Viral glycoprotein variants and uses thereof

Abstract

The present disclosure relates to viral glycoprotein variants with reduced (e.g., abolished) binding to low-density lipoprotein receptor (LDL-R) compared to a reference viral glycoprotein. Recombinant viruses pseudotyped with viral glycoprotein variants described herein and optionally an envelope surface-bound targeting molecule (e.g., an anti-CD3 scFv), methods of producing thereof, and methods of using thereof, are also provided.

Description

VIRAL GLYCOPROTEIN VARIANTS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority of U. S. Patent Application No. US 63 / 712,846 filed on October 28, 2024, the content of which is incorporated herein by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing 761422004841seqlist.xml; Size: 138,600 bytes; and Date of Creation: October 27, 2025) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure relates to viral glycoprotein variants with reduced binding to low-density lipoprotein receptor (LDLR) family (e.g., LDL-R) compared to a reference viral glycoprotein. Also provided are recombinant viruses pseudotyped with a glycoprotein variant described herein, methods of producing thereof, and methods of using thereof.BACKGROUND

[0004] Viral vectors have become an important tool for gene therapy. In particular, lentiviral vectors (LVVs) are attractive tools as they can stably integrate large gene fragments into target cells, and they have the ability to transduce both dividing and non-dividing cells. LVVs are typically produced using packaging vectors encoding viral structural proteins, an envelope vector encoding a viral glycoprotein, and a transfer vector encoding the transgene of interest. The cells that LVVs target can be adapted by pseudotyping glycoproteins from different viruses which have varying cell tropism and target different receptors.

[0005] Glycoprotein is responsible for both viral attachment and fusion within endosomal vesicles. The most widely used glycoprotein in LVVs is vesicular stomatitis virus (VSV) glycoprotein (VSV-G). VSV-G serves dual function of a) attachment of the virion to the cell surface receptor leading to virion endocytosis and b) membrane fusion triggered by low pH encountered during endosomal trafficking (Duverge A. and Negroni M. (2020) Viruses. 12(11); Naldini L. (1998) Curr Opin Biotechnol. 9(5), 457-463). VSV-G enhances gene transfer into multiple cell types including immune cells. Because VSV-G-pseudotyped LVV is stable and results in high titers, VSV-G-pseudotyped LVVs are used in current approved ex vivo CAR-Tproducts. However, VSV-G-pseudotyped LVVs show limitations, such as inefficient transduction in quiescent blood cells, sensitivity to trypsin, and serum inactivation. Cocal virus (COV) is in the Vesiculovirus genus, and is a causative agent of vesicular stomatitis in mammals. The cocal virus glycoprotein (COV-G) shares around 74% identity at the amino acid level with VSV-G of the Indiana serotype. Phylogenetic comparison of the envelope gene of vesiculoviruses shows that Cocal virus is serologically distinct from, but most closely related to, VSV-G Indiana among the vesiculoviruses. Maraba virus (MARAV) is another species in the Vesiculovirus genus, and the Maraba virus glycoprotein (MARAV-G) shares around 80% identity at the amino acid level with VSV-G Indiana.

[0006] An associated risk of CAR-T cell production directly in vivo is the transduction of other cell types with the transgene induced by, e.g., the interaction between VSV-G and its receptor LDL-R. The use of integrating vectors with broad cell tropism, e.g., LVV-pseudotyped with a VSV-G envelope protein, can represent a rare though serious safety concern. Two main challenges for in vivo LVV gene therapy include: (1) lack of cell-specific targeting strategy; and (2) serum inactivation of the virus envelope glycoprotein mediated by the complement system.

[0007] The disclosure of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated by reference in their entirety.BRIEF SUMMARY OF THE INVENTION

[0008] The present application in one aspect provides a viral glycoprotein variant of a reference viral glycoprotein, wherein the viral glycoprotein variant comprises one or more mutations (e.g., one or more mutations at any of the amino acid position equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a wildtype cocal virus glycoprotein (COV-G) sequence of SEQ ID NO: 1), and wherein the viral glycoprotein variant has reduced infectivity of its pseudotyped virus compared to the reference viral glycoprotein. In some embodiments, the reference viral glycoprotein is COV-G, vesicular stomatitis virus glycoprotein (VSV-G), or Maraba virus glycoprotein (MARAV-G).

[0009] In some embodiments according to any of the viral glycoprotein variants described above, the viral glycoprotein variant is a variant of COV-G, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356, and wherein the amino acid position is in reference to the wildtype COV-G sequence of SEQ IDNO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions selected from the group consisting of W14A, V17A, M41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, F148A, C153A, C158A, V182E, and L356A. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-47, 74, 77 and 80-83, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-47, 74, 77 and 80-83.

[0010] In some embodiments according to any of the viral glycoprotein variants described above, the viral glycoprotein variant is a variant of VSV-G, wherein the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356, and wherein the amino acid position is in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions selected from the group consisting of W14A, V17A, L41A or I41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 48-59, 73, 84-86, 89, 91 and 93, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 48-59, 73, 84-86, 89, 91 and 93.

[0011] In some embodiments according to any of the viral glycoprotein variants described above, the viral glycoprotein variant is a variant of MARAV-G, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356, and wherein the amino acid position is in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, L148A, C153A, C158A, A182E, and L356A. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103.

[0012] The present application in another aspect provides a viral glycoprotein variant of a reference viral glycoprotein, wherein the viral glycoprotein variant comprises a mutation (e.g., mutation at amino acid position equivalent to 50 or 51 in reference to a wildtype cocal virus glycoprotein (COV-G) sequence of SEQ ID NO: 1), and wherein the viral glycoprotein variant has reduced binding to low-density lipoprotein receptor (LDL-R) and optionally retained fusogenic activity compared to the reference viral glycoprotein. In some embodiments, the reference viral glycoprotein is COV-G, vesicular stomatitis virus glycoprotein (VSV-G), or Maraba virus glycoprotein (MARAV-G).

[0013] In some embodiments according to any of the viral glycoprotein variants described above, the viral glycoprotein variant is a variant of COV-G, wherein the COV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50 or 51, and wherein the amino acid position is in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) a substitution selected from the group consisting of K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, and A51P. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 44-47, 74, 77 and 80-83, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 44-47, 74, 77 and 80-83.

[0014] In some embodiments according to any of the viral glycoprotein variants described above, the viral glycoprotein variant is a variant of COV-G, wherein the COV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50, and wherein the amino acid position is in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) a substitution selected from the group consisting of K50Q, K50A, K50I, and K50E. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 44-47, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 44-47.

[0015] In some embodiments according to any of the viral glycoprotein variants described above, the viral glycoprotein variant is a variant of VSV-G, wherein the VSV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50 or 51, and wherein the amino acid position is in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) a substitution selected from the group consisting of K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, and A51P. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 48-59, 84-86, 89, 91 and 93, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 48-59, 84-86, 89, 91 and 93.

[0016] In some embodiments according to any of the viral glycoprotein variants described above, the viral glycoprotein variant is a variant of VSV-G, wherein the VSV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50, and wherein the amino acid position is in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) a substitution selected from the group consisting of K50Q, K50A, K50I, and K50E. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 48-59, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 48-59.

[0017] In some embodiments according to any of the viral glycoprotein variants described above, the viral glycoprotein variant is a variant of MARAV-G, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50 or 51, and wherein the amino acid position is in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) a substitution selected from the group consisting of K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, and A51P. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103.

[0018] In some embodiments according to any of the viral glycoprotein variants described above, the viral glycoprotein variant is a variant of MARAV-G, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50, and wherein the amino acid position is in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) a substitution selected from the group consisting of K50Q, K50A, K50I, and K50E. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 60-

[0019] In some embodiments according to any of the viral glycoprotein variants described above, wherein the viral glycoprotein variant has a combination of two mutations with the substitution at amino acid position 50 and 51 selected from the group consisting of K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, and A51P.

[0020] In some embodiments according to any of the viral glycoprotein variants described above, the viral glycoprotein variant has one single mutation with the substitution at amino acid position 50 or 51 selected from the group consisting of K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, and A51P.

[0021] Also provided are first nucleic acids comprising a nucleic acid sequence encoding any of the viral glycoprotein variants described above. Also provided are vectors comprising any of the first nucleic acids described herein.

[0022] The present application in another aspect provides recombinant viruses comprising any of the viral glycoprotein variants described above. In some embodiments, the recombinant virus is a lentivirus.

