 includes the S134A substitution of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 135 includes the L135A substitution of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 136 includes the K136A substitution of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 137 includes the Q137A substitution of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 225 includes the K225A substitution of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 226 includes the E226A substitution of SEQ ID NO: 1.

[0166] Payload In various aspects of the engineered viral vectors provided herein, in some embodiments, the engineered viral vector further comprises a payload. In some embodiments, the payload encodes a transgenic or gene of interest. In some embodiments, the payload encodes at least one therapeutic agent. In some embodiments, at least one therapeutic agent comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises DNA, RNA, or a combination thereof. In some embodiments, the RNA comprises mRNA. In some embodiments, at least one therapeutic agent comprises at least one therapeutic polypeptide. In some embodiments, at least one therapeutic polypeptide comprises a suicide protein. In some embodiments, the suicide protein comprises thymidine kinase, cytosine deaminase, IL-12, IL-2, nitroreductase (NR), carboxylesterase, β-glucuronidase, cytochrome p450, β-galactosidase, diphtheria toxin A chain (DT-A), carboxypeptidase G2 (CPG2), purine nucleoside phosphorylase (PNP), or deoxycytidine kinase (dCK).

[0167] Thymidine kinase is a rescue enzyme that phosphorylates natural nucleoside substrates and nucleoside analogs. Thymidine kinase can convert prodrugs into active ingredients for treating diseases or conditions. In some embodiments, a prodrug refers to any inactive compound that can later be converted into an active form. In some embodiments, the active form includes a toxic product or suicide protein that can kill target cells, such as tumors or cancer cells. Thymidine kinases (such as viral thymidine kinase) can be therapeutically utilized by administering nucleoside analogs (such as ganciclovir or acyclovir) to cells expressing viral thymidine kinase, wherein the viral thymidine kinase phosphorylates the nucleoside analog, thereby producing a toxic product capable of killing cells. The viral thymidine kinase of this disclosure can be derived from a variety of viral thymidine kinases. In some embodiments, the thymidine kinase is derived from herpes simplex virus (HSV-TK) or vesicular stomatitis virus (VSV-TK). In some embodiments, the thymidine kinase is modified. In some embodiments, the thymidine kinase is derived from thymidine kinase of the Herpesviridae family, including but not limited to those derived from both primate and non-primate herpesviruses (such as avian herpesviruses). Representative examples of suitable herpesviruses include, but are not limited to, herpes simplex virus (HSV) type 1, herpes simplex virus type 2, varicella-zoster virus, marmosetan herpesvirus, feline herpesvirus type 1, pseudorabies virus, equine herpesvirus type 1, bovine herpesvirus type 1, turkey herpesvirus, Marek's disease virus, squirrel monkey herpesvirus, or Epstein-Barr virus. In some aspects, the thymidine kinase described herein may be a mutated thymidine kinase, wherein the mutated thymidine kinase contains at least one amino acid mutation. In some embodiments, the mutated thymidine kinase is described, for example, in U.S. Patent No. 9,925,276, which is incorporated herein by reference in its entirety.

[0168] In some cases, at least one therapeutic agent may be a nucleic acid. Nucleic acids that can be delivered to an individual using the methods described herein include, but are not limited to, non-translated RNAs such as antisense RNA, ribozymes, RNAi, and siRNA. In some cases, the nucleic acid includes heterologous nucleic acids.

[0169] Figure 1A A diagram illustrates modifications introduced into the Sindbis (SB) viral envelope protein to increase the targeting efficiency of the engineered viral vector generated by the methods described herein. The wild-type Sindbis envelope protein can be engineered to have a ZZ protein domain (ZZ Sindbis). For example, the ZZ protein domain can be introduced into the E2 domain. In some aspects, the wild-type Sindbis envelope protein can be modified to have an m168 modification, which includes three modifications introduced at the positions indicated by the arrows, such as... Figure 1A As shown in the diagram. The m1 modification is located between the E2 and E3 boundaries, and the m1 modification is the deletion of amino acids 61-64 of E3 in SEQ ID NO: 1. The m6 modification is located in the E2 domain, and this is an amino acid substitution from K225E226 to A225A226. Finally, the m8 modification is also located in the E2 domain, and this modification includes amino acid substitutions from S134L135K136Q137 to A134A135A136A137. In some aspects, the modified Sindbis envelope protein can be encoded by a 2.2 (2.2-SG) plasmid, in which mutations from A226K227 to S226G227 are introduced into the E1 domain. In some aspects, the modified Sindbis envelope protein can be engineered to introduce or remove protease (e.g., furin protease) cleavage sites (in... Figure 1A (Represented as "F" in Chinese). As shown in engineered plasmids such as m168, 2.2, 2.2-AK, or GVO2.2, furin cleavage sites have been removed compared to wild-type Sindbis envelope protein. Figure 1A As shown, "tm" or the targeting portion can be a ZZ protein domain replaced by a nucleic acid sequence encoding an antigen-binding fragment, such as a single-stranded variable domain (scFv), a dual antibody (DB), or a single variable domain (V) on the heavy chain of a nucleic acid sequence derived from a targeting antibody. H H).

[0170] In one aspect, this disclosure provides a system comprising an engineered viral vector as described herein. In some cases, the system is a cell. In some embodiments, the cell comprises an engineered viral vector. In some embodiments, the cell may be a packaging cell line used to generate the engineered viral vector by transfection with one or more plasmids as described herein. In some cases, the cell may be a target cell, wherein the engineered viral vector binds to and fuses with the cell membrane of the target cell.

[0171] Pharmaceutical Composition In another aspect, this disclosure provides a pharmaceutical composition comprising an engineered viral vector, cell, or system as described herein. In some embodiments, the composition comprises at least one additional active ingredient. In some embodiments, the composition comprises at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition stimulates an immune response in a subject.

[0172] This document describes a pharmaceutical composition comprising a therapeutic agent (e.g., an engineered viral vector or cells comprising an engineered viral vector). In some aspects, cells in contact with the engineered viral vector described herein express at least one therapeutic agent. For example, the cells may express the mutant thymidine kinase described herein.

[0173] In some aspects, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient, or diluent. In some aspects, the pharmaceutical composition described herein comprises at least one additional active agent besides the engineered viral vector described herein. In some aspects, at least one additional active agent is a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitor, an anti-hormonal agent, an anti-angiogenic agent, or a checkpoint inhibitor.

[0174] In some embodiments, the pharmaceutical composition further comprises one or more pH adjusters or buffers, such as 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(hydroxymethyl)aminomethane; amino acids, such as histidine, arginine, and glycine; and buffers, such as citrate / glucose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are added in amounts necessary to maintain the pH of the composition within an acceptable range.

[0175] In some embodiments, the pharmaceutical composition further comprises one or more salts in an amount required to bring the osmotic pressure of the pharmaceutical composition within an acceptable range. Such salts include, but are not limited to, those having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0176] method In some embodiments, methods using the engineered viral vectors described herein are described. In one aspect, this disclosure provides methods for delivering a therapeutic agent to target cells in a subject. In some embodiments, the method includes administering a pharmaceutical composition comprising an engineered viral vector to a subject via intravenous, subcutaneous, intraperitoneal, intramuscular, or a combination thereof. In some embodiments, the method includes administering a pharmaceutical composition comprising an engineered viral vector to a subject via bronchoalveolar lavage, sublingual, intravenous, intraarterial, oral, parenteral, buccal, local, percutaneous, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration routes. In some embodiments, the pharmaceutical composition comprising an engineered viral vector can be administered to the subject via oral, bronchoalveolar lavage, sublingual, intratumoral, parenteral, intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, subarachnoid, intraocular, intrasternal, ocular, endothelial, local, intranasal, intrapulmonary, rectal, intraarterial, intrasheath, inhalation, intralesional, intradermal, epidural, intracystic, subcapsular, intracardiac, tracheal, subepidermal, or intraspinal administration, for example, by injection or infusion. In some embodiments, the pharmaceutical composition comprising an engineered viral vector can be administered to the subject via absorption through the epithelial or mucocutaneous layer (e.g., oral mucosa, rectal mucosa, and intestinal mucosa). In some embodiments, the pharmaceutical composition is delivered via multiple routes of administration.

[0177] In some aspects, this disclosure provides methods for inducing cell-killing activity in cancer cells of a subject. In some embodiments, the method includes administering to the subject an engineered viral vector carrying thymidine kinase, which can convert a prodrug into an active ingredient. In some embodiments, a prodrug refers to any inactive compound that can be converted into an active form, for example, by an enzyme (such as thymidine kinase). In some embodiments, the active form includes a toxic product or suicide protein that can kill target cells, such as tumors or cancer cells. In some embodiments, the method includes administering to the subject an engineered viral vector carrying thymidine kinase, which converts a prodrug into a toxic product, thereby inducing cell-killing activity.

[0178] In various aspects, this disclosure provides methods for inducing an immune response in a subject. In some embodiments, the method includes administering an engineered viral vector carrying an immunogenic protein to the subject, thereby inducing an immune response triggered by target cells in the subject.

[0179] In some embodiments, the method includes treating a disease or condition in a subject by administering an engineered vector or a pharmaceutical composition comprising an engineered viral vector as described herein. In some embodiments, the cells contacted with the engineered viral vector are autologous cells. For example, the cells may first be isolated from the subject and optionally cultured or expanded prior to contact with the engineered viral vector. In some embodiments, the method includes administering a pharmaceutical composition comprising an engineered viral vector or cells comprising an engineered viral vector to the subject. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, non-human animals include cows, mice, rats, rabbits, guinea pigs, chickens, fish, birds, reptiles, camels, cattle, chimpanzees, sheep, goats, dogs, cats, horses, or non-human primates. In some embodiments, the disease or condition includes cancer.

[0180] In another embodiment, this disclosure relates to the use of the engineered viral vectors provided herein for the preparation of medicaments. In yet another embodiment, this disclosure relates to the use of engineered viral vectors for the preparation of medicaments for the treatment or prevention of cancer, chronic infections (e.g., HIV infection), hereditary single-gene diseases, cardiovascular diseases, and neurodegenerative diseases. In yet another embodiment, the cancer is selected from leukemia, myeloid leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia, lymphomas such as Hodgkin's disease and non-Hodgkin's disease, fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, Ewing's tumor, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, liver cancer, Wilms' tumor, cervical cancer, uterine cancer, etc. Cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, oligodendroglioma, melanoma, neuroblastoma, retinoblastoma, dysplasia and hyperplasia, prostate cancer, prostatitis, benign prostatic hyperplasia (BPH), prostatic paraganglioma, prostatic adenocarcinoma, prostatic intraepithelial neoplasia, prostatic-rectal fistula, Merkel cell carcinoma, angiosarcoma, cutaneous T-cell lymphoma, cutaneous B-cell lymphoma, dermatofibrosarcoma protuberans, sebaceous gland carcinoma, cutaneous neuroendocrine carcinoma, and atypical prostatic stromal lesions. In another embodiment, the pharmaceutical composition of the present invention is administered alone or in combination with other types of cancer treatments.

[0181] In some embodiments, engineered viral vectors can be used to vaccinate subjects. For example, an engineered viral vector can encode an antigen that triggers an immune response in the subject, thus conferring immunity to the antigen. In some embodiments, a method for treating or preventing a disease or condition in a subject by vaccinating the subject is described herein, the method comprising administering to the subject an engineered viral vector, cells containing an engineered viral vector, or a pharmaceutical composition described herein.