[0023] In some embodiments according to the recombinant viruses described above, the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell. In some embodiments, the envelope surface-bound targeting molecule is encoded by a second nucleic acid. In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain (e.g., a transmembrane domain derived from CD8a, CD4, CD28, 4-1BB, CD80, CD86, CD152, and PD-1, such as CD8a) or membrane-anchoring domain. In some embodiments, the target cell is an immune cell, such as an immune cell selected from the group consisting of a monocyte, a dendritic cell, a macrophage, a B cell, a T cell, a natural killer (NK) cell, or a combination thereof. In some embodiments, the immune cell is a T cell. In some embodiments, the cell surface protein is selected from the group consisting of CD3, CD5, CD2, TCR, CD4, CD8 and CD7, such as CD3. In some embodiments, the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein on the target cell. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab, a Fab’, a (Fab’)2, an Fv, an scFv, and an sdAb, such as scFv. In some embodiments, the envelope surface-bound targeting molecule is an anti-CD3 scFv. In some embodiments, the envelope surface-bound targeting molecule is a T-cell activation molecule or a co-stimulation molecule. In some embodiments, the T-cell activation or co-stimulation molecule comprises CD86, CD80, CD137E, CD58, ICOSE, anti-CD28 antibody or antigen-binding fragment, anti-CD2 antibody or antigen-binding fragment, anti-ICOS antibody or antigen-binding fragment, or anti-4- IBB antibody or antigen-binding fragment.

[0024] In some embodiments according to any of the recombinant viruses described above, the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a heterologous molecule (e.g., heterologous protein or RNA). In some embodiments, the heterologous molecule is an engineered receptor, such as a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), a chimeric TCR (cTCR), aT cell antigen coupler (TAC), or a TAC-like engineered receptor. In some embodiments, the engineered receptor is a CAR, wherein the CAR comprises an extracellular binding domain specifically recognizing a cancer-associated antigen, a cancer- specific antigen, or an autoimmune disease-associated antigen. In some embodiments, the extracellular binding domain specifically recognizes cancer-associated antigen or cancer-specific antigen selected from the group consisting of CD19, CD20, CD22, BCMA, DLL3, B7H3, PD-L1, PD-L2, CLL1, GPC3, GU2CYC, CD7, CD38, CD41, CD123, Claudin 18.2, Claudin 6, NKG2D, GPRC5D, CD70, and any combination thereof.

[0025] In some embodiments according to any of the recombinant viruses described above, the first nucleic acid and the second nucleic acid are on the same vector. In some embodiments, the first nucleic acid and the second nucleic acid are under the control of a same promoter. In some embodiments, the first nucleic acid and the second nucleic acid are connected via a linking nucleic acid encoding a cleavable linker, such as a 2A peptide (e.g., P2A, T2A, etc.). In some embodiments, the first nucleic acid and the second nucleic acids are on two separate vectors. In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid are on different vectors.

[0026] The present application in another aspect provides methods of specifically delivering a heterologous nucleic acid into a target cell, comprising contacting the target cell with a recombinant virus, wherein the recombinant virus comprises the heterologous nucleic acid to be delivered into the target cell and any of the viral glycoprotein variants described above. In some embodiments, the heterologous nucleic acid encodes a heterologous molecule, such as a CAR, an engineered TCR, a cTCR, a TAC, or a TAC-like engineered receptor.

[0027] The present application in another aspect provides methods of producing a recombinant virus comprising any of the viral glycoprotein variants described above, the method comprising introducing a first nucleic acid comprising a nucleic acid sequence encoding the viral glycoprotein variant and a packaging vector comprising one or more nucleic acids encoding one or more packaging proteins into a producer cell, thereby producing the recombinant virus.In some embodiments, the method further comprises introducing a second nucleic acid comprising a nucleic acid sequence encoding an envelope surface-bound targeting molecule into the producer cell, wherein the envelope surface-bound targeting molecule (e.g., scFv) specifically recognizes a cell surface protein on a target cell (e.g., CD3 on a T cell). In some embodiments, the method further comprises introducing a third nucleic acid comprising a nucleic acid sequence encoding a heterologous molecule (e.g., CAR) into the producer cell.

[0028] Also provided are pharmaceutical compositions comprising any of the recombinant viruses described above, and a pharmaceutically acceptable excipient.

[0029] Further provided are methods of treating a disease or disorder (e.g., cancer or an autoimmune disease) in an individual (e.g., human), the method comprising administering to the individual a therapeutically effective amount of any of the pharmaceutical compositions described herein (e.g., pharmaceutical compositions comprising recombinant viruses expressing CAR).BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 shows the transduction of Jurkat cells with a titration of lentiviruses (LVs) pseudotyped with a WT or mutant COV-G, encoding a CD20+CD19 dual CAR transgene. Transduction was assessed by measuring the percentage of CAR+Jurkat cells by flow cytometry.

[0031] FIG. 2A shows a schematic diagram of an engineered CD3scFv+COVG lentivirus encoding a CAR.

[0032] FIG. 2B shows the rescue of Jurkat cell transduction by LVs pseudotyped with a WT or mutant COV-G along with an anti-CD3 specific scFv. Transduction was assessed by measuring the percentage of CAR+Jurkat cells by flow cytometry.

[0033] FIG. 2C shows rescued functional infectious titer in Jurkat cells measured by transducing units (TU) / mL.

[0034] FIG. 3A compares the transduction of LVs pseudotyped with various K50 COV-G variants with or without CD3 scFv in Jurkat cells. Transduction was assessed by measuring the percentage of CAR+Jurkat cells by flow cytometry.

[0035] FIG. 3B compares the functional infectious titer rescued of LVs pseudotyped with various K50 COV-G variants with or without CD3 scFv in Jurkat cells, measured by TU / mL.

[0036] FIG. 3C shows the minimal transduction of LVs pseudotyped with COVGm42 in Nalm6 cells. Transduction was assessed by measuring the percentage of CAR+Nalm6 cells by flow cytometry.

[0037] FIG. 4A shows transduction by LVs pseudotyped with a WT or mutant VSV-G with or without an anti-CD3 specific scFv in Jurkat cells. Transduction was assessed by measuring the percentage of CAR+Jurkat cells by flow cytometry.

[0038] FIG.4B shows transduction by LVs pseudotyped with mutant VSV-G without an anti-CD3 specific scFv in Nalm6 cells. Transduction was assessed by measuring the percentage of CAR+Nalm6 cells by flow cytometry.

[0039] FIG. 5 shows transduction by LVs pseudotyped with a WT or mutant COVGm38, COVGm300, COVGm301, or COVGm303, with or without an anti-CD3 specific scFv in Jurkat cells. Transduction was assessed by measuring the percentage of CAR+Jurkat cells by flow cytometry.

[0040] FIG.6 shows transduction by LVs pseudotyped with a mutant COVGm45, COVGm46, COVGm47, COVGm48, COVGm49, COVGm51, or COVGm52, with or without an anti-CD3 specific scFv in Jurkat cells. Transduction was assessed by measuring the percentage of CAR+Jurkat cells by flow cytometry.

[0041] FIG.7A shows transduction by LVs pseudotyped with mutant VSVGm32, VSVGm33, VSVGm34, or VSVGm35, with or without an anti-CD3 specific scFv in Jurkat cells.

[0042] FIG.7B shows transduction by LVs pseudotyped with mutant VSVGm36, VSVGm37, VSVGm38, VSVGm39, VSVGm40, or VSVGm41, with or without an anti-CD3 specific scFv in Jurkat cells.

[0043] FIG. 8 shows serum inactivation level LVs pseudotyped with a COVG WT, mutant COVG, VSVG WT, or mutant VSVG with or without an anti-CD3 specific scFv in Jurkat cells.

[0044] FIG. 9 shows transduction of T cells by CD3scFv+COVG mutant LV in resting human PBMC. Untransduced (unT) cells were used as a negative control. Transduction was assessed by measuring the percentage of CAR+T cells by flow cytometry.

[0045] FIG. 10 shows the efficacy of CD3scFv+COVGm38 LVs to kill tumors in mice. Tumor-free mice are used as a control (Group 1 control (G1 Cont.)).

[0046] FIG. 11 shows an amino acid sequence alignment between COV-G, VSV-G, and MARAV-G. “COVG” indicates wildtype COV-G protein (SEQ ID NO: 1). “VSVG” indicates reference VSV synthetic (syn)-G protein (SEQ ID NO: 2). “MARAV-G” indicates wildtype MARAV-G protein (SEQ ID NO: 5).DETAILED DESCRIPTION

[0047] The present application in one aspect provides viral glycoprotein variants of a reference viral glycoprotein (e.g., COV-G, VSV-G, or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., one or more mutations at any of the amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356) in reference to a wildtype COV-G sequence of SEQ ID NO: 1. The present application in another aspect provides viral glycoprotein variants of a reference viral glycoprotein (e.g., COV-G, VSV-G, or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) a mutation at amino acid position equivalent to 50 in reference to a wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the viral glycoprotein variant has reduced (e.g., abolished) binding to LDL-R compared to a reference viral glycoprotein (e.g., a reference viral glycoprotein not containing the one or more mutations). In some embodiments, the viral glycoprotein has reduced (e.g., abolished) fusogenic activity. In some embodiments, the glycoprotein variant has reduced (e.g., abolished) binding to LDL-R but retains (e.g., retains at least about 20%) fusogenic activity compared to a reference viral glycoprotein. In some embodiments, the viral glycoprotein variant has reduced (e.g., abolished) binding to LDL-R and reduced (e.g., abolished) fusogenic activity compared to a reference viral glycoprotein. In some embodiments, the viral glycoprotein variant does not have reduced binding to LDL-R and optionally has reduced (e.g., abolished) fusogenic activity compared to a reference viral glycoprotein. In some embodiments, the viral glycoprotein variant results in a reduced (e.g., abolished) infectivity of its pseudotyped virus compared to a reference viral glycoprotein (e.g., a corresponding wildtype viral glycoprotein).

[0048] Also provided are recombinant viruses pseudotyped with any of the viral glycoprotein variants disclosed herein. In some embodiments, the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell (e.g., CD3 on T cells), such as an envelope surface-bound antibody or antigenbinding fragment thereof (e.g., an anti-CD3 scFv). In some embodiments, the recombinant virus further comprises a nucleic acid comprising a nucleic acid sequence encoding a heterologous molecule (e.g., heterologous protein or RNA), such as an engineered receptor (e.g., a CAR). Pharmaceutical compositions comprising any of the recombinant viruses described herein, methods of making thereof, and methods of using thereof are also provided.

[0049] The broad tropism of LDL-R can lead to the non-specific transduction of target cells from recombinant viruses pseudotyped with glycoproteins such as VSV-G, COV-G, and MARAV-G. In order to improve target cell specificity of viral vectors for efficient and specific transduction, inventors of the present application identified mutations in viral glycoproteins that can reduce binding to LDL-R. When LVVs were pseudotyped with these glycoprotein variants, non-specific transduction efficiency was greatly reduced, indicating these mutations were effective in reducing binding affinity to LDL-R.

[0050] Upon further engineering the recombinant virus to display an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) specific for the target cell of interest (e.g., T cells), it was observed that the recombinant viruses exhibited target cell-specific transduction, the efficiency of some of which was comparable or even higher than recombinant viruses pseudotyped with a reference viral glycoprotein but without the envelope surface-bound targeting molecule. These glycoprotein variants were impaired in LDL-R binding but have rescuable fusogenic activity. It was also observed that the nucleic acid of interest (e.g., encoding a CAR) delivered by the recombinant viruses pseudotyped with glycoprotein variants described herein and expressing an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) were able to specifically transduce human T cells and treat tumors in a human PBMC reconstituted NOD scid gamma (NSG) mouse tumor model.

[0051] These results demonstrate the superior efficacy and safety of viral glycoprotein variants described herein, recombinant viruses comprising thereof with or without expressing an envelope surface-bound targeting molecule described herein, suggesting their promising applications in gene therapy.

[0052] Thus, the present application in one aspect provides a viral glycoprotein variant comprising one or more mutations as compared to a reference viral glycoprotein and having reduced binding to LDL-R compared to the reference viral glycoprotein. Also provided is a viral glycoprotein variant comprising one or more mutations as compared to a reference viral glycoprotein and having reduced binding to LDL-R but retained fusogenic activity compared to the reference viral glycoprotein.

[0053] In another aspect, there is provided a recombinant virus comprising any of the viral glycoprotein variants described herein, with or without expressing an envelope surface-bound targeting molecule described herein (e.g., anti-CD3 scFv).

[0054] Also provided are recombinant viruses that further comprise a nucleic acid comprising a nucleic acid sequence encoding a heterologous molecule described herein, such as a therapeutic protein (e.g., an engineered receptor, such as a CAR).I. Definitions

[0055] Techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3d ed. 2001); Lentivirus Gene Engineering Protocols (2ndedition, 2003); Fields Virology (6thedition, 2013); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003). The contents of each of which are incorporated herein by reference in their entirety.

[0056] ‘ ‘Percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0057] The term “variant” when used in reference to a polypeptide means a polypeptide that has one or more substitutions, deletions, or insertions relative to a parent or reference polypeptide. The term may analogously be applied to polynucleotides that have one or more substitutions, deletions, or insertions relative to a parent or reference polynucleotide.

[0058] A “pseudotyped” recombinant virus (e.g., lentivirus) is a recombinant viral particle having one or more envelope glycoproteins that are encoded by a virus that is distinct from the recombinant virus (e.g., lentiviral) genome. The envelope glycoprotein may be modified, mutated, or engineered as described herein.

[0059] ‘ ‘Recombinant viruses” are those that are generated to incorporate modification(s) introduced into a specific gene / locus (or multiple loci) of the viral genome. A recombinant virus may occur naturally or be produced by recombining pieces of DNA using recombinant DNA technology.

[0060] The term “specificity” refers to selective recognition of an antigen binding protein (such as a CAR or an antibody) for a particular epitope of an antigen. Natural antibodies, for example,are monospecific. The term "multispecific" as used herein denotes that an antigen binding protein has two or more antigen-binding sites of which at least two bind different epitopes. "Bispecific" as used herein denotes that an antigen binding protein has two different antigenbinding specificities. The term "monospecific" as used herein denotes an antigen binding protein that has one or more binding sites each of which bind the same epitope.

[0061] The term “antibody” herein is used in its broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity.

[0062] An “antibody” may refer to an immunoglobulin molecule or a fragment thereof which is able to specifically bind to a specific epitope of an antigen. Antibodies can be intact immunoglobulins derived from natural sources, or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, antigen-binding fragments (such as Fv, Fab, Fab’, F(ab)2 and F(ab’)2), as well as single chain antibodies (scFv), heavy chain antibodies, such as camelid antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0063] The term “epitope” as used herein refers to the specific group of atoms or amino acids on an antigen to which an antibody or antibody moiety binds.

[0064] A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). CDR boundaries for antibodies and antigen-binding fragments may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved inantigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of a, 5, a, y, and p heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgGl (yl heavy chain), lgG2 (y2 heavy chain), lgG3 (y3 heavy chain), lgG4 (y4 heavy chain), IgAl (al heavy chain), or lgA2 (a2 heavy chain).

[0065] The term “antigen-binding fragment” as used herein refers to an antibody fragment including, for example, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (ds diabody), a single-chain Fv (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a single domain antibody (sdAb) (e.g., a camelized single domain antibody), a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds.

[0066] “Fv” is the minimum antibody fragment, which contains a complete antigenrecognition and -binding site. This fragment consists of a dimer of one heavy- and one lightchain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0067] “Single-chain Fv,” also abbreviated as “sFv” or “scFv,” are antibody fragments that comprise the VH and VE antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pliickthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994).

[0068] “Single domain antibody” or “sdAb” as used herein refers to a single monomeric variable antibody domain and which is capable of antigen binding. Single domain antibodies include VHH domains as described herein. Examples of single domain antibodies include, but are not limited to, antibodies naturally devoid of light chains such as those from Camelidaespecies (e.g., llama), single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies (e.g., VHH domains) may be derived from any species including, but not limited to mouse, human, camel, llama, goat, rabbit, and bovine. For example, a single domain antibody can be derived from antibodies raised in Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain; VHHs derived from such other species are within the scope of the disclosure. The single domain antibody (e.g., VHH domain) provided herein has a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single domain antibodies may be genetically fused or chemically conjugated to another molecule (e.g., an agent). Single domain antibodies may be part of a bigger binding molecule (e.g., a multispecific antibody or an engineered receptor).

[0069] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites. Heavy-chain only antibodies from the Camelidae species have a single heavy chain variable region, which is referred to as “VHH”. VHH is thus a special type of VH.

[0070] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or posttranslation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-497 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory Press, 2nded. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N. Y., 1981)), recombinant DNA methods (see, e.g., U. S. Pat. No. 4,816,567), phage-display technologies (see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Sidhu etal., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U. S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild etal., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol.14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).