[0182] In some implementations, an engineered viral vector, a cell or pharmaceutical composition containing an engineered viral vector is administered to increase the local concentration of a therapeutic agent (such as thymidine kinase, e.g., mutant HSV1-TK) in the cell or microenvironment associated with a disease or condition (e.g., cancer).

[0183] In some embodiments, the method includes delivering a therapeutic agent to target cells in a subject by administering an engineered viral vector, a cell containing an engineered viral vector, or a pharmaceutical composition to the subject. In some embodiments, the specificity or efficiency of therapeutic delivery to target cells via an engineered viral vector, a cell containing an engineered viral vector, or a pharmaceutical composition is increased by at least 2.0-fold, 5.0-fold, 10.0-fold, or more compared to the specificity or efficiency of therapeutic delivery to target cells via a wild-type viral vector.

[0184] As described herein, in some cases, engineered viral vectors generated from modified SB envelope plasmids have increased the targeting efficiency or therapeutic delivery to target cells by at least 1.0-fold, 2.0-fold, 5.0-fold, 10.0-fold, 50.0-fold or more compared to targeting efficiency or therapeutic delivery mediated by viral vectors generated from wild-type SB envelope plasmids.

[0185] definition The use of absolute or sequential terms, such as “will,” “will not,” “shall,” “shall not,” “must,” “must not,” “first,” “initially,” “next,” “subsequently,” “before,” “after,” “lastly,” and “finally,” is not intended to limit the scope of the embodiments of the invention disclosed herein, but is merely an example.

[0186] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, in the context of the terms “including,” “includes,” “having,” “has,” “with,” or variations thereof, such terms are intended to be included in a manner similar to the term “comprising.”

[0187] As used herein, the phrases “at least one,” “one or more,” and “and / or” are open-ended expressions that have both conjunction and disjunction in the operation. 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.

[0188] As used herein, “or” can mean “and,” “or,” or “and / or,” and can be used exclusively and inclusively. For example, the term “A or B” can mean “A or B,” “A but not B,” “B but not A,” and “A and B.” In some cases, the context can prescribe a specific meaning.

[0189] As used herein, the term "about" when referring to a number or range of values ​​means that the number or range of values ​​mentioned is an approximation within experimental variability (or statistical experimental error), and that the number or range of values ​​may vary, for example, from 1% to 15% of the number or range of values. In the example, the term "about" means ±10% of the number or value.

[0190] As used herein, the terms “increased,” “increasing,” or “increase” are used to generally mean an increase that is statically significant. In some respects, the terms “increased” or “increase” mean an increase of at least 10% compared to a reference level, such as 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 up to and including an increase of 100% or any increase between 10% and 100%. Other examples of “increase” include increases of at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 1000, or more than 1000 times compared to a reference level.

[0191] As used herein, the terms “decreased,” “decreasing,” or “decrease” are generally used to mean a reduction in a statistically significant amount. In some respects, the terms “decreased” or “decrease” mean a reduction of at least 10% compared to a reference level, such as 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 up to and including a reduction of 100% (e.g., a level that does not exist or is undetectable compared to the reference level) or any reduction between 10% and 100%. In the context of a biomarker or symptom, these terms mean a statistically significant reduction at such levels. A 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 recognized within the normal range for individuals without a given disease.

[0192] As used herein, the terms “vector,” “plasmid,” or “construct” are used interchangeably to refer to a nucleic acid sequence that can be used to clone and amplify a genetic sequence or a gene encoding a protein of interest. In some cases, a “vector” is a nucleic acid capable of transporting another nucleic acid sequence. In some cases, a “vector” is a small circular DNA molecule used to carry genetic information, and the nucleic acid sequence on the vector can be modified. However, as used herein, “viral vector” or “viral particle” refers to a viral vector generated by transfecting packaging cells with one or more plasmids, including envelope plasmids, payload plasmids, or one or more GAGPol plasmids.

[0193] As used herein, the term "pseudotype" refers to a viral particle or viral vector in which the envelope or capsid is derived from a heterologous viral protein. A "pseudotyped" virus or "pseudotyped" viral vector has an envelope protein derived from a virus other than the one from which the genome originates. The envelope protein can be a natural envelope protein or a modified, mutated, recombinant, or engineered envelope protein as described herein.

[0194] As used herein, the term "chimeric" refers to an antibody or antibody-derived molecule in which (a) a constant region or a portion thereof is altered, replaced, or exchanged such that the antigen-binding site (variable region) is connected to a constant region of a different or altered class, effector function, and / or species, or to a constant region of a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) that imparts new properties to the chimeric antibody; or (b) a variable region or a portion thereof is altered, replaced, or exchanged by a variable region having a different or altered antigen specificity.

[0195] Example The following embodiments are provided for the purpose of illustrating various embodiments of this disclosure and are not intended to limit the invention in any way. The embodiments of the invention and the methods described herein are representative preferred embodiments and are exemplary, and are not intended to limit the scope of this disclosure. Variations and other uses thereof will be apparent to those skilled in the art, and are covered within the spirit of this disclosure as defined by the scope of the claims.

[0196] Example 1. Transduction of GVO2.2 plasmid and GVO2.2 pseudotyped viral vector.

[0197] In this embodiment, GVO2.2 plasmids encoding Sindbis (SB) virus envelope proteins and other modified GVO2.2 plasmids were designed and generated.

[0198] Materials and methods Molecular cloning Plasmids were sourced from commercially available GVO samples and were either subcloned or synthesized using GenScript. Standard molecular cloning techniques were employed to generate novel plasmids, including restriction enzyme digestion, agarose gel purification, enzyme-mediated ligation, drug selection, and mutagenesis. Plasmid preparation was performed using commercially available kits, such as the QIAGEN kit. All GVO plasmids were confirmed in Primordium by restriction enzyme digestion and plasmid sequencing.

[0199] Tissue culture Human and mouse cell lines were maintained in their respective culture media using a 5% CO2 humidified tissue culture incubator. DNA transfection and vector transduction experiments were performed using 293T cells. 293T cells were cultured in 10% FBS / DMEM supplemented with sodium pyruvate, GlutaMAX®, and pen / strep.

[0200] Production of engineered viral vectors Recombinant or engineered viral vectors such as bitropic MLV and SB pseudotyped MLV are generated by plasmid DNA transfection using calcium phosphate. Briefly, on day 0, 293T cells are seeded in tissue culture plates or flasks. On day 2, 20–24 hours post-seedling, a transfection mixture containing plasmid DNA [encoding MLV gagpol, envelope (i.e., A4070 for bitropic MLV, GVO construct for SB pseudotyped MLV), and payload (a plasmid with firefly luciferase as a reporter molecule or otherwise)], MgCl2 and CaCl2 solutions, and 2×HBS (HEPES-buffered saline) is added to the seeded cells in 6% FBS / DMEM (1 g / L glucose) (day 1). On day 2, 16 hours post-transfection, the transfection medium is replaced with 10% FBS / DMEM (4.5 g / L glucose) for 9 hours, and the cells are transferred to final prepared medium (FFM) until harvest. Collect the culture supernatant 36-40 hours after FFM placement, filter it through a 0.45 μm PES filter, divide it into equal portions, and store it at -80℃.

[0201] Viral titration Vector titration was performed, and the physical titer (RNA genome copies / mL) was estimated by RT-qPCR. Briefly, vector RNA was extracted from a source (e.g., cell culture supernatant from transfected cells) using a commercially available kit. Following DNase treatment, RT-qPCR was performed on the psi-packaged sequence of the MLV using primers and probes for the payload vector. Genome copy titer was used in most of the experiments described herein.

[0202] Preparation of SB pseudotyped viral vectors When engineered viral vectors generated from GVO2.2 or GVO2.2F plasmids are conjugated with antibodies prior to transduction, a predetermined amount of engineered viral vector, as determined by RT-qPCR, is mixed with antibodies at a concentration of 1–10 μg / mL. The mixture is allowed to stand for 15–60 minutes before use for transduction. Antibodies used for conjugation include mAbs against HLA, CD47, HER2, EGFR, and PSMA. Antibodies were purchased from their respective vendors. Several antibodies were submitted for protein sequencing for use in the design of scFv or dual-antibody vector constructions, as described in subsequent examples.

[0203] Viral vector transduction Bitropic MLVs or various SB pseudotyped viral vectors were used to transduce cells to assess the transducibility of engineered viral vectors; that is, whether they could bind, internalize, reverse transcribe their genome, and express the gene of interest as a reporter protein. Cells for transduction were selected based on the purpose of transduction. For example, SB vectors conjugated with HLA antibodies could be used for most cell lines; however, anti-EGFR and anti-HER2 antibodies were used for cell lines that (over)expressed those target molecules. The transduction procedure was as follows: On day 0, cells were seeded 20–24 hours before transduction. Twenty-four (24)-well plates were primarily used. On day 2, cells were transduced with a vector containing a luciferase reporter molecule and incubated for 3 days. For fluorescent protein reporter molecules, 12 or 6-well plates were used and incubated for 3 to 5 days. For GCV-mediated cell killing assays using HSV1-TK as a transgene, 96-well plates were used. In general, the carrier was added to the culture medium at a predetermined concentration in the presence of 6-8 μg / mL polybrene.

[0204] Reporter gene assay Most of the transduction experiments described above were performed using luciferase reporter molecules. In short, after incubation with the vector for 3 days, the culture supernatant was removed by aspiration. Then, luciferase cell lysis buffer (Thermo) was added to each well to lyse the cells. After incubation at room temperature for 5–10 minutes, the lysates were transferred to a luciferase assay plate and mixed with luciferase substrate (Perkin Elmer). The luminescence of the samples was read and analyzed using a SpectraMax M5 Molecular Devices reader.

[0205] The SB virus envelope gene encodes four compartments from the N-terminus to the C-terminus: E3, E2, 6K, and E1. The SB envelope protein, containing these four compartments, is synthesized as a single 108 kDa polypeptide, which is then proteolytically separated to form a quaternary trimer. Each of these four compartments of the SB envelope protein has its own function. The E3 domain separates after the E2 domain matures. The E2 domain acts as a binding domain, while the E1 domain regulates fusion. Therefore, modifications to the E2 domain should not affect the structure and function of the E1 domain.

[0206] SB envelope proteins can be like Figure 1AThe modified region shown has the ZZ protein domain, which serves as the IgG binding domain of bacterial protein A, added to the E2 domain of the wild-type SB envelope protein (Ohno et al., Nat Biotechnol, 1997, 15:763-7; Morizono et al., J. Virol., 2001, 75:8016-20; Yang et al., PNAS, 2006, 103:11479-84; Löwenadler et al., Gene, 1987, 58:87-97). This is the ZZ Sindbis construct. Furthermore, the m168 construct encoding the modified SB envelope protein has three modifications: m1, m6, and m8 (Morizono et al., 2001; Morizono et al., Nat. Med., 2005, 11:346-52; Pariente et al., Mol. Ther, 2007, 15:1973-81). The m1 modification is located between the E2 and E3 boundaries and is a deletion of amino acids 61-64 in E3. The m6 modification is located in the E2 domain and is an amino acid substitution from K159E160 to A159A160. Finally, the m8 modification is also located in the E2 domain and includes an amino acid substitution from S68L69K70Q71 to A68A69A70A71. The construct named 2.2 has similar modifications to the m168 construct, with additional modifications in the E1 domain, consisting of amino acid substitutions from A226K227 to S226G227. In this embodiment, the base construct named GVO2.2 has a different substitution in the E1 domain, and this substitution is modified to Y226G227. The amino acid sequence of the E1 subunit in the GVO2.2 plasmid is shown below. Figure 1B (SEQ ID NO: 19). In general, compared with the wild-type SB envelope protein, the GVO2.2 plasmid has m1, m6, m8 modifications, the addition of the ZZ domain, and the substitution of Y226G227 in the E1 domain.