[0071] CDR regions are well known to those skilled in the art and have been defined by well-known numbering systems. For example, the Kabat CDRs are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., supra; Nick Deschacht et al., J Immunol 2010; 184:5696-5704). Chothia refers instead to the location of the structural loops see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35 A is present, the loop ends at 33; if both 35 A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (see, e.g., Antibody Engineering Vol. 2 (Kontermann and Diibel eds., 2d ed. 2010)). The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. Another universal numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol.27(l):55-77 (2003)). IMGT is an integrated information system specializing in immunoglobulins (IG), T-cell receptors (TCR), and major histocompatibility complex (MHC) of human and other vertebrates. Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the “location” of the CDRswithin the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pliickthun, J. Mol. Biol. 309: 657-70 (2001). Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra). The residues from each of these hypervariable regions or CDRs are exemplified in Table A below.Table A. Exemplary CDRs According to Various Numbering Systems

[0072] The boundaries of a given CDR may vary depending on the scheme used for identification. Thus, unless otherwise specified, the terms “CDR” and “complementary determining region” of a given antibody or region thereof, such as a variable region, as well as individual CDRs (e.g., CDR-H1, CDR-H2) of the antibody or region thereof, should be understood to encompass the complementary determining region as defined by any of the known schemes described herein above. In some instances, the scheme for identification of a particular CDR or CDRs is specified, such as the CDR as defined by the IMGT, Kabat, Chothia, or Contact method. In other cases, the particular amino acid sequence of a CDR is given. It should be noted CDR regions may also be defined by any combination of various numberingsystems, e.g., a combination of Kabat and Chothia numbering systems, a combination of Kabat and AbM numbering systems, or a combination of Kabat and IMGT numbering systems. Therefore, the term such as “a CDR1 as set forth in a specific VH” includes any CDR1 as defined by the exemplary CDR numbering systems described above, but is not limited thereby. Once a variable region e.g., a VH or VL) is given, those skilled in the art would understand that CDRs within the region can be defined by different numbering systems or combinations thereof.

[0073] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure.

[0074] As used herein, the term “conservative amino acid substitution” refers to substitutions in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Antibody variants having one or more conservative amino acid substitutions may be provided. Conservative substitutions are shown in Table B under the heading of “Preferred Substitutions”, while more substantial changes are provided in Table B under the heading “Exemplary Substitutions”. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity (e.g., retained / improved antigen binding).Table B: Amino acid substitutions

[0075] Amino acids may be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Amino acids that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0076] ‘ ‘Chimeric antigen receptor” or "CAR" as used herein refers to genetically engineered receptors, which can be used to graft one or more antigen specificity onto immune effector cells, such as T cells. Some CARs are also known as “artificial T-cell receptors,” “chimeric T cell receptors,” or “chimeric immune receptors.” The CAR may comprise an extracellular antigen binding domain specific for one or more antigens (such as tumor antigens), a transmembrane domain, and an intracellular signaling domain of aT cell and / or other receptors. “CAR-T cell” refers to a T cell that expresses a CAR.

[0077] The terms “polypeptide” and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, including but not limited to, unnatural amino acids, as well as other modifications known in the art. It is understood that, because the polypeptides of this disclosure may be based upon antibodies or other members of the immunoglobulin superfamily, a “polypeptide” can occur as a single chain or as two or more associated chains.

[0078] “Polynucleotide” or “nucleic acid,” as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. “Oligonucleotide,” as used herein, refers to short, generally single-stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides. A cell that produces a binding molecule of the present disclosure may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced. Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of double- stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3’ to the 3’ end of the RNA transcript are referred to as “downstream sequences.”

[0079] An “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or a mixed nucleic acids, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule may include isolated forms of the molecule.

[0080] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or anRNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).

[0081] The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0082] As used herein, the term “operatively linked,” and similar phrases (e.g., genetically fused), when used in reference to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequence, respectively, placed in functional relationships with each other. For example, an operatively linked promoter, enhancer elements, open reading frame, 5’ and 3’ UTR, and terminator sequences result in the accurate production of a nucleic acid molecule (e.g., RNA). In some embodiments, operatively linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operatively linked peptide is one in which the functional domains are placed with appropriate distance from each other to impart the intended function of each domain.

[0083] The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding a binding molecule (e.g., an antibody) as described herein, in order to introduce a nucleic acid sequence into a host cell. A vector for use can be expression vectors, plasmids, and viral vectors, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., both an antibody heavy and light chain or an antibody VH and VL), both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methodsinclude, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0084] The term “host” as used herein can refer to any organism that can receive administration of a recombinant virus, such as an animal (e.g., a mammal, such as a human).

[0085] The term “host cell” as used herein refers to a particular subject cell that may be transduced with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transduced with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0086] The term “producer cell” as used herein refers to a particular subject cell that is capable of producing recombinant viral particles when the transfer vector, packaging vector(s), and envelope vector(s) are introduced into the cells. Various methods of introducing the plasmids into the cells may be used, including transfection or electroporation.

[0087] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0088] “Allogeneic” refers to a graft derived from a different individual of the same species.

[0089] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0090] The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in United States Pharmacopeia, European Pharmacopeia, or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

[0091] The terms “effective amount” or “therapeutically effective amount” as used herein refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction (e.g., reducing at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, or 100%) and / or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system.

[0092] The terms “subject,” “individual,” and “patient” may be used interchangeably. A subject or an individual can be a mammal, such as a non-primate or a primate (e.g., human). In some embodiments, the individual is a human. In one embodiment, the individual is a mammal, e.g., a human, diagnosed with a disease or disorder, or at risk of developing a disease or disorder.

[0093] As used herein, “treat,” “treatment” or “treating” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and / or prolonging survival.

[0094] As used herein, “delaying” development of a disease means to defer, hinder, slow, retard, stabilize, suppress and / or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.

[0095] “Preventing” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in an individual that may be predisposed to the disease but has not yet been diagnosed with the disease.

[0096] The term “activation”, as used herein, refers to the state of a cell following sufficient cell surface moiety ligation to induce a noticeable biochemical or morphological change. Within the context of T cells, such activation refers to the state of a T cell that has been sufficiently stimulated to induce cellular proliferation. Activation of a T cell may also induce cytokine production and performance of regulatory or cytolytic effector functions. Within the context of other cells, this term infers either up or down regulation of a particular physicochemical process. The term “activated T cells” indicates T cells that are currently undergoing cell division, cytokine production, performance of regulatory or cytolytic effector functions, and / or has recently undergone the process of “activation.”

[0097] The terms “about” and “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, or less of a given value or range.

[0098] As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.

[0099] The term “between” as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B.

[0100] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in aphrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).IL Viral glycoprotein variants

[0101] The present application in one aspect provides novel viral glycoprotein variants. In some embodiments, the viral glycoprotein variant results in a reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) infectivity of its pseudotyped virus compared to a reference viral glycoprotein (e.g., a corresponding wildtype viral glycoprotein). The viral glycoprotein variants described herein in some embodiments comprise (and in some embodiments consist of or consisting essentially of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) fusogenic activity compared to a reference viral glycoprotein (e.g., a corresponding wildtype viral glycoprotein). In some embodiments, the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to a viral glycoprotein receptor (e.g., LDLR family receptor, such as LDL-R), compared to a reference viral glycoprotein (e.g., a corresponding wildtype viral glycoprotein). In some embodiments, the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to a viral glycoprotein receptor (e.g., LDLR family receptor, such as LDL-R), and has retained (e.g., retained by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or100%) fusogenic activity, compared to a reference viral glycoprotein (e.g., a corresponding wildtype viral glycoprotein). The viral glycoprotein variants described herein in some embodiments comprise (and in some embodiments consist of or consisting essentially of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the viral glycoprotein variant only contains a mutation at one or more amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to SEQ ID NO: 1. In some embodiments, the viral glycoprotein variant further contains one or more mutations outside the one or more amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to SEQ ID NO: 1. In some embodiments, the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more) binding to a viral glycoprotein receptor (e.g., LDLR family receptor), compared to the reference viral glycoprotein. In some embodiments, the viral glycoprotein variant has abolished binding (i.e., no binding, or no detectable binding) to a viral glycoprotein receptor (e.g., LDLR family receptor). In some embodiments, the viral glycoprotein receptor is LDL-R.

[0102] Hence in some embodiments, there is provided a viral glycoprotein variant of a reference viral glycoprotein, wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 41, 46, 50, 51, and 182 (e.g., equivalent to 50) in reference to a wildtype COV-G sequence of SEQ ID NO: 1, and wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to a viral glycoprotein receptor (e.g., LDL-R) compared to the reference viral glycoprotein. In some embodiments, the viral glycoprotein variant has retained (e.g., retained by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to the reference viral glycoprotein.

[0103] In some embodiments, there is provided a viral glycoprotein variant of a reference viral glycoprotein, wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 44, 46, 144, 145, 146, 148, 153, 158, and 356 in reference to SEQ ID NO: 1, and wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, ormore, or abolished) binding to a viral glycoprotein receptor (e.g., LDL-R) compared to the reference viral glycoprotein.

[0104] In some embodiments, the viral glycoprotein variant is derived from a virus of the Rhabdoviridae family, such as the Vesiculovirus genus. Vesiculovirus is a genus of negativesense single-stranded (ss) RNA viruses. In some embodiments, the viral glycoprotein variant is derived from Cocal virus (COV), or a serotype thereof. In some embodiments, the viral glycoprotein variant is derived from vesicular stomatitis virus (VSV), or a serotype thereof. In some embodiments, the viral glycoprotein variant is derived from a strain of the VSV Indiana serotype, such as VSV Orsay strain (VSV Orsay) or VSV San Juan strain (VSV SJ). In some embodiments, the viral glycoprotein variant is derived from a synthetic vesicular stomatitis virus (VSV syn). In some embodiments, the viral glycoprotein variant is derived from Maraba virus (MARAV), or a serotype thereof.