[0207] In some cases, modified SB envelope proteins include a targeting moiety (tm) but not a ZZ domain. Different modifications of the modified SB envelope protein are also shown in... Figure 1AIn the figure, GVO2.2 with A226K227 is designated as 2.2-AK, and GVO2.2 with S226G227 is designated as GVO2.2-SG (amino acid numbering based on SEQ ID NO: 19). GVO2.2F refers to a modified SB envelope protein with a furin cleavage site located between the E3 and E2 domains. ZZ refers to the IgG-binding ZZ domain; tm refers to the targeting moiety. Different modifications of the GVO2.2 construct are also referred to as modified GVO2.2 constructs.

[0208] To generate engineered viral vectors with SB envelope proteins (such as pseudotyped MLV vectors), GVO2.2-enveloped pseudotyped MLV vectors were generated by transiently transfecting 293T cells with the following three plasmids: GVO2.2 SB envelope plasmid or modified GVO2.2 envelope plasmid, pCGPN plasmid ( gagpol The payload plasmid was used for both the MLV ditropic vector and the SB pseudotyped MLV vector. The pseudotyped MLV vector with the SB envelope protein produced a physical titer similar to that of the recombinant MLV vector with the ditropic (4070A) MLV envelope protein. Since the same payload plasmid was used for both the ditropic MLV vector and the SB pseudotyped MLV vector, the physical titer of the pseudotyped vector was measured using a physical titer method based on RNA genome copy number by RT-qPCR. For the payload, the transduction efficiency of the SB pseudotyped MLV vector was examined using a firefly luciferase reporter plasmid (pCL-Gm-luc2 reporter plasmid).

[0209] To determine whether the ZZ protein domain of the engineered viral vector generated from the GVO2.2 plasmid is functional, an anti-human leukocyte antigen (HLA) antibody was conjugated to the viral envelope of an SB pseudotyped MLV vector (or a GVO2.2 pseudotyped MLV vector), and human cells were transduced with the antibody-conjugated SB pseudotyped MLV vector. The payload used here was firefly luciferase. HLA is a cell surface molecule type expressed on the surface of most cells in the human body. Therefore, it served as a positive control for the transduction assay of the engineered viral vector generated from the modified GVO2.2 envelope plasmid. Anti-HLA monoclonal antibodies (mouse, ThermoFisher) were conjugated to the GVO2.2 pseudotyped MLV vector at various concentrations ranging from 0.156 to 10 mcg / mL (in conjugation). The negative control was the GVO2.2 pseudotyped MLV vector without antibody conjugation.

[0210] For the conjugation of the antibody with the pseudotyped viral vector, after incubating the engineered viral vector and antibody together at room temperature for 30 minutes, the engineered viral vector was added to 293T cells plated in 24-well plates in the presence of 8 mcg / mL polybrene. Three days later, cells were harvested and luciferase expression was measured. Figure 2 As shown, the negative control (without antibody) only exhibited baseline activity. A dose-dependent response up to 5 mcg / mL antibody was demonstrated by transduction of 293T cells with an engineered viral vector conjugated to an anti-HLA antibody, generated from a modified GVO2.2 plasmid.

[0211] After confirming the transduction capacity of the GVO2.2 pseudotyped MLV vector, optimization experiments were conducted to determine the optimal transfection conditions for physical titer and infectivity. GVO2.2 pseudotyped MLV vectors were generated under conditions with different plasmid ratios in the transfection mixture. The results showed no significant difference in physical titer, but 10 mcg of plasmid per T80 flask exhibited the highest transduction efficiency via luciferase activity. Total plasmid levels exceeding 10 mcg did not increase transduction efficiency.

[0212] Transduction of recombinant MLVs with amphophilic MLV envelope proteins is mediated by Pit-2, while transduction of GVO2.2 pseudotyped vectors is not mediated by Pit-2. Therefore, the presence or absence of Pit-2 should not affect the infectivity of GVO2.2 pseudotyped MLV vectors. Figure 3A As shown, the engineered viral vector conjugated with anti-HLA antibodies, generated from the GVO2.2 plasmid, exhibited antibody dose-dependent transduction in both wild-type A375 cells (hollow bars) and Pit-2 knockout A375 cells (solid bars). Conversely, in Figure 3B In this study, regardless of whether anti-HLA antibodies were conjugated, the bitropic MLV vector could not transduce Pit-2-KO A375 cells. RVE stands for bitropic envelope protein. Luciferase (Luc) was used as a reporter molecule. Various antibody concentrations were used in the conjugation and various antibody concentrations were examined for transduction.

[0213] These results demonstrate that transduction of antibody-conjugated engineered viral vectors generated from GVO2.2 plasmids occurs via the target molecule but not through the Pit-2 receptor, which in this experiment was HLA. Because the GVO2.2 pseudotyped MLV vector can transduce cells lacking the Pit-2 receptor, this result suggests that, compared to conventional bitropic MLV vectors, the GVO2.2 pseudotyped MLV vector is advantageous for targeting Pit-2-deficient cells (such as cancer cells) by using antibodies that target the protein of interest.

[0214] In summary, the results show that i) the MLV vector can be pseudotyped with a modified GVO2.2 SB envelope protein while retaining an MLV core, ii) the GVO2.2 pseudotyped MLV vector can be conjugated with antibodies targeting proteins of interest, and iii) the GVO2.2 pseudotyped MLV vector exhibits antibody dose-dependent transduction. Even in the absence of the Pit-2 receptor, transduction of this GVO2.2 pseudotyped MLV vector can occur in conjunction with the targeting antibody, allowing for the transduction of this engineered viral vector into another type of cell.

[0215] Example 2. Using antibody conjugation to cell surface proteins to transduce cancer cell lines.

[0216] In this embodiment, antibodies targeting various cell surface proteins (such as HLA, CD47, CEA, HER2, EGFR, PSMA, nectin-4, Pit-2, and TfR) were examined on various cancer cell lines. The transduction activity of engineered viral vectors against conjugated antibodies (e.g., GVO2.2 pseudotyped MLV vectors conjugated with antibodies) was examined on various human cancer cell lines. In this embodiment, a luciferase payload was used as a reporter molecule to examine the transduction of the engineered viral vectors. The method was performed as described in Example 1.

[0217] CD47 is an integrin-associated protein expressed on the cell surface. Many cancer cells can evade macrophages of the immune system by expressing CD47. Therefore, CD47 is considered a therapeutic target on cancer cells. Several different monoclonal antibodies (mAbs) were used to examine whether they could mediate the transduction of engineered viral vectors (e.g., GVO2.2 pseudotyped viral vectors).

[0218] Since CD47 is expressed in most cancer cell lines, engineered viral vectors conjugated with anti-CD47 antibodies, generated from GVO2.2 plasmids, showed transduction ability in most tested cancer cell lines. Figure 4A Representative data from transduction experiments are shown. Figure 4AIn this study, SKBR3 human breast cancer cells were used to test the transduction efficiency of GVO2.2 pseudotyped MLV vectors conjugated with antibodies. Unlike anti-HLA antibodies, the amount of anti-CD47 mAb conjugation did not proportionally increase transduction. For anti-CD47 mAb conjugation, the range of 0.1 to 1.1 mcg / mL showed peak activity, and this result differed from that of anti-HLA conjugation (which showed the highest transduction efficiency at the highest concentration tested, 10 mcg / mL). The same results were observed in 293T cells. When the antibody was conjugated with an engineered viral vector generated using a GVO2.2 envelope plasmid, the transduction efficiency varied among different CD47 mAbs, indicating that the ability of antibodies to mediate transduction can vary. Several anti-CD47 antibodies were tested, and the anti-CD47 mAb obtained from clone BRIC126 showed the highest transduction.

[0219] The transduction capacity of GVO 2.2 pseudotyped viral vectors conjugated with anti-HER2 antibodies was also investigated. HER2 is a receptor tyrosine kinase, also known as ERBB-2. HER2 is an oncogene and a member of the epidermal growth factor receptor. It is amplified or overexpressed in many cancer types, such as breast cancer. Several antibody-based therapeutics have been commercialized as antibody therapies, such as pertuzumab and trastuzumab. Therefore, this characteristic of the protein is attractive as a targeting molecule. SKBR3 is a human breast cancer cell line that overexpresses HER2. Figure 4B As shown, SKBR3 cells exhibited excellent transduction with a GVO 2.2 pseudotyped viral vector conjugated to a monoclonal antibody against HER2 (clone 191924). Anti-HLA mAb was used as a positive control.

[0220] EGFR is involved in cell growth, making it a reasonable target for cancer cells as a targeting vector. The transduction of engineered viral vectors conjugated with anti-EGFR antibodies was investigated in EGFR-overexpressing cancer cells. Similar to other target molecules, the transduction level of the conjugated viral vectors depended on the Ab clone used for conjugation. For anti-EGFR antibodies, anti-EGFR clone 528 was the best-performing mAb tested.

[0221] HCT-15 (human colorectal cancer cell line) is a representative cell line that indicates the specificity of antibody-mediated transduction because HCT-15 cells lack HLA expression (Gattoni-Celli et al., Cancer Res, 1992, 52:1201-4). Figure 4CAs shown, HCT-15 cells exhibited transduction when the engineered viral vector was conjugated with an anti-EGFR antibody; however, when the engineered viral vector was conjugated with an anti-HLA antibody, transduction was negligible. This is because the HCT-15 cell line lacks HLA expression. The anti-HLA mAb was clone W6 / 32. This clearly demonstrates that transduction via engineered viral vectors conjugated with HLA mAbs depends on the expression of the target molecule HLA.

[0222] like Figure 4D As shown, HepG2 (human hepatocellular carcinoma cell line) was transduced using engineered viral vectors conjugated with either anti-HLA mAb or anti-EGFR mAb, and similar results were observed. The transduction levels of the GVO2.2 pseudotyped viral vector conjugated with anti-EGFR mAb were significantly higher than those conjugated with anti-HLA mAb.

[0223] In summary, this demonstrates that antibody-conjugated GVO2.2 pseudotyped viral vectors can bind to target molecules. Targeted transduction of specific targets using specific targets on target cells or lesions is feasible. The results support the functionality and usefulness of engineered viral vectors (e.g., GVO2.2 pseudotyped viral vectors) conjugated to a variety of cell surface molecules, as a single vector can be used for multiple targets by modifying the antibody used for conjugation.

[0224] Example 3. Generation and testing of GVO2.2-scFv vector.

[0225] Based on results from engineered viral vectors from previous embodiments, a targeting portion (such as a single-chain variable fragment (scFv)) is introduced into the SB envelope plasmid to replace the ZZ protein domain. Although the ZZ protein domain specifically binds to the Fc domain of IgG, the binding affinity of the ZZ protein domain to the Fc domain of each IgG varies. Therefore, predicting how stable the binding is (especially in vivo) is quite challenging. Furthermore, incorporating monoclonal antibodies into drug manufacturing requires complex procedures and regulations. Therefore, incorporating scFv into the envelope protein derived from the SB virus may be an excellent option for solving the above problems. Thus, replacing the ZZ domain with scFv can simplify the production process and increase stable target binding ability.