[0105] In some embodiments, a wildtype viral glycoprotein is used as a reference viral glycoprotein. In some embodiments, a synthetic viral glycoprotein is used as a reference viral glycoprotein. In some embodiments, the synthetic viral glycoprotein has the same sequence as a wildtype viral glycoprotein. In some embodiments, the synthetic viral glycoprotein has a different sequence than a wildtype viral glycoprotein.

[0106] In some embodiments, the reference viral glycoprotein is wildtype COV-G comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the reference viral glycoprotein is wildtype VSV Orsay-G comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the reference viral glycoprotein is wildtype VSV SJ-G comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the reference viral glycoprotein is VSV syn-G comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the reference viral glycoprotein is wildtype MARAV-G comprising the amino acid sequence of SEQ ID NO: 5.

[0107] The most common glycoprotein pseudotyped on viral vectors is VSV-G. Unless otherwise indicated, “VSV-G” described herein refers to VSV-Gs in general, including all those VSV-Gs known in the art, either wildtype or synthetic. Exemplary VSV-G include, but are not limited to, VSV Orsay-G (e.g., SEQ ID NO: 3), VSV SJ-G (e.g., SEQ ID NO: 4), and VSV syn-G (e.g., SEQ ID NO: 2). VSV-G is a 511 amino acid protein made up of a fusion domain, pleckstrin homology domain, and trimerization domain. It has recently been demonstrated that VSV-G from the Indiana serotype is able to independently bind two distinct cysteine-rich (CR) domains (CR2 and CR3) of LDL-R, with VSV-G in complex with those domains reported in crystal structures (Nikolic J, Belot L, Raux H, Legrand P, Gaudin Y, AAlbertini A. Nat Commun. 9(1): 1029 (2018)). In addition, specific segments of VSV-G were identified as important for binding, specifically residues 8 to 10 and 350 to 354 in the trimerization domain, certain residues in the pleckstrin homology domain, and residues 47 to 50 in segment S2 (PCT EP2018 / 075824; Nikolic J, Belot L, Raux H, Legrand P, Gaudin Y, A Albertini A. Nat Commun. 9(1): 1029 (2018)). VSV-G forms trimers on the viral envelope, and upon binding to host receptors, mediates host cell entry through clathrin-mediated endocytosis (Rehman S, Bishnoi S, Roy R, Kumari A, Jayakumar H, Gupta S, Kar P, Pattnaik AK, Nayak D. ACS Omega. 7(37):32840- 32848 (2022)).

[0108] COV-G and MARAV-G can also be used for pseudotyping viral vectors. Within the Vesiculovirus genus, COV and MARAV are serologically distinct species from VSV, where COV-G and VSV-G share around 74% sequence identity, and MARAV-G and VSV-G share around 80% sequence identity at the amino acid level. While VSV-G, COV-G, and MARAV-G all exhibit a broad tropism and good stability, COV-G has been shown to have increased serum resistance (Trobridge GD, Wu RA, Hansen M, Ironside C, Watts KL, Olsen P, Beard BC, Kiem HP. Mol Ther. 18(4):725-33 (2010)). VSV, COV, and MARAV are all negative-stranded RNA viruses in the Rhabdoviridae family and utilize LDL-R as the major entry receptor (Gutierrez-Guerrero A, Cosset FL, Verhoeyen E. Viruses. 2020 Sep 11;12(9): 1016 (2020); Jayawardena N, Burga LN, Poirier JT, Bostina M. Oncolytic Virother. 8:39-56 (2019)).

[0109] Receptors that bind viral glycoproteins include, but are not limited to, phosphatidylserine, nicotinic acetylcholine receptor, NPC1 (Niemann-Pick Cl), TfRl (transferrin receptor 1), ACE2 (angiotensin-converting enzyme 2), sialic acid, neuraminidase, and members of the LDLR family. In some embodiments, viral glycoprotein variants described herein have reduced (e.g., reducing at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) binding to one or more receptors that bind viral glycoproteins, such as LDL-R.

[0110] LDL-R is a type I transmembrane protein in the LDLR gene family involved in maintaining cholesterol homeostasis. This receptor binds cholesterol-rich lipoprotein ligands and transports them to the endosomal pathway (Go GW, Mani A. Yale J Biol Med. 85(1): 19-28 (2012)). The extracellular ligand binding domain consists of seven LDLR type A repeats, which are each comprised of 40 amino acid stretches that contain two internal disulfide bonds, mediated by six cysteines (Pedersen NB, Wang S, Narimatsu Y, Yang Z, Halim A, Schjoldager KT, Madsen TD, Seidah NG, Bennett EP, Levery SB, Clausen H. J Biol Chem. 289(25): 17312-24 (2014)). Viral glycoproteins such as VSV-G binds LDL-R in the cysteine-rich regions of this extracellular ligand binding domain (Nikolic J, Belot L, Raux H, Legrand P, Gaudin Y, AAlbertini A. Nat Commun. 9(l):1029 (2018); Finkelshtein D, Werman A, Novick D, Barak S, Rubinstein M. Proc Natl Acad Sci U S A. 110(18):7306-l 1 (2013)). LDL-R also contains epidermal growth factor (EGF)-like domains and a P-propeller domain, which are involved in allowing the release of bound lipoprotein ligands in the endosome in a pH-dependent manner (Zhao Z, Michaely P. J Biol Chem. 283(39):26528-37 (2008); Arias-Moreno X, Velazquez-Campoy A, Rodriguez JC, Pocovl M, Sancho J. J Biol Chem. 283(33):22670-9 (2008)). LDL-R further contains an O-linked sugar domain next to the 22-amino acid single-pass transmembrane domain (Go GW, Mani A. Yale J Biol Med. 85(l):19-28 (2012); Siidhof TC, Goldstein JL, Brown MS, Russell DW. Science. 228(4701):815-22 (1985)). The cytoplasmic domain contains an NPxY motif important in signaling for endocytosis (Chen WJ, Goldstein JL, Brown MS. J Biol Chem. 265(6):3116-23 (1990)).

[0111] While VSV-G can bind other family members in the LDLR family, LDL-R is the primary receptor (Finkelshtein D, Werman A, Novick D, Barak S, Rubinstein M. Proc Natl Acad Sci U SA. 2013 Apr 30; 110(18):7306-l 1 (2013)). LDL-R has low levels of expression on un-activated T cells, B cells and hematopoietic stem cells (Amirache F, Levy C, Costa C, Mangeot PE, Torbett BE, Wang CX, Negre D, Cosset FL, Verhoeyen E. Blood. 2014 Feb 27;123(9): 1422-4 (2014)). This can result in low transduction efficiency in un-activated target cells using LVVs pseudotyped with VSV-G (Gutierrez-Guerrero A, Cosset FL, Verhoeyen E. Viruses. 2020 Sep 11;12(9): 1016 (2020)). For example, activation of T cells can result in increased expression of LDL-R on the cell surface, which can influence transduction efficiency of LVVs (Bonacina F, Moregola A, Svecla M, Coe D, Uboidi P, Fraire S, Beretta S, Beretta G, Pellegatta F, Catapano AL, Marelli-Berg FM, Norata GD..7 Cell Biol. 221(ll):e2022020ll (2022)).

[0112] In some embodiments, the viral glycoprotein variant results in reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) infectivity of its pseudotyped virus compared to a reference viral glycoprotein (e.g., a corresponding wildtype viral glycoprotein). In some embodiments, the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the viral glycoprotein variant comprises (or consists essentially of, orconsists of) one or more mutations at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a wildtype MARAV-G sequence of SEQ ID NO: 5. Amino acid positions “equivalent to” 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to any of SEQ ID NOs: 1-5 in a viral glycoprotein X are amino acid positions in viral glycoprotein X that are homologous to the amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in any of SEQ ID NOs: 1-5, hereinafter also referred to as “equivalent amino acid positions”. The equivalent amino acid positions in viral glycoprotein X: (i) can be at amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in viral glycoprotein X as well, or can be at a different location; and / or (ii) can have the same, partially same, or different amino acid sequences compared to amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in any of SEQ ID NOs: 1-5. Methods of identifying such equivalent amino acid positions in a viral glycoprotein X are well-known in the art, such as by amino acid sequence alignment (e.g., see FIG. 11). For example, 1182 in VSV-G is considered as an “equivalent amino acid position” in reference to V182 of the wildtype COV-G sequence of SEQ ID NO: 1. Alignment for purposes of determining the equivalent amino acid positions of a viral glycoprotein or a variant thereof can be achieved using any tool available in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN™ (DNASTAR), or HMMER.

[0113] In some embodiments, there is provided a viral glycoprotein variant comprising (or consisting essentially of, or consisting of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a reference sequence of any of SEQ ID NOs: 1-5, wherein the viral glycoprotein results in reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) infectivity of its pseudotyped virus compared to a corresponding reference viral glycoprotein comprising the reference sequence of any of SEQ ID NOs: 1-5. In some embodiments, the viral glycoprotein variant comprises (or consists essentially of, or consists of) a 14A, 17A, 41A, 44E, 46A, 46T,46Q, 5OT, 5OQ, 5OA, 501, 50E, 50D, 50H, 50S, 5 IE, 5 ID, 5 IP, 144A, 144E, 144Q, 145 A, 146A, 148A, 153A, 158A, 182E, and / or 356A substitution, wherein the equivalent amino acid position is in reference to a reference sequence of any of SEQ ID NOs: 1-5. The viral glycoprotein variant may or may not comprise further mutation(s) in addition to the one or more mutations at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356. The further mutation(s) can be at or in addition to the amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356. In some embodiments, the viral glycoprotein variant that comprises any of 14A, 17A, 41A, 44E, 46A, 46T, 46Q, 50T, 50Q, 50A, 501, 50E, 50D, 50H, 50S, 5 IE, 5 ID, 5 IP, 144A, 144E, 144Q, 145A, 146A, 148A, 153A, 158A, 182E, and / or 356A substitutions further comprises one or more additional mutations outside of amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a reference sequence of any of SEQ ID NOs: 1-5. In some embodiments, the viral glycoprotein variant that comprises any of 14A, 17A, 41 A, 44E, 46A, 46T, 46Q, 50T, 50Q, 50A, 501, 50E, 50D, 50H, 50S, 5 IE, 5 ID, 5 IP, 144A, 144E, 144Q, 145 A, 146A, 148A, 153A, 158A, 182E, and / or 356A substitutions does not comprise any other mutations outside the amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a reference sequence of any of SEQ ID NOs: 1-5.

[0114] In some embodiments, there is provided a viral glycoprotein variant comprising (or consisting essentially of, or consisting of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to a reference sequence of any of SEQ ID NOs: 1-5, wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity, compared to a corresponding reference viral glycoprotein comprising the reference sequence of any of SEQ ID NOs: 1-5. In some embodiments, the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more substitutions at amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to areference sequence of any of SEQ ID NOs: 1-5. In some embodiments, the viral glycoprotein variant comprises (or consists essentially of, or consists of) a 41A, 46A, 50T, 50Q, 50A, 501, 50E, 50D, 50H, 50S, 5 IE, 5 ID, 5 IP, and / or 182E substitution, wherein the equivalent amino acid position is in reference to a reference sequence of any of SEQ ID NOs: 1-5. In some embodiments, the viral glycoprotein variant comprises (or consists essentially of, or consists of) a 50T substitution,wherein the equivalent amino acid position is in reference to a reference sequence of any of SEQ ID NOs: 1-5. The viral glycoprotein variant may or may not comprise further mutation(s) in addition to the one or more mutations at amino acid positions equivalent to 41, 46, 50, 51, and 182. In some embodiments, the viral glycoprotein variant that comprises any of 41A, 46A, 50T, 50Q, 50A, 501, 50E, 50D, 50H, 50S, 51E, 51D, 51P, and / or 182E substitutions further comprises one or more additional mutations outside the amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to a reference sequence of any of SEQ ID NOs: 1-5. In some embodiments, the viral glycoprotein variant that comprises any of 41A, 46A, 50T, 50Q, 50A, 501, 50E, 50D, 50H, 50S, 5 IE, 5 ID, 5 IP, and / or 182E substitutions does not comprise any other mutations outside the amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to a reference sequence of any of SEQ ID NOs: 1-5.

[0115] In some embodiments, there is provided a viral glycoprotein variant comprising (or consisting essentially of, or consisting of) a mutation (e.g., insertion, deletion, and / or substitution) at amino acid position equivalent to 50 in reference to a reference sequence of any of SEQ ID NOs: 1-5, wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity, compared to a corresponding reference viral glycoprotein comprising the reference sequence of any of SEQ ID NOs: 1-5. In some embodiments, the viral glycoprotein variant comprises (or consists essentially of, or consists of) a substitution at amino acid position equivalent to 50 in reference to a reference sequence of any of SEQ ID NOs: 1-5. In some embodiments, the viral glycoprotein variant comprises (or consists essentially of, or consists of) a K50Q, K50A, K50I, or K50E substitution, wherein the equivalent amino acid position is in reference to a reference sequence of any of SEQ ID NOs: 1-5. The viral glycoprotein variant may or may not comprise further mutation(s) in addition to the mutation at amino acid position equivalent to 50 in reference to a reference sequence of any of SEQ ID NOs: 1-5. In some embodiments, the viral glycoprotein variant that comprises a 50Q, 50A, 501, or 50E substitution further comprises one or more additional mutations outside of the amino acid position equivalent to 50 in reference to a reference sequence of any of SEQ ID NOs: 1-5. In some embodiments, the viral glycoprotein variant that comprises a 50Q, 50A, 501, or 50E substitution does not comprise any other mutations outside the amino acid position equivalent to 50 in reference to a reference sequence of any of SEQ ID NOs: 1-5.

[0116] In some embodiments, there is provided a viral glycoprotein variant comprising (or consisting essentially of, or consisting of) a mutation (e.g., insertion, deletion, and / or substitution) at amino acid position equivalent to 51 in reference to a reference sequence of any of SEQ ID NOs: 1-5, wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity, compared to a corresponding reference viral glycoprotein comprising the reference sequence of any of SEQ ID NOs: 1-5. In some embodiments, the viral glycoprotein variant comprises (or consists essentially of, or consists of) a substitution at amino acid position equivalent to 51 in reference to a reference sequence of any of SEQ ID NOs: 1-5. In some embodiments, the viral glycoprotein variant comprises (or consists essentially of, or consists of) a A51E, A51D, or A5 IP substitution, wherein the equivalent amino acid position is in reference to a reference sequence of any of SEQ ID NOs: 1-5. The viral glycoprotein variant may or may not comprise further mutation(s) in addition to the mutation at amino acid position equivalent to 50 in reference to a reference sequence of any of SEQ ID NOs: 1-5. In some embodiments, the viral glycoprotein variant that comprises a A5 IE, A5 ID, or A5 IP substitution further comprises one or more additional mutations outside of the amino acid position equivalent to 51 in reference to a reference sequence of any of SEQ ID NOs: 1-5. In some embodiments, the viral glycoprotein variant that comprises a A51E, A51D, or A51P substitution does not comprise any other mutations outside the amino acid position equivalent to 51 in reference to a reference sequence of any of SEQ ID NOs: 1-5.

[0117] In some embodiments, the reference viral glycoprotein is COV-G. Hence, in some embodiments, there is provided a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, and wherein the COV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) infectivity of its pseudotyped virus compared to wildtype COV-G. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356, wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1. The COV-G variant may or may not comprise further mutation(s) in additionto the one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356. The further mutation(s) can be at or in addition to the amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions W14, V17, M41, K44, P46, K50, A51, 1144, D145, S146, F148, C153, C158, V182, and L356. The amino acid at any one of positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 can be substituted for any other amino acid (preferably an amino acid with different side chain property), such as a hydrophilic amino acid (such as Cys, Ser, Thr, Asn, or Gin), an acidic amino acid (such as Asp or Glu), a basic amino acid (such as His, Lys, or Arg), an aromatic amino acid (such as Trp, Tyr, or Phe), an amino acid that influences chain orientation (such as Gly or Pro), or a hydrophobic amino acid (e.g., Norleucine, Met, Ala, Vai, lie, Leu). In some embodiments, the amino acid of any of positions 14, 17, 41, 46, 50, 144, 145, 146, 148, 153, 158, and / or 356 are substituted for a hydrophobic amino acid, such as an alanine (A). In some embodiments, the amino acid of position 50 is substituted for a hydrophobic amino acid, such as an isoleucine (I). In some embodiments, the amino acid of any of positions 44, 50, 144, and / or 182 are substituted for an acidic amino acid, such as a glutamic acid (E). In some embodiments, the amino acid of any of positions 46 and / or 50 are substituted for a hydrophilic amino acid, such as a threonine (T). In some embodiments, the amino acid of any of positions 46, 50, and / or 144 are substituted for a hydrophilic amino acid, such as a glutamine (Q). Hence in some embodiments, there is provided a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of W14A, V17A, M41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, H44E, I144Q, D145A, S146A, F148A, C153A, C158A, V182E, and L356A, wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, there is provided a COV-G variant comprising (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7-17, 20, 24, 33, 36, and 41-47, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 7-17, 20, 24, 33, 36, and 41-47. In some embodiments, the COV-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, M41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, I144A, I144E, I144Q, D145A, S146A, F148A, C153A, C158A, V182E, and L356A further comprises one or more additionalmutations at one or more amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, M41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, F148A, C153A, C158A, V182E, and L356A further comprises one or more additional mutations outside of amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, M41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, F148A, C153A, C158A, V182E, and L356A does not comprise any other mutations outside the amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-47, 74, 77, and 80-83.