[0226] Perform the method as described in Example 1.

[0227] First, antibodies were screened for adequate transduction. The amino acid sequences of antibodies working in the conjugation system described in previous examples were determined. The resulting heavy and light chain amino acid sequences were used to assemble the scFv. Then, the heavy chain (V) encoding the base linker, variable fragment, was synthesized in GenScript. H ), intermediate joints, and variable segment light chains (V LThe sequence contains nucleic acid sequences of a base linker and another base linker. These sequences are flanked by BstEII sites, which are also present on the GVO2.2 envelope plasmid. A scFv containing the BstEII fragment is cloned into the GVO2.2 plasmid to replace the ZZ domain in the GVO2.2 plasmid. As shown in Figure 1, the targeting portion (tm) used in this embodiment is the scFv. Further modifications can be made based on the GVO2.2 envelope with tm.

[0228] To incorporate the nucleic acid sequence encoding scFv into the SB envelope gene of the GVO2.2 envelope plasmid, seven different adapter configurations, including basal and intermediate adapters, were designed, as shown in Table 1, since different adapter structures or lengths can significantly affect transduction efficiency. The five basal adapters tested were: GGGGSGGGGS (SEQ ID NO: 2), Whitlow adapter (SEQ ID NO: 3) (Whitlow et al., Protein Eng, 1993, 6:989-95), the IgG4 hinge region between Fab and Fc (SEQ ID NO: 4), the hIgG1 hinge region (SEQ ID NO: 5), and AAGHVG (SEQ ID NO: 6). The intermediate linkers tested comprised different combinations of sequences of 14 or 18 amino acids, namely SEQ ID NO: 7 for the 14-amino acid linker and SEQ ID NO: 8 for the 18-amino acid linker (Aires da Silva et al., Hum. GeneTher., 2005, 16:223-34). Table 2 shows the sequence alignments of all seven constructs integrated with different linker constructs (SEQ ID NO: 9-15). Human CD47 scFv was tested as a proof of concept.

[0229] Table 1. Amino acid sequences of the linker of the SB envelope protein with scFv as the targeting part. Table 2. Amino acid alignment of constructs #1 to #7 of the GVO2.2 plasmid with CD47-scFv Engineered viral vectors containing CD47-modified scFv (as the target portion) are transmitted via a modified GVO2.2 plasmid carrying scFv (GVO2.2-scFv plasmid) and pCGPN ( gagpolThe engineered viral vector was generated by transient transfection of the plasmid and payload plasmid together. The method for generating the engineered viral vector was described in previous examples. The physical titer of the engineered viral vector was measured by RT-qPCR, the engineered viral vector was aliquoted, and stored at ≤ -65°C until use. This engineered viral vector with scFv as the targeting motif was then tested with CD47-positive cell lines to examine targeted transduction. Luciferase reporter plasmids were used in most experiments to determine transduction efficiency.

[0230] The transduction of various cancer cell lines was examined using a GVO2.2 pseudotyped viral vector (GVO2.2-scFv-C-1) embedded with human CD47 scFv. For example... Figure 5A As shown, the GVO2.2 pseudotyped viral vector embedding CD47 scFv exhibits transduction activity, but the level depends on the adapter structure and configuration. In this figure, the numbers (#) correspond to the configurations shown in Table 1. Numbers #8 to #14 have the same adapter as #1 to #7; however, #8 to #14 have a reverse V L , its in Figure 5B This trend has also been observed in other cell lines, as shown in the diagram. Figure 6 This demonstrates the transduction of various cancer cell lines using a GVO2.2 pseudotyped MLV vector (GVO2.2-scFv-C-1) embedded with human CD47 scFv or a bitropically encapsulated (RVE) pseudotyped MLV vector. Figure 6 As shown, luciferase expression differs across different cell lines. This is likely due to variations in the expression levels of surface CD47 in different cell lines.

[0231] Adapter #6, containing the basal adapter of SEQ ID NO: 6 and the intermediate adapter of SEQ ID NO: 8, was selected for use in other GVO2.2-scFv plasmids because this adapter showed the highest transduction in the cells tested. Various SB-scFv plasmids with different scFv targets (such as mCD47, CD47, CEA, HER2, EGFR, PSMA, and nectin-4) were generated using different basal and intermediate adapters.

[0232] Currently, the relationship between viral vector transduction and the type and length of the base and intermediate adapters remains unclear. However, for the intermediate adapter, viral vectors with longer intermediate adapters exhibit higher transduction, while for the base adapter, viral vectors with shorter base adapters show relatively higher transduction. Further research is needed to determine the relationship between adapter type and length and transduction.

[0233] In summary, this experiment demonstrates that targeting moieties such as scFv can be embedded in SB envelope proteins and can replace the ZZ domain. Furthermore, different conformations of the intermediate and basal linkers play a crucial role in the transduction efficiency of engineered viral vectors.

[0234] Example 4. Generation and testing of GVO2.2-dual antibody vector.

[0235] Following the successful application of GVO2.2 plasmids incorporating scFv, a biantibody (DB, a non-covalent dimer of scFv) was designed and incorporated into the SB envelope protein of the GVO2.2 envelope plasmid. In this embodiment, this DB replaces scFv as the targeting component. Within the DB, each V... H The structural domain is connected to a V via a short connector. L The domain forms two antigen-binding sites. With twice the antigen-binding capacity, DB can exhibit higher target binding and thus perform stronger vector transduction. One challenge compared to scFv, which is an approximately 25 kDa insert, is that DB has a molecular weight of approximately 55 kDa, which is almost the same size as the E2 domain of the SB envelope protein or about half the size of the entire SB envelope polypeptide. Therefore, adding DB to the SB envelope gene of GVO2.2 is considered challenging.

[0236] Perform the method as described in Example 1.

[0237] Figure 7 This shows a schematic conformation of scFv (left inset) and DB (right inset) in the SB envelope protein. In short, within the E2 region of the SB envelope protein (where the ZZ domain is located), the ZZ domain is replaced with the targeting portion scFv or DB. In the left inset, the scFv-SB envelope protein construct contains a linker V H and V L It contains two basal linkers and one intermediate linker. In the inset on the right, the DB-SB envelope protein construct comprises two basal linkers, three intermediate linkers, and two sets of V... H and V L .based on Figure 1A The diagram shows the insertion of scFv or DB at the target region (tm). For plasmids embedding DB, the sequence is from the N-terminus to the C-terminus of the SB envelope protein, followed by the basal linker after the first BstEII site at the N-terminus, and then V. H 18 amino acid linkers, V L 18 amino acid linkers, V H The linker consists of 18 amino acids and another basal linker preceding the second BstEII site at the C-terminus. L .

[0238] Discovery of furin cleavage sites in the scFv or DB targeting moiety of HER2 First, the SB envelope gene was engineered to incorporate a nucleic acid sequence encoding a dual antibody against HER2. This GVO2.2-DB-H-1 plasmid was coupled with two distinct intermediate linkers between two scFvs using V... H -V L -Connector-V H -V L HER2-targeted engineered viral vectors are engineered in the following directions. The first intermediate linker (linker 1) has a 15-mer of SSSSGSSSSGSSSSG (SEQ ID NO: 16), while the second intermediate linker (linker 2) has an 18-mer of SSGGGGSGGGGGGGSSRSS (SEQ ID NO: 8). These HER2-targeted engineered viral vectors are generated using the method described in the previous embodiments. The HER2-targeted engineered viral vectors result in low transduction, with the engineered viral vector having linker 2 exhibiting slightly higher activity. Figure 8 As shown, Western blot analysis using a rabbit anti-E2 polyclonal antibody (manufactured in-house by GenScript) was performed on concentrated vector particle samples and cell samples at the end of vector production. This analysis was run to determine the cause of the low transduction. Compared to the engineered viral vector generated from the GVO2.2-scFv-C-1 plasmid described in Example 3, the HER2-targeting engineered viral vectors generated from the GVO2.2-scFv-H-1 and GVO2.2-DB-H-1 plasmids both exhibited unexpectedly small fragments of approximately 20 kDa. When the engineered viral vectors were generated in the presence of a furin protease inhibitor, the intensity of the approximately 20 kDa fragments became very low, while the predicted bands of scFv or DB became more prominent. Therefore, the presence of furin protease cleavage sites within scFv-H-1 or DB-H-1 is suspected to be the cause of the low transduction.

[0239] like Figure 8 As shown, the predicted sizes of each SB envelope construct are: GVO2.2-scFv-C-1 (targeting CD47), 81 kDa; GVO2.2-scFv-H-1 (targeting HER2), 81.6 kDa; GVO2.2-DB-H-1 (targeting HER2), 110 kDa. Assuming the small fragments are due to furin cleavage, then for GVO2.2-scFv-H-1, the small fragment size is 15.6 kDa and the rest is 65.8 kDa; for GVO2.2-DB-H-1, the small fragments are 15.6 kDa and 28.5 kDa, and the rest is 65.9 kDa.

[0240] Potential furin cleavage sites were estimated using the ProP-1.0 website (services.healthtech.dtu.dk / services / ProP-1.0 / ), which predicts arginine and lysine propeptide cleavage sites in eukaryotic protein sequences. The amino acid sequence of HER2 antibodies was examined, and potential furin cleavage sites were identified that could interfere with the generation of functional HER2-targeting scFv or DB-engineered viral vectors. The results indicate that potential furin cleavage sites exist in both the H-1 (HER2-targeted) GVO2.2 scFv construct and the dual-antibody construct.

[0241] Frin is a protease that cleaves specific peptide sequences and contributes to the activation of many protein processing and signaling molecules. Frin is known to play a crucial role in viral envelope processing and in the maturation of viral envelope proteins to be functionalized. Based on the hypothesis that furin can digest a shared sequence within the scFv-H-1 and DB-H-1 sequences, leading to the failure of engineered viral vector transduction in cells, a furin inhibitor was added during the production of a biantibody-engineered viral vector. As a result, the biantibody-engineered viral vector generated in the presence of the furin inhibitor successfully showed significantly increased transduction levels in the HER2-overexpressing cell line SKBR3. In response to this observation, a potential furin cleavage site was mutated. Based on amino acid sequence analysis, a lysine residue was mutated to histidine, and this mutation removes the furin cleavage site from both HER2's scFv and DB. This modification involves the coding nucleotide from AAG to CAC, and is therefore termed a K2H (K to H; lysine to histidine) modification.

[0242] like Figure 9As shown, engineered viral vectors generated from modified HER2-targeting constructs (both scFv(K2H) and DB(K2H)) exhibited excellent transduction improvements. The transduction levels of the engineered viral vectors generated using the scFv(K2H)-H-1 or DB(K2H)-H-1 plasmids depended on HER2 expression on the cells used for transduction. In this figure, scFv-H-1 and scFv-H-2 are based on two different antibodies (H-1 or H-2), which resulted in different transduction levels. scFv-H-1 refers to a HER2-targeting scFv derived from the H-1 mAb sequence; scFv(K2H)-H-1 refers to a HER2-targeting scFv derived from the H-1 mAb sequence and also includes a modification to remove the furin cleavage site located in the HER2 scFv sequence; scFv-H-2 refers to a HER2-targeting scFv derived from the H-2 mAb sequence; scFv(K2H)-H-2 refers to a HER2-targeting scFv derived from the H-2 mAb sequence and also includes a modification to remove the furin cleavage site located in the HER2 scFv sequence; DB(K2H)-H-1 refers to a HER2-targeting DB derived from the H-1 mAb sequence with a modification to remove the furin cleavage site located in the HER2 DB sequence. H-1 represents anti-HER2 mAb trastuzumab. H-2 represents anti-HER2 mAb obtained from clone 191924.