[0118] In some embodiments, the reference viral glycoprotein is COV-G. Hence, in some embodiments, there is provided a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, and wherein the COV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to wildtype COV-G. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions 41, 46, 50, 51, and 182, wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1. The COV-G variant may or may not comprise further mutation(s) in addition to the one or more substitutions at any of amino acid positions of 41, 46, 50, 51, and 182. The further mutation(s) can be at or in addition to the amino acid positions equivalent to 41, 46, 50, and 182 in reference to SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions M41, P46, K50, A51, and V182. The aminoacid at any one of positions 41, 46, 50, 51, and 182 can be substituted for any other amino acid (preferably an amino acid with different side chain property), such as a hydrophilic amino acid (such as Cys, Ser, Thr, Asn, or Gin), an acidic amino acid (such as Asp or Glu), a basic amino acid (such as His, Lys, or Arg), an aromatic amino acid (such as Trp, Tyr, or Phe), an amino acid that influences chain orientation (such as Gly or Pro), or a hydrophobic amino acid (e.g., Norleucine, Met, Ala, Vai, lie, Leu). In some embodiments, the amino acid of any of positions 41, 46, 50, and / or 51 are substituted for a hydrophobic amino acid, such as an alanine (A). In some embodiments, the amino acid of position 50 is substituted for a hydrophobic amino acid, such as an isoleucine (I). In some embodiments, the amino acid of any of positions 50 and / or 182 are substituted for an acidic amino acid, such as a glutamic acid (E). In some embodiments, the amino acid of position 50 is substituted for a hydrophilic amino acid, such as a threonine (T). In some embodiments, the amino acid of positions 50 is substituted for a hydrophilic amino acid, such as a glutamine (Q). Hence in some embodiments, there is provided a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of M41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A5 IE, A5 ID, A5 IP, and V182E, wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) a K50T mutation, wherein the amino acid position is in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, there is provided a COV-G variant comprising (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7, 17, 20, 24, and 44-47, 74, 77, and 80-83, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 7, 17, 20, 24, and 44-47, 74, 77, and 80-83. In some embodiments, the COV-G variant that comprises one or more mutations selected from the group consisting of M41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and V182E further comprises one or more additional mutations at one or more amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant that comprises one or more mutations selected from the group consisting of M41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and V182E further comprises one or more additional mutations outside of amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant that comprises one or more mutations selected from the group consisting of M41A, P46A,K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and V182E does not comprise any other mutations outside the amino acid positions 41, 46, 50, 51, and 182 in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7, 17, 20, 24, 44-47, and 74, 77 and 80-83.

[0119] In some embodiments, the reference viral glycoprotein is COV-G. Hence, in some embodiments, there is provided a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) a mutation (e.g., insertion, deletion, and / or substitution) at amino acid position equivalent to 50 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, and wherein the COV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to wildtype COV-G. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50, wherein the amino acid position is in reference to the wildtype COV-G sequence of SEQ ID NO: 1. The COV-G variant may or may not comprise further mutation(s) in addition to the substitution at amino acid position 50. The further mutation(s) can be at or in addition to the amino acid position equivalent to 50 in reference to SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position K50. The lysine at position 50 can be substituted for any other amino acid (preferably an amino acid with different side chain property), such as a hydrophilic amino acid (such as Cys, Ser, Thr, Asn, or Gin), an acidic amino acid (such as Asp or Glu), a basic amino acid (such as His, Lys, or Arg), an aromatic amino acid (such as Trp, Tyr, or Phe), an amino acid that influences chain orientation (such as Gly or Pro), or a hydrophobic amino acid (e.g., Norleucine, Met, Ala, Vai, lie, Leu). In some embodiments, the lysine at position 50 is substituted for a hydrophobic amino acid, such as an alanine (A). In some embodiments, the lysine at position 50 is substituted for a hydrophobic amino acid, such as an isoleucine (I). In some embodiments, the lysine at position 50 is substituted for an acidic amino acid, such as a glutamic acid (E). In some embodiments, the lysine at position 50 is substituted for a hydrophilic amino acid, such as a glutamine (Q). Hence in some embodiments, there is provided a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) a mutation selected from the group consisting of 50Q, 50A, 501, and 50E, wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, there is provided a COV-G variant comprising (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 44-47, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 44-47. In some embodiments, the COV-G variant that comprises a mutation selected from the group consisting of 50Q, 50A, 501, and 50E further comprises one or more additional mutations outside amino acid position 50 in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant that comprises a mutation selected from the group consisting of 50Q, 50A, 501, and 50E does not comprise any other mutations outside amino acid position 50 in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 44-47.

[0120] In some embodiments, the reference viral glycoprotein is VSV-G, such as VSV Orsay-G, VSV SJ-G, or VSV syn-G. Hence, in some embodiments, there is provided a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to areference VSV-G sequence of any of SEQ ID NOs: 2-4, and wherein the VSV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) infectivity of its pseudotyped virus compared to a reference VSV-G (e.g., VSV Orsay-G, VSV SJ-G, or VSV syn-G). In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356, wherein the amino acid positions are in reference to a reference VSV-G sequence of any of SEQ ID NOs: 2-4. The VSV-G variant may or may not comprise further mutation(s) in addition to the one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356. The further mutation(s) can be at or in addition to the amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to any of SEQ ID NOs: 2-4. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions W14, V17, L41 or 141, K44, P46, K50, A51, V144, D145, S146, F148, C153, C158, 1182, and L356. The amino acid at any one of positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 can be substituted for any other amino acid (preferably an amino acid with different side chain property), such as a hydrophilic amino acid (such as Cys, Ser, Thr, Asn, or Gin), an acidicamino acid (such as Asp or Glu), a basic amino acid (such as His, Lys, or Arg), an aromatic amino acid (such as Trp, Tyr, or Phe), an amino acid that influences chain orientation (such as Gly or Pro), or a hydrophobic amino acid (e.g., Norleucine, Met, Ala, Vai, lie, Leu). In some embodiments, the amino acid of positions 14, 17, 41, 46, 50, 51, 144, 145, 146, 148, 153, 158, and / or 356 are substituted for a hydrophobic amino acid, such as an alanine (A). In some embodiments, the amino acid of position 50 is substituted for a hydrophobic amino acid, such as an isoleucine (I). In some embodiments, the amino acid of any of positions 44, 50, 144, and / or 182 are substituted for an acidic amino acid, such as a glutamic acid (E). In some embodiments, the amino acid of any of positions 46 and / or 50 are substituted for a hydrophilic amino acid, such as a threonine (T). In some embodiments, the amino acid of any of positions 46, 50, and / or 144 are substituted for a hydrophilic amino acid, such as a glutamine (Q). In some embodiments, the VSV-G variant comprises one or more mutations selected from the group consisting of W14A, V17A, L41A or 141 A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A in reference to a reference VSV-G sequence of any of SEQ ID NOs: 2-4. Hence in some embodiments, there is provided a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A, wherein the amino acid positions are in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A further comprises one or more additional mutations at one or more amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A further comprises one or more additional mutations outside of amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, there is provided a VSV Orsay-Gvariant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 52-55. In some embodiments, the VSV Orsay-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A does not comprise any other mutations outside the amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of W14A, V17A, I41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A, wherein the amino acid positions are in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, there is provided a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 56-59. In some embodiments, the VSV SJ-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, 141 A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A further comprises one or more additional mutations at one or more amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, I41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A further comprises one or more additional mutations outside of amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A,I41A, K44E, P46A, P46T, P46Q, K5OT, K5OQ, K5OA, K5OI, K5OE, K5OD, K5OH, K5OS, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A does not comprise any other mutations outside the amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, there is provided a VSV syn-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A, wherein the amino acid positions are in reference to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, there is provided a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93. In some embodiments, the VSV syn-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A further comprises one or more additional mutations at one or more amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, the VSV syn-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A further comprises one or more additional mutations outside of amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, the VSV syn-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A does not comprise any other mutations outside the amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the reference VSV syn-G sequence ofSEQ ID NO: 2. In some embodiments, the VSV syn-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93.