[0243] In addition to the HER2 biantibody, an EGFR biantibody construct was also developed based on mAb cloning. This construct exhibited high transduction in various cell lines in both conjugated and scFv forms. This biantibody construct was generated in a similar manner to the HER2-DB construct. However, the transduction ability of engineered viral vectors coated with EGFR-DB was very poor. Engineered viral vectors with EGFR-DB modification showed lower transduction than their corresponding scFv vectors. Therefore, testing each modification of the GVO2.2 construct is necessary to determine the transduction ability of engineered viral vectors.

[0244] In summary, this experiment demonstrates that targeting regions such as DB can be embedded in SB envelope proteins. Furthermore, this embodiment shows that the presence of protease cleavage sites (such as furin cleavage sites) within the targeting region can affect the transduction efficiency of engineered viral vectors.

[0245] Example 5. GVO2.2 was modified into a GVO2.2F carrier.

[0246] Although engineered viral vectors generated from the GVO2.2 plasmid showed targeted transduction upon conjugation to various antibodies, their absolute transduction levels were lower compared to viral vectors with a biphile envelope (RVE). One of the biggest issues regarding the mutations in the m168 construct is concerning m1, specifically the deletion at the E3-E2 boundary (amino acids 61-64) (amino acid numbering based on SEQ ID NO: 1). This mutation removes the amino acid sequence RSKR at the C-terminus of the E3 domain, which is the recognition sequence for the furin cleavage site. This furin cleavage site is present in the wild-type sequence of the SB envelope protein. Therefore, this m1 mutation prevents furin cleavage of the construct, causing the E3 domain (which normally separates from the E2 domain) to remain as part of the E2 domain. Since this appears to contradict the original process of envelope protein maturation, the furin recognition sequence was reintroduced by inserting the original sequence into the GVO2.2 plasmid, and this new plasmid with the furin cleavage site at the E3-E2 boundary was named GVO2.2F. In summary, when compared with wild-type SB envelope protein, GVO2.2F has m6 mutation, m8 mutation, addition of ZZ protein domain, Y226G227 substitution in E1 domain, and furin cleavage site at E3-E2 boundary (replacing m1 modification in GVO2.2).

[0247] Perform the method as described in Example 1.

[0248] like Figure 10 As shown, engineered viral vectors generated with antibody-conjugated modified GVO2.2F envelope plasmids (e.g., GVO2.2F-YG, GVO2.2F-SG, and GVO2.2F-AK) exhibited significantly increased transduction under each antibody examined, averaging 10-20 times higher than GVO2.2 with antibodies. The detailed mechanism of action remains unclear, but dissociation of the E3 domain from the E2 domain may play a key role in this strong transduction. Importantly, this modification showed only a small increase in background transduction, such as that without conjugated antibodies. Furthermore, the transduction levels of each engineered viral vector differed when conjugated with antibodies using engineered viral vectors generated with either GVO2.2 or GVO2.2F plasmids. Engineered viral vectors generated with GVO2.2F-AK and GVO2.2F-YG plasmids showed the highest transduction in SKBR3 cells, followed by the GVO2.2F-SG plasmid. Each cancer cell line showed different outcomes under different mutations.

[0249] In summary, these results show that the transduction efficiency of engineered viral vectors generated from GVO2.2F plasmid is improved by reintroducing the furin cleavage site into the E3-E2 boundary of the modified SB envelope protein compared to the transduction efficiency of engineered viral vectors generated from GVO2.2 plasmid.

[0250] Example 6: Modify the targeting portion of GVO2.2F with scFv or a double antibody.

[0251] The GVO2.2F construct from Example 5 can be further modified by inserting an scFv fragment or a double antibody fragment into the GVO2.2F plasmid to replace the ZZ domain. Since all scFv and DB fragments have BstEII sites at both ends, these constructs can be easily generated by inserting scFv or DB fragments with BstEII restriction enzyme sites flanking the GVO2.2F plasmid.

[0252] Perform the method as described in Example 1.

[0253] like Figure 11 As shown, when compared with GVO2.2 constructs having K2H modification [see GVO2.2-scFv(K2H)-H-1 and GVO2.2-DB(K2H)-H-1] or without K2H modification [see GVO2.2-scFv-H-1 and GVO2.2-DB-H-1], the HER2-targeting scFv construct with GVO2.2F and the HER2-targeting dual antibody constructs (GVO2.2F-(K2H) [see GVO2.2F-scFv(K2H)-H-1 and GVO2.2F-DB(K2H)-H-1] with the furin cleavage site removed from the targeting portion (described in Example 4) showed improved transduction in SKBR3 cells.

[0254] Example 7: Specificity of the targeting portion of the GVO2.2F vector.

[0255] As described in Example 6, engineered viral vectors were generated using a GVO2.2F construct with a targeting moiety, demonstrating significant transduction levels in cells expressing the target molecule. However, it remains unclear whether the targeting moiety specifically binds to the target molecule prior to the transduction process (including the internalization of the vector particles). To investigate this hypothesis, cancer cell lines from similar cancer types but with different levels of target receptor expression were used in this example.

[0256] Perform the method as described in Example 1.

[0257] Breast cancer cell lines SKBR3 and MDA-MB-468 express different levels of HER2 on their cell surface (confirmed using FACS via a fluorescently labeled HER2 antibody). Figure 12 As shown, the engineered viral vector generated from the GVO2.2F-DB(K2H)-H-1 plasmid exhibited high transduction in HER2-overexpressing SKBR3 cells, while low transduction was observed in MDA-MB-468 cells with low HER2 expression.

[0258] In addition, such as Figure 13A As shown, engineered viral vectors with two different adapters (configurations #5 and #6 in Table 1), generated using GVO2.2F-scFv-N-1 (nectin-4), can specifically bind to and transduce nectin-4-positive cell lines (such as the MDA-MB-468 cell line), but not in low nectin-4 expression cell lines (such as the PC-3 and A375 cell lines). This transduction efficiency is related to the expression level of nectin-4 on the cell surface, which is... Figure 13B As shown in the diagram. NTC refers to a negative control (only non-transduced cells) in the well, where no transduced cells were present.

[0259] Alternatively, CHO-K1 cells were used to examine the specificity of the targeting vector. This cell line is insensitive to the bitropic pseudotyped MLV vector. CHO-K1 cells were transduced with lenti-EGFR-puro or lenti-HER2-puro vectors to establish stable EGFR or HER2 expression cell lines. Next, engineered viral vectors generated from the GVO2.2F-scFv-E-1 plasmid showed transduction in the CHO-K1-EGFR cell line, but not in parental CHO-K1 and CHO-K1-HER2 cells. Similarly, engineered viral vectors generated from the GVO2.2F-scFv(K2H)-H-1 plasmid showed transduction in CHO-K1-HER2 cells, but not in other cell lines. Based on these data, engineered viral vectors with scFv or DB targeting motifs showed good specificity.

[0260] Example 8: GCV-mediated cytotoxicity of GVO2.2F vector with HSV1-TK To study the function of expressed proteins when transduced into target cells using engineered viral vectors, herpes simplex virus 1-thymidine kinase (HSV1-TK) was incorporated as a transgene, and ganciclovir (GCV)-mediated cytotoxicity was examined in test cells. The mutant HSV1-TK exhibits a higher substrate-binding affinity for GCV compared to the natural substrate (thymidine), and its intracellular localization is altered through genetic modifications as presented in US9925276.

[0261] Perform the method as described in Example 1.

[0262] GCV-mediated cell killing assay (also known as relative potency assay) SKBR3 cells plated in 96-well plates were transduced with RDRS (a biphilic pseudotyped MLV vector with an HSV1-TK mutation, purified from GVO) and GVO2.2F-scFv-H-1 or GVO202F-DB-H-1 at the same genomic copy titer in the presence of polyglobulin (8 μg / mL). The vectors were serially diluted 2-fold, starting at 4e7 RNA genome copies / well and increasing to 1.56e5 RNA genome copies / well. The medium was replaced the following day with medium containing 20 μM GCV. Since SKBR3 cells are relatively slow-growing, they were cultured for 5 days after adding GCV, compared to 3 days for A375 cells (cell doubling time less than 1 day). Cell viability was determined using PrestoBlue, and cell killing was analyzed using 4PL curves (SpectraMax M5).

[0263] Several breast cancer cell lines with high HER2 expression (such as SKBR3 and SKOV3 cells) were examined using engineered viral vectors generated from GVO2.2F-scFv(K2H)-H-1 or GVO2.2F-DB(K2H)-H-1 plasmids carrying payload genes for expressing human granulocyte-macrophage colony-stimulating factor (GM-CSF) and HSV1-TK. Cell killing assays were performed accordingly to evaluate transduction efficiency compared to previously qualified reference standards (Research and Development Reference Standard (RDRS) 20A) carrying the same payload genes.

[0264] like Figures 14A-14B As shown, the GVO2.2F-scFv(K2H)-H-1 plasmid ( Figure 14A ) and GVO2.2F-DB(K2H)-H-1 plasmid ( Figure 14B The engineered viral vectors generated exhibited significant cytotoxic activity in SKBR3 cells. Specifically, in SKBR3 cells, the engineered viral vectors derived from the scFv(K2H)-H-1 or DB(K2H)-H-1 plasmids showed higher cytotoxicity than the RDRS20A reference standard. In SKOV3 cells, the cytotoxicity of the engineered viral vectors generated from the scFv(K2H)-H-1 or DB(K2H)-H-1 plasmids was slightly lower than that of RDRS20A.

[0265] Example 9: The receptor ligand was incorporated as the targeting component into the GVO2.2F vector.

[0266] Based on the successful results obtained from incorporating the scFv or DB targeting portion into the SB envelope protein, pseudotyped engineered viral vectors (e.g., pseudotyped MLV vectors with the SB envelope protein) should be able to incorporate other ligands of cell surface receptors and can be used as binding mechanisms to initiate transduction into target cells. These ligands include, but are not limited to, G protein-coupled receptor ligands (such as endorphins and oxytocin), ligand peptides of GPR78, small peptide ligands of EGFR, and the C-terminal domain of platelet-reactive proteins targeting CD47. This system has the potential to be applied to many other ligand-receptor relationships.

[0267] Example 10: Applying GVO2.2 or GVO2.2F vectors to vaccine products.

[0268] Recently, new types of vaccine products targeting SARS-CoV-2 have been approved, including mRNA and adenovirus vectors. These vaccines are introduced into cells and generate immunogens to trigger an immune response. This is significantly different from traditional vaccine products that consist of their own immunogens. Therefore, retroviral vectors can also function as vaccines, where the RNA genome is reverse transcribed into complementary DNA, which is then transcribed into multiple mRNA copies over a longer period. Thus, compared to vaccines that only deliver mRNA, there is the potential for longer-lasting immunogen expression and associated immune responses. These considerations are independent of the envelope proteins expressed on the surface of the virus or vector particles; retroviral vectors carrying wild-type or engineered envelope proteins (or mixtures thereof), including those carrying the Sindbis envelope protein, all have the potential to serve as vaccines, through the delivery of a genome encoding an immunogenic payload.

[0269] GVO-wtSB vector Wild-type SB (wtSB) exhibits broad tropism. Therefore, envelope proteins derived from wtSB are strong candidates for use in retroviral vectors intended for vaccines. To generate GVO-wtSB, a portion of the wild-type sequence was synthesized to replace the mutated sequence in GVO2.2 (SEQ ID NO: 20). Transduction experiments were performed using various cell lines. Absolute transduction levels were lower than with conjugated antibody vectors, scFv vectors, or biantibody vectors; however, most cell lines showed sensitivity to the GVO-wtSB vector.

[0270] GVO-E160G-SB carrier In addition to the wild-type construct, a mutant GVO-E160G-wtSB (SEQ ID NO: 22) with a single amino acid substitution of the wild-type construct was generated to preferentially target antigen-presenting cells (Gardner et al. J. Virol. 2000, 74: 11849-57). Antigen-presenting cells are the gateway to the immune response; therefore, targeting antigen-presenting cells (such as dendritic cells) can induce an immune response. The construct GVO-E160G-wtSB was generated by mutagenesis using the GVO-wtSB plasmid (SEQ ID NO: 21) as a template. A vector was generated and tested on cancer cell lines. However, the transduction level was lower than that of the engineered viral vector generated using the GVO-wtSB plasmid. The transduction efficiency was also very low when the vector with this envelope was examined in mouse dendritic cells.

[0271] GVO2.2F-CoVec(N) vector The E2 region of the SB envelope protein has the capacity to incorporate additional components, regardless of their size. Therefore, portions or entire viral proteins, such as the envelope or spike protein, can be inserted into the E2 domain. In previous embodiments, the use of the SB envelope protein as a scaffold to incorporate the IgG Fc binding domain (ZZ protein domain), scFv, and biantibody was described. These different components were inserted into the BstEII site in the E2 region of the GVO2.2 construct. Information regarding size limitations is currently insufficient; however, the capacity appears to be quite high, given that the biantibody insert is approximately 55 kDa in length, encoding a 531-amino acid polypeptide, while the wtSB has only a small number of amino acids in the corresponding region (the entire wtSB is approximately 108 kDa, and the E2 region is approximately 47 kDa).

[0272] As an example of an immunogenic protein, the N-terminal domain (NTD) of the SARS-CoV-2 spike protein was inserted into the BstEII site to generate the GVO2.2F-CoVec(N) construct (SEQ ID NO: 23). A pseudotyped MLV vector, SB-CoVec(N), was generated by conventional triple plasmid transfection. This vector has an MLV core and an SB envelope protein incorporating the NTD of the SARS-CoV-2 spike protein. The MLV core is capable of carrying any gene of interest comparable in size to the full-length spike of the SARS-CoV-2 virus or a specific region. The vector was then assembled and examined in animal models for an immune response against SARS-CoV-2. This vector provides proteins on its surface as immunogens in addition to the MLV payload. This engineered viral vector is suitable for evaluation in animal models to investigate whether the SARS-CoV-2 envelope spike protein stimulates an immune response.

[0273] Example 11: Combination therapy with two GVO pseudotyped carriers BT474 cell transduction was examined using a single agent (GVO2.2-scFv-C-1, GVO2.2-scFv-H-1, or GVO2.2-DB(K2H)-H-1 vector) or a combination of two GVO pseudotype vectors.

[0274] Perform the method as described in Example 1.

[0275] like Figure 15A As shown, the combination of the two GVO pseudotyped vectors, GVO2.2-scFv-C-1 and GVO2.2-scFv-H-1, exhibited increased transduction efficacy in BT474 cells compared to single-agent GVO2.2-scFv-C-1 or GVO2.2-scFv-H-1. Figure 15B As shown, the combination of the two GVO pseudotyped vectors, GVO2.2-scFv-C-1 and GVO2.2-DB(K2H)-H-1, exhibited increased transduction efficacy in BT474 cells compared to single agents GVO2.2-scFv-C-1 or GVO2.2-DB(K2H)-H-1. These results suggest that different combinations of GVO pseudotyped vectors can be used to target two different surface molecules in the same cell population.

[0276] Example 12: In vivo transduction via the GVO2.2F-DB(K2H)-H-1 vector was more efficient than that via the RVE control vector. body like Figure 16A As shown, human ovarian cancer SKOV3ip-fLuc2 cells were intraperitoneally injected into immunodeficient mice. Five days post-injection, each group of mice (n=6) was intraperitoneally injected every 5 days (4 times) with either a GFP-expressing GVO2.2F-DB(K2H)-H-1 vector, a GFP-expressing RVE control vector, or control culture medium. For each injection, each mouse received the same infection titer of vector daily. Tumor growth was tracked by bioluminescence imaging, and overall growth was monitored by animal body weight (no differences between groups, data not shown). On day 23, animals were sacrificed, and tumors were harvested from the peritoneum. The collected cells were isolated and subjected to flow cytometry, where SKOV3ip-fLuc2 human tumor cells were identified using an antibody against human β2 macroglobulin (huβ2M).

[0277] By GFP + / huβ2M + The percentage of tumor cells indicates the transduction of the corresponding vector. For example... Figure 16B As shown, GFP-positive human tumor cells (GFP) + / huβ2M +The percentage of GFP-positive cells was significantly higher in the GVO2.2F-DB(K2H)-H-1 group than in the control and RVE control vector groups. Meanwhile, the percentage of tumor cells in the GFP-positive population was comparable between the GVO2.2F-DB(K2H)-H-1 group and the RVE control vector group. Figure 16C (As shown in the figure). These results indicate that in vivo transduction via the GVO2.2F-DB(K2H)-H-1 vector is more efficient than that via the RVE control vector.

[0278] Example 13: EGFR targeting vector effectively and specifically transduces target cells expressing EGFR. EGFR targeting vectors were constructed using the EGF core domain (53 amino acids) incorporated into a modified Sindbis envelope. These EGFR targeting vectors were used to transduce SKBR3 cells. The physical titer of GVO2.2F-EGF adapter 5 used for transduction was 4.6e6 vector genome copies / well. The physical titer of GVO2.2F-EGF adapter 6 used for transduction was 5.2e6 vector genome copies / well. Figure 17A and Figure 17B The results showed that the EGFR-targeting vector successfully transduced SKBR3 cells using EGF ligand targeting. EGFR-targeting vectors containing adapter 5 transduced cells better under these conditions.

[0279] HT29-Luc cells were transduced using EGFR-targeting vectors. The physical titer of the RVE control vector used for transduction was 2.75e8 vector genome copies / well. The physical titer of the GVO2.2F-scFv-E-1 vector used for transduction was 2.6e8 vector genome copies / well. The physical titer of the GVO2.2F-EGF adapter 5 vector used for transduction was 6.6e7 vector genome copies / well. NTC cells were used as non-transduced control cells. Figure 18 The results showed that, when taking into account differences in physical titers used for transduction, EGF ligand targeting had similar relative infectivity in HT-29-Luc cells compared to the RVE vector and GVO2.2F-scFv-E-1.

[0280] EGFRvIII is a constitutively active EGFR mutant that cannot bind EGF ligands due to extracellular truncation. EGFRvIII contains deletions of domain I (L1) and domain II (CRI) or deletions of amino acids 6-273 of EGFR. Transduction of EGFR targeting vectors was evaluated using U87MG wild-type (U87MGwt) cells and U87MG-EGFRvIII cells. Figure 19 The results showed that both the GVO2.2F-scFv-E-1 vector and the GVO2.2F-EGF adapter 5 vector could transduce U87-EGFRvIII cells.

[0281] The transduction specificity of the EGFR-targeting vector was evaluated by transducing wild-type 293T cells and EGFR-expressing 293T cells at two different multiples of infection (MOI). Figure 19 The 5x indicates that more than 5 times the amount of vector was used in the experiment. The physical titer of all vectors used for transduction is 5e7 vector genome copies / well. Figure 20 The binding specificity of the targeting vectors was demonstrated, including the GVO2.2F-scFv-E-1 vector and the GVO2.2F-EGF linker 5 vector.

[0282] Example 14: Contains anti-PD-L1 V H The PD-L1 targeting vector H was successfully transduced into PD-L1 expressing cells.

[0283] PD-L1 targeting vectors are retroviral vectors constructed to contain anti-PD-L1 V. H H-modified Sindbiose capsule. PD-L1-expressing cells, including RKO cells and HT1080 cells, were transduced using PD-L1-targeting vectors. orig and delG are two different anti-PD-L1 V... H H vector. The amino acid sequence was optimized in delG. Figure 21 The results showed that the PD-L1 targeting vector was successfully transduced in both RKO and HT1080 cells. Since PD-L1 expression was higher in RKO cells compared to HT1080 cells, the transduction level was also higher in RKO cells.

[0284] The effects of various connectors on the transduction efficiency of the target carrier were evaluated. The connector and its configuration are v1: GGGGS-AAGHVG-V H H-GVHGAA, v2: AAGHVG-V H H-GVHGAA-GGGGS, v3: GGGGS-AAGHVG-V H H-GVHGAA-GGGGS, v4: GGGGS-GGGGS-AAGHVG-V H H-GVHGAA, v5: AAGHVG-V H H-GVHGAA-GGGGS-GGGGS, v6: GGGGS-GGGGS-AAGHVG-V H H-GVHGAA-GGGGS-GGGGS. Figure 22 and Figure 23 The results show that, apart from v4 in SKBR3, no build exhibits a performance higher than delG-optimized V. H The H construct showed higher transduction. Furthermore, a consistent pattern was observed in both cell lines. For example, v2 and v5 exhibited lower transduction levels.

[0285] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from this disclosure. It should be understood that various alternatives to the embodiments described herein may be employed. The following claims are intended to define the scope of this disclosure and thereby cover the methods and structures within the scope of these claims and their equivalents.

[0286] sequence

Claims

1. An engineered viral vector, the engineered viral vector comprising a modified envelope protein, wherein the modified envelope protein comprises: a) at least one target portion, and b) At least one modification in the wild-type protein sequence (SEQ ID NO: 1); The modified envelope protein, compared to a viral vector without the modified envelope protein, increases the transduction efficiency or specificity of the engineered viral vector for target cells.

2. The engineered viral vector of claim 1, wherein the at least one modification is in the E2 domain.

3. The engineered viral vector as described in claim 1 or claim 2, wherein the engineered viral vector includes an engineered retroviral vector.

4. The engineered viral vector as described in claim 3, wherein the engineered retroviral vector includes an engineered gamma retroviral vector.

5. The engineered viral vector of claim 4, wherein the engineered gamma retroviral vector comprises an engineered murine leukemia virus (MLV) vector.

6. The engineered viral vector according to any one of claims 1-5, wherein the modified envelope protein comprises a recombinant viral envelope protein derived from a DNA virus.

7. The engineered viral vector according to any one of claims 1-5, wherein the modified envelope protein comprises a recombinant viral envelope protein derived from an RNA virus.

8. The engineered viral vector of claim 7, wherein the RNA virus comprises an alphavirus.

9. The engineered viral vector of claim 8, wherein the alphavirus comprises Sindbis virus.

10. The engineered viral vector of claim 9, wherein the modified envelope protein derived from the Sindbis virus comprises an E3 domain, an E2 domain, a 6K domain, an E1 domain, or a combination thereof.

11. The engineered viral vector according to any one of claims 1-10, wherein the modified envelope protein further comprises a protease cleavage site located between the E3 and E2 domains.

12. The engineered viral vector of claim 11, wherein the protease cleavage site includes a furin protease cleavage site.

13. The engineered viral vector according to any one of claims 1-12, wherein the at least one targeting portion is located within the E2 domain.

14. The engineered viral vector according to any one of claims 1-13, wherein the at least one targeting portion includes a conjugation portion.

15. The engineered viral vector of claim 14, wherein the conjugation portion comprises an IgG binding domain of a bacterial protein.

16. The engineered viral vector of claim 15, wherein the bacterial protein is bacterial protein A.

17. The engineered viral vector of claim 15 or claim 16, wherein the IgG binding domain of the bacterial protein is a ZZ protein domain.

18. The engineered viral vector according to any one of claims 14-17, wherein the engineered viral vector is further conjugated with an antibody or an antigen-binding fragment thereof.

19. The engineered viral vector of claim 18, wherein the antibody or its antigen-binding fragment comprises IgG-scFv, a single variable domain (V) on the heavy chain, and so on. H H), nanobodies, BiTE, biantibodies, DART, TandAb, sc biantibodies, sc biantibodies-CH3, triantibodies, microantibodies, microantibodies, TriBi microantibodies, scFv-CH3 KIH, Fab-scFv-Fc KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc biantibodies-Fc, biantibodies-Fc, tandem scFv-Fc, intracellular antibodies, their binding fragments, their chemically modified derivatives, heavy chains of variable fragments (V H ), light chains of variable segments (V L (or more) combinations.

20. The engineered viral vector of claim 18 or claim 19, wherein the antibody or its antigen-binding fragment binds to the target ligand of the target cell.

21. The engineered viral vector according to any one of claims 1-20, wherein the target cell is a cancer cell.

22. The engineered viral vector of claim 21, wherein the cancer cells include human cancer cells.

23. The engineered viral vector of claim 21 or claim 22, wherein the cancer cells include leukemia cells, myeloid cells, promyeloid cells, myeloid mononuclear cells, mononuclear cells, erythroleukemia cells, chronic myeloid (granulocytic) leukemia cells, chronic lymphocytic leukemia cells, lymphoma cells such as Hodgkin's and non-Hodgkin's lymphoma cells, fibrosarcoma cells, myoma cells, liposarcoma cells, chondrosarcoma cells, osteosarcoma cells, angiosarcoma cells, endothelial sarcoma cells, Ewing's tumor cells, colon cancer cells, pancreatic cancer cells, breast cancer cells, ovarian cancer cells, prostate cancer cells, squamous cell carcinoma cells, basal cell carcinoma cells, adenocarcinoma cells, renal cell carcinoma cells, liver cancer cells, Wilms' tumor cells, cervical cancer cells, uterine cancer cells, testicular tumor cells, and lung cancer cells. Small cell lung cancer cells, bladder cancer cells, epithelial cancer cells, glioma cells, astrocytoma cells, oligodendroglioma cells, melanoma cells, neuroblastoma cells, retinoblastoma cells, dysplastic and hyperplastic cells, prostatitis cells, benign prostatic hyperplasia (BPH) cells, prostatic paraganglioma cells, prostate adenocarcinoma cells, prostate intraepithelial neoplasia cells, prostate-rectal fistula cells, atypical prostatic stromal lesion cells, bladder cancer cells, lung cancer cells, head and neck cancer cells, non-small cell lung cancer cells, renal cancer cells, glioblastoma cells, Merkel cell carcinoma cells, angiosarcoma cells, cutaneous T-cell lymphoma cells, cutaneous B-cell lymphoma cells, dermatofibrosarcoma protuberans cells, sebaceous gland cancer cells, or cutaneous neuroendocrine cancer cells.

24. The engineered viral vector of any one of claims 20-23, wherein the target ligand comprises a cell surface marker.

25. The engineered viral vector of claim 24, wherein the cell surface markers include cancer cell markers.

26. The engineered viral vector of claim 25, wherein the cancer cell markers comprise CD47, CEA, HER2, EGFR, PSMA, TfR, nectin-4, muc-1, endothelin, EphA2, EphB2, folate receptor, GRP78, IGF-1R, cMET / HGFR, Tn antigen, EpCAM, ανβ3 integrin, CD44, mesothelin, PSCA, uPAR, CAIX, CD13, FAP-α, matriptase, MT-1 MMP, MT6-MMP, KDR, KIT, CD276, CD-83, or combinations thereof.

27. The engineered viral vector of claim 20, wherein the target ligands include CD33, CD30, HER2, CD22, CD79b, nectin-4, Trop-2, BCMA, CD19, EGFR, folate receptor α, CD3, CD38, CD20, BCMA, CEA, PSMA, OX40, 4-1BB, CD16A, PD-1, PD-L1, CTLA-4, LAG-3, TIM3, VEGF, and CD4+.

7. ICOS, MET, LGR5, IGF-1R, HER3, ANG2, DLL4, FAP, DR5, fibronectin ED-B, tendinin C, EpCAM, TNFα, digoxin, GPIIb / IIIa, rattlesnake venom, scorpion venom, CA6, PLVAP, 5T4, EDB, EGFRvIII, 6B11, CXCR4, MSLN, FN1, GPC2, GPC3, VCAN1, COL11A1, or MMR.

28. The engineered viral vector of claim 20, wherein the target ligand of the target cell comprises an immune checkpoint protein or an immune checkpoint receptor.

29. The engineered viral vector of claim 28, wherein the target ligand of the target cell is an immune checkpoint protein.

30. The engineered viral vector of claim 29, wherein the immune checkpoint protein comprises PD-L1, CTLA-4, B7RP1, HVEM, CD137L, OX40L, CD40, CD70, GAL9, MHCII, CD47, VISTA, or GITR.

31. The engineered viral vector of claim 28, wherein the target ligand of the target cell is an immune checkpoint receptor.

32. The engineered viral vector of claim 31, wherein the immune checkpoint receptor comprises PD-1, ICOS (CD278), BTLA, CD137 (4-IBB), OX40 (CD134), CD40L, CD27, TIM3, CD20, or LAG3.

33. The engineered viral vector according to any one of claims 28-32, wherein the target cell is an immune cell.

34. The engineered viral vector of claim 33, wherein the immune cells include T cells, B cells, macrophages, natural killer (NK) cells, or dendritic cells.

35. The engineered viral vector of claim 20, wherein the target ligand of the target cell comprises a cell surface antigen.

36. The engineered viral vector of claims 1-13, wherein the at least one targeting portion includes a binding portion.

37. The engineered viral vector of claim 36, wherein the binding portion comprises a single-stranded variable fragment (scFv), a single variable domain (V) on a biantibody or heavy chain. H H).

38. The engineered viral vector of claim 37, wherein the binding portion comprises the scFv.

39. The engineered viral vector of claim 38, wherein the scFv is encoded by a nucleotide sequence in the E2 domain, wherein the nucleotide sequence encodes at least one basal linker, the heavy chain (V) of the variable fragment. H ), intermediate joints or light chains of the variable segments (V L ).

40. The engineered viral vector of claim 39, wherein the V encoding the scFv H or the V L The nucleic acid sequences described herein are derived from the same antibody.

41. The engineered viral vector of claim 39, wherein the V encoding the scFv H or the V L The nucleic acid sequences described are derived from different antibodies.

42. The engineered viral vector according to any one of claims 39-41, wherein the nucleotide sequence further comprises at least one restriction enzyme site.

43. The engineered viral vector of claim 42, wherein the at least one restriction enzyme site is located upstream, downstream, or a combination thereof of the nucleotide sequence in the E2 domain.

44. The engineered viral vector of claim 42 or claim 43, wherein the at least one restriction enzyme site includes the BstEII site.

45. The engineered viral vector according to any one of claims 39-43, wherein the at least one base connector comprises SEQ ID NO: 2-6.

46. ​​The engineered viral vector according to any one of claims 39-44, wherein the intermediate linker comprises at least 5 amino acids.

47. The engineered viral vector according to any one of claims 39-44, wherein the intermediate linker comprises up to 25 amino acids.

48. The engineered viral vector according to any one of claims 39-44, wherein the intermediate linker comprises between 5 and 25 amino acids.

49. The engineered viral vector according to any one of claims 39-48, wherein the intermediate linker comprises a linker of 14 amino acids or a linker of 18 amino acids.

50. The engineered viral vector according to any one of claims 39-49, wherein the intermediate connector comprises SEQ ID NO: 7-8.

51. The engineered viral vector according to any one of claims 39-50, wherein the V L It's the opposite.

52. The engineered viral vector of claim 37, wherein the binding portion comprises the dual antibody.

53. The engineered viral vector of claim 52, wherein the biantibody comprises a dimer of a single-chain variable fragment (scFv).

54. The engineered viral vector of claim 52 or claim 53, wherein the dual antibody is encoded by a nucleotide sequence in the E2 domain, wherein the nucleotide sequence encodes at least one basal linker and at least one heavy chain (V) of the variable fragment. H ), at least one intermediate joint or at least one light chain (V) of the variable segment L ).

55. The engineered viral vector of claim 54, wherein the at least one intermediate linker comprises at least 5 amino acids.

56. The engineered viral vector of claim 54, wherein the at least one intermediate linker comprises up to 25 amino acids.

57. The engineered viral vector of claim 54, wherein the at least one intermediate linker comprises between 5 and 25 amino acids.

58. The engineered viral vector according to any one of claims 54-57, wherein the at least one intermediate linker comprises a linker of 18 amino acids.

59. The engineered viral vector according to any one of claims 54-58, wherein the at least one intermediate connector comprises SEQ ID NO: 7-8.

60. The engineered viral vector of claim 53, wherein the dimer of the scFv is derived from the same antibody.

61. The engineered viral vector of claim 53, wherein the dimer of the scFv is derived from different antibodies.

62. The engineered viral vector of claim 54, wherein the at least one V H They originate from the same antibody.

63. The engineered viral vector of claim 54, wherein the at least one V H They originate from different antibodies.

64. The engineered viral vector of claim 54, wherein the at least one light chain (V) of the variable fragment L They originate from the same antibody.

65. The engineered viral vector of claim 54, wherein the at least one light chain (V) of the variable fragment L () originates from different antibodies.

66. The engineered viral vector according to any one of claims 36-65, wherein the binding portion further comprises at least one modification relative to the sequence of the wild-type binding portion.

67. The engineered viral vector of claim 66, wherein the at least one modification comprises an amino acid substitution that removes a protease cleavage site from the binding moiety.

68. The engineered viral vector of claim 67, wherein the protease cleavage site is a furin protease cleavage site.

69. The engineered viral vector of claim 67 or claim 68, wherein the at least one modification comprises a substitution of lysine to histidine.

70. The engineered viral vector of claim 37, wherein the V H The connecting portion of H includes the following: connector 1-V H H-Connector 2.

71. The engineered viral vector of claim 70, wherein the connector 1 is selected from (GGGGS)nAAGHVG, AAGHVG(GGGGS)n, (GGGGS)nAAGHVG(GGGGS)n, (GGGGS)nGVHGAA, GVHGAA(GGGGS)n and (GGGGS)nGVHGAA(GGGGS)n.

72. The engineered viral vector of claim 70 or 71, wherein the connector 2 is selected from (GGGGS)nAAGHVG, AAGHVG(GGGGS)n, (GGGGS)nAAGHVG(GGGGS)n, (GGGGS)nGVHGAA, GVHGAA(GGGGS)n and (GGGGS)nGVHGAA(GGGGS)n.

73. The engineered viral vector of any one of claims 36-72, wherein the binding portion comprises a nucleotide sequence encoding its antigen-binding fragment, wherein the antigen-binding fragment binds to a target ligand of the target cell.

74. The engineered viral vector of claim 73, wherein the target cell is a cancer cell.

75. The engineered viral vector of claim 74, wherein the cancer cells include human cancer cells.

76. The engineered viral vector of claim 74 or claim 75, wherein the cancer cells include breast cancer cells, colon cancer cells, bladder cancer cells, lung cancer cells, head and neck cancer cells, squamous cell carcinoma, pancreatic cancer cells, prostate cancer cells, melanoma cells, non-small cell lung cancer cells, renal cancer cells, glioblastoma cells, Merkel cell carcinoma cells, angiosarcoma cells, cutaneous T-cell lymphoma cells, cutaneous B-cell lymphoma cells, dermatofibrosarcoma protuberans cells, sebaceous gland cancer cells, or cutaneous neuroendocrine cancer cells.

77. The engineered viral vector of any one of claims 73-76, wherein the target ligand comprises a cell surface marker.

78. The engineered viral vector of claim 77, wherein the cell surface markers include cancer cell markers.

79. The engineered viral vector of claim 78, wherein the cancer cell markers comprise CD47, CEA, HER2, EGFR, PSMA, TfR, nectin-4, muc-1, endothelin, EphA2, EphB2, folate receptor, GRP78, IGF-1R, cMET / HGFR, Tn antigen, EpCAM, ανβ3 integrin, CD44, mesothelin, PSCA, uPAR, CAIX, CD13, FAP-α, matriptase, MT-1 MMP, MT6-MMP, KDR, KIT, CD276, CD-83, or combinations thereof.

80. The engineered viral vector of claim 73, wherein the target ligand comprises CD33, CD30, HER2, CD22, CD79b, nectin-4, Trop-2, BCMA, CD19, EGFR, folate receptor α, CD3, CD38, CD20, BCMA, CEA, PSMA, OX40, 4-1BB, CD16A, PD-1, PD-L1, CTLA-4, LAG-3, TIM3, VEGF, CD4+.

7. ICOS, MET, LGR5, IGF-1R, HER3, ANG2, DLL4, FAP, DR5, fibronectin ED-B, tendinin C, EpCAM, TNFα, digoxin, GPIIb / IIIa, rattlesnake venom, scorpion venom, CA6, PLVAP, 5T4, EDB, EGFRvIII, 6B11, CXCR4, MSLN, FN1, GPC2, GPC3, VCAN1, COL11A1, or MMR.

81. The engineered viral vector of claim 73, wherein the target ligand of the target cell comprises an immune checkpoint protein or an immune checkpoint receptor.

82. The engineered viral vector of claim 81, wherein the target ligand of the target cell is an immune checkpoint protein.

83. The engineered viral vector of claim 82, wherein the immune checkpoint protein comprises PD-L1 or CTLA-4.

84. The engineered viral vector of claim 81, wherein the target ligand of the target cell is an immune checkpoint receptor.

85. The engineered viral vector of claim 84, wherein the immune checkpoint receptor comprises PD-1.

86. The engineered viral vector according to any one of claims 81-85, wherein the target cell is an immune cell.

87. The engineered viral vector of claim 86, wherein the immune cells include T cells, B cells, macrophages, natural killer (NK) cells, or dendritic cells.

88. The engineered viral vector of claim 73, wherein the target ligand of the target cell comprises a cell surface antigen.

89. The engineered viral vector according to any one of claims 1-88, further comprising, relative to the wild-type protein sequence (SEQ ID NO: 1), at least one amino acid modification in the E1 domain.

90. The engineered viral vector of claim 89, relative to the wild-type protein sequence (SEQ ID NO: 1), wherein the at least one amino acid modification in the E1 domain comprises at least one amino acid substitution.

91. The engineered viral vector of claim 90, wherein the at least one amino acid substitution comprises an amino acid substitution at position 226, an amino acid substitution at position 227, or a combination thereof, wherein the amino acid position is based on the numbering of the amino acid on the E1 domain.

92. The engineered viral vector of claim 91, wherein the amino acid substitution at position 226 comprises A226S or A226Y substitution.

93. The engineered viral vector of claim 91 or claim 92, wherein the amino acid substitution at position 227 includes a K227G substitution.

94. The engineered viral vector according to any one of claims 90-93, wherein the at least one amino acid substitution further comprises an amino acid substitution at position 160.

95. The engineered viral vector of claim 91, wherein the amino acid substitution at position 160 includes an E160G substitution.

96. The engineered viral vector according to any one of claims 1-95, wherein the at least one modification in the E2 domain, relative to the wild-type protein sequence (SEQ ID NO: 1), comprises the substitution of one or more amino acids.

97. The engineered viral vector according to any one of claims 1-96, wherein the substitution of one or more amino acids comprises an amino acid substitution at position 68, an amino acid substitution at position 69, an amino acid substitution at position 70, an amino acid substitution at position 71, an amino acid substitution at position 159, an amino acid substitution at position 160, or a combination thereof, wherein the amino acid position is based on the numbering of the amino acid on the E2 domain.

98. The engineered viral vector of claim 97, wherein the amino acid substitution at position 68 includes an S68A substitution.

99. The engineered viral vector of claim 97 or claim 98, wherein the amino acid substitution at position 69 includes an L69A substitution.

100. The engineered viral vector according to any one of claims 97-99, wherein the amino acid substitution at position 70 includes a K70A substitution.

101. The engineered viral vector according to any one of claims 97-100, wherein the amino acid substitution at position 71 includes a Q71A substitution.

102. The engineered viral vector according to any one of claims 97-101, wherein the amino acid substitution at position 159 includes a K159A substitution.

103. The engineered viral vector according to any one of claims 97-99, wherein the amino acid substitution at position 160 includes an E160A substitution.

104. The engineered viral vector according to any one of claims 1-13, wherein the at least one targeting portion comprises a ligand of a cell surface receptor.

105. The engineered viral vector of claim 104, wherein the ligand is a hormone, neurotransmitter, cytokine, ion, or a subunit thereof.

106. The engineered viral vector of claim 104 or 105, wherein the ligand comprises a G protein-coupled receptor ligand or a small peptide ligand.

107. The engineered viral vector of any one of claims 104-106, wherein the ligand is encoded by a nucleotide sequence inserted into the E2 domain.

108. The engineered viral vector of claim 106 or 107, wherein the G protein-coupled receptor ligand comprises endorphin or oxytocin.

109. The engineered viral vector according to any one of claims 106-108, wherein the small peptide ligand comprises a ligand of a GPR78, EGFR, PD-1, or CD47 receptor.

110. The engineered viral vector according to any one of claims 106-109, wherein the small peptide ligand is epidermal growth factor (EGF) or a subunit of EGF.

111. The engineered viral vector of claim 110, wherein the subunit of EGF is an EGF core domain.

112. The engineered viral vector of claim 111, wherein the EGF core domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO:

24.

113. The engineered viral vector of claim 111 or 112, wherein the EGF core domain comprises SEQ ID NO:

24.

114. The engineered viral vector according to any one of claims 104-113, wherein the targeting portion comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 25 or 26.

115. The engineered viral vector according to any one of claims 104-113, wherein the targeting portion comprises SEQ ID NO: 25 or 26.

116. The engineered viral vector according to any one of claims 104-115, wherein the cell surface receptor is EGFR or an EGFR mutant.

117. The engineered viral vector according to any one of claims 104-115, wherein the target cell is a cancer cell.

118. The engineered viral vector of any one of claims 1-13, wherein the at least one targeting portion comprises an immunogenic protein.

119. The engineered viral vector of claim 118, wherein the immunogenic protein comprises a viral protein.

120. The engineered viral vector of claim 119, wherein the viral protein comprises viral envelope protein, spike protein, or a combination thereof.

121. The engineered viral vector of claim 119 or claim 120, wherein the viral protein is derived from a virus.

122. The engineered viral vector of claim 121, wherein the virus comprises a respiratory virus.

123. The engineered viral vector of claim 122, wherein the respiratory virus includes SARS-CoV-2 or influenza virus.

124. The engineered viral vector of claim 119, wherein the viral protein comprises the N-terminal domain of the SARS-CoV-2.

125. The engineered viral vector of claim 119, wherein the viral protein comprises the hemagglutinin (HA) of the influenza virus.

126. The engineered viral vector according to any one of claims 1-125, wherein the engineered viral vector further comprises a payload vector.

127. The engineered viral vector of claim 126, wherein the payload vector encodes at least one therapeutic agent.

128. The engineered viral vector of claim 127, wherein the at least one therapeutic agent comprises a nucleic acid molecule.

129. The engineered viral vector of claim 128, wherein the nucleic acid molecule comprises DNA, RNA, or a combination thereof.

130. The engineered viral vector of claim 129, wherein the RNA comprises mRNA.

131. The engineered viral vector of claim 127, wherein the at least one therapeutic agent comprises at least one therapeutic polypeptide.

132. The engineered viral vector of claim 131, wherein the at least one therapeutic polypeptide comprises a suicide protein.

133. The engineered viral vector of claim 132, wherein the suicide protein comprises thymidine kinase, cytosine deaminase, IL-2, nitroreductase (NR), carboxylesterase, β-glucuronidase, cytochrome p450, β-galactosidase, diphtheria toxin A chain (DT-A), carboxypeptidase G2 (CPG2), purine nucleoside phosphorylase (PNP), or deoxycytidine kinase (dCK).

134. The engineered viral vector of claim 133, wherein the thymidine kinase is derived from herpes simplex virus (HSV-TK) or vesicular stomatitis virus (VSV-TK).

135. The engineered viral vector according to any one of claims 1-134, wherein the engineered viral vector is generated by transient transfection of a cell line.

136. The engineered viral vector according to any one of claims 1-135, wherein the engineered viral vector is an integrated engineered viral vector.

137. The engineered viral vector according to any one of claims 1-135, wherein the engineered viral vector is a non-integrated engineered viral vector.

138. A cell comprising an engineered viral vector as claimed in any one of claims 1-137.

139. A system comprising an engineered viral vector as claimed in any one of claims 1-137.

140. A pharmaceutical composition comprising an engineered viral vector as described in any one of claims 1-137, a cell as described in claim 138, or a system as described in claim 139.

141. The pharmaceutical composition of claim 140, wherein the pharmaceutical composition comprises at least one additional active ingredient.

142. The pharmaceutical composition of claim 140 or claim 141, wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable excipient.

143. The pharmaceutical composition of any one of claims 141-142, wherein the pharmaceutical composition stimulates an immune response in the subject.

144. A method of delivering a therapeutic agent to target cells in a subject, the method comprising administering, for example by injection or infusion, the pharmaceutical composition of any one of claims 140-143 to the subject via oral administration, bronchoalveolar lavage, sublingual, intratumoral, parenteral, intravenous, subcutaneous, intramuscular, intradermal, intraperitoneal, intracerebral, subarachnoid, intraocular, intrasternal, ocular, endothelial, local, intranasal, intrapulmonary, rectal, intraarterial, intrasheathal, inhalation, intralesional, intradermal, epidural, intracapsular, subcapsular, intracardiac, tracheal, subepidermal, or intraspinal administration.

145. A method for inducing cell-killing activity in cancer cells of a subject, the method comprising administering to the subject an engineered viral vector as described in any one of claims 1-137, thereby inducing cell-killing activity in the cancer cells.

146. A method for inducing an immune response in a subject, the method comprising administering to the subject an engineered viral vector as described in any one of claims 1-137, thereby inducing an immune response triggered by target cells in the subject.

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