[0121] In some embodiments, the reference viral glycoprotein is VSV-G, such as VSV Orsay-G, VSV SJ-G, or VSV syn-G. Hence, in some embodiments, there is provided a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to a reference VSV-G sequence of any of SEQ ID NOs: 2-4, and wherein the VSV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to a reference VSV-G (e.g., VSV Orsay-G, VSV SJ-G, or VSV syn-G). In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions equivalent to 41, 46, 50, 51, and 182, wherein the amino acid positions are in reference to a reference VSV-G sequence of any of SEQ ID NOs: 2-4. The VSV-G variant may or may not comprise further mutation(s) in addition to the one or more substitutions at any of amino acid positions of 41, 46, 50, 51, and 182. The further mutation(s) can be at or in addition to the amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to any of SEQ ID NOs: 2-4. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions L41 or 141, P46, K50, A51, and 1182. The amino acid at any one of positions 41, 46, 50, 51, and 182 can be substituted for any other amino acid (preferably an amino acid with different side chain property), such as a hydrophilic amino acid (such as Cys, Ser, Thr, Asn, or Gin), an acidic amino acid (such as Asp or Glu), a basic amino acid (such as His, Lys, or Arg), an aromatic amino acid (such as Trp, Tyr, or Phe), an amino acid that influences chain orientation (such as Gly or Pro), or a hydrophobic amino acid (e.g., Norleucine, Met, Ala, Vai, He, Leu). In some embodiments, the amino acid of positions 41, 46, 50, and / or 51 are substituted for a hydrophobic amino acid, such as an alanine (A). In some embodiments, the amino acid of position 50 is substituted for a hydrophobic amino acid, such as an isoleucine (I). In some embodiments, the amino acid of any of positions 50 and / or 182 are substituted for an acidic amino acid, such as a glutamic acid (E). In some embodiments, the amino acid of position 50 is substituted for a hydrophilic amino acid, such as a threonine (T). In some embodiments, the amino acid of position 50 is substituted for ahydrophilic amino acid, such as a glutamine (Q). In some embodiments, the VSV-G variant comprises one or more mutations selected from the group consisting of L41A or 141 A, P46A, K50T, K50Q, K50A, K50I, K50E, and I182E in reference to a reference VSV-G sequence of any of SEQ ID NOs: 2-4. Hence in some embodiments, there is provided a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E, wherein the amino acid positions are in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant that comprises one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E further comprises one or more additional mutations at one or more amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant that comprises one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E further comprises one or more additional mutations outside of amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, there is provided a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 52-55. In some embodiments, the VSV Orsay-G variant that comprises one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E does not comprise any other mutations outside the amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of 141 A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E, wherein the amino acid positions are in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, there is provided a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59, or a variant thereof having at least about 70% (e.g., at least aboutany of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 56-59. In some embodiments, the VSV SJ-G variant that comprises one or more mutations selected from the group consisting of I41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E further comprises one or more additional mutations at one or more amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant that comprises one or more mutations selected from the group consisting of I41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E further comprises one or more additional mutations outside of amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant that comprises one or more mutations selected from the group consisting of 141 A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A5 IE, A5 ID, A5 IP, and I182E does not comprise any other mutations outside the amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, there is provided a VSV syn-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E, wherein the amino acid positions are in reference to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, there is provided a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93. In some embodiments, the VSV syn-G variant that comprises one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E further comprises one or more additional mutations at one or more amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, the VSV syn-G variant that comprises one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E further comprises one or more additional mutations outside of amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to the reference VSV syn-G sequence of SEQ ID NO: 2. Insome embodiments, the VSV syn-G variant that comprises one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E does not comprise any other mutations outside the amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, the VSV syn-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93.

[0122] In some embodiments, the reference viral glycoprotein is VSV-G, such as VSV Orsay-G, VSV SJ-G, or VSV syn-G. Hence, in some embodiments, there is provided a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) a mutation (e.g., insertion, deletion, and / or substitution) at amino acid position 50 in reference to a reference VSV-G sequence of any of SEQ ID NOs: 2-4, and wherein the VSV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained atleast about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to a reference VSV-G (e.g., VSV Orsay-G, VSV SJ-G, or VSV syn-G). In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50, wherein the amino acid position is in reference to a reference VSV-G sequence of any of SEQ ID NOs: 2-4. The VSV-G variant may or may not comprise further mutation(s) in addition to the substitution at amino acid position 50. The further mutation(s) can be at or in addition to the amino acid position equivalent to 50 in reference to any of SEQ ID NOs: 2-4. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position K50. The amino acid at position 50 can be substituted for any other amino acid (preferably an amino acid with different side chain property), such as a hydrophilic amino acid (such as Cys, Ser, Thr, Asn, or Gin), an acidic amino acid (such as Asp or Glu), a basic amino acid (such as His, Lys, or Arg), an aromatic amino acid (such as Trp, Tyr, or Phe), an amino acid that influences chain orientation (such as Gly or Pro), or a hydrophobic amino acid (e.g., Norleucine, Met, Ala, Vai, He, Leu). In some embodiments, the lysine at position 50 is substituted for a hydrophobic amino acid, such as an alanine (A). In some embodiments, the lysine at position 50 is substituted for a hydrophobic amino acid, such as an isoleucine (I). In some embodiments, the lysine at position 50 is substituted for an acidic amino acid, such as a glutamic acid (E). In some embodiments, the lysine at position 50 is substituted for a hydrophilic amino acid, such as a glutamine (Q). In some embodiments, theVSV-G variant comprises one or more mutations selected from the group consisting of K50Q, K50A, K50I, and K50E in reference to a reference VSV-G sequence of any of SEQ ID NOs: 2-4. Hence in some embodiments, there is provided a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E, wherein the amino acid positions are in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant that comprises a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E further comprises one or more additional mutations outside amino acid position 50 in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, there is provided a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 52-55. In some embodiments, the VSV Orsay-G variant that comprises a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E does not comprise any other mutations outside amino acid position 50 in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E, wherein the amino acid positions are in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, there is provided a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 56-59. In some embodiments, the VSV SJ-G variant that comprises a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E further comprises one or more additional mutations outside amino acid position 50 in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant that comprises a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E does not comprise any other mutations outside amino acid position 50 in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, there is provided a VSV syn-G variant comprising (or consisting essentially of, or consisting of) a mutation selected from the groupconsisting of K50Q, K50A, K50I, and K50E, wherein the amino acid positions are in reference to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, there is provided a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 48-51. In some embodiments, the VSV syn-G variant that comprises a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E further comprises one or more additional mutations outside amino acid position 50 in reference to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, the VSV syn-G variant that comprises a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E does not comprise any other mutations outside amino acid position 50 in reference to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, the VSV syn-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 48-51.

[0123] In some embodiments, the reference viral glycoprotein is MARAV-G. Hence, in some embodiments, there is provided a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a wildtype MARAV-G sequence of SEQ ID NO: 5, and wherein the MARAV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) infectivity of its pseudotyped virus compared to wildtype MARAV-G. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356, wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. The MARAV-G variant may or may not comprise further mutation(s) in addition to the one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356. The further mutation(s) can be at or in addition to the amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356in reference to SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at amino acid positions W14, V17, L41, K44, P46, K50, A51, 1144, D145A, S146, L148, C153, C158, A182, and L356. The amino acid at any one of positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 can be substituted for anyother amino acid (preferably an amino acid with different side chain property), such as a hydrophilic amino acid (such as Cys, Ser, Thr, Asn, or Gin), an acidic amino acid (such as Asp or Glu), a basic amino acid (such as His, Lys, or Arg), an aromatic amino acid (such as Trp, Tyr, or Phe), an amino acid that influences chain orientation (such as Gly or Pro), or a hydrophobic amino acid (e.g., Norleucine, Met, Ala, Vai, He, Leu). In some embodiments, the amino acid of positions 14, 17, 41, 46, 50, 51, 144, 145, 146, 148, 153, 158, and / or 356 are substituted for a hydrophobic amino acid, such as an alanine (A). In some embodiments, the amino acid of position 50 is substituted for a hydrophobic amino acid, such as an isoleucine (I). In some embodiments, the amino acid of any of positions 44, 50, 51, 144, and / or 182 are substituted for an acidic amino acid, such as a glutamic acid (E). In some embodiments, the amino acid of any of positions 46 and / or 50 are substituted for a hydrophilic amino acid, such as a threonine (T). In some embodiments, the amino acid of any of positions 46, 50, and / or 144 are substituted for a hydrophilic amino acid, such as a glutamine (Q). Hence in some embodiments, there is provided a MARAV-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, L148A, C153A, C158A, A182E, and L356A, wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, there is provided a MARAV-G variant comprising (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103. In some embodiments, the MARAV-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, L148A, C153A, C158A, A182E, and L356A further comprises one or more additional mutations at one or more amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, L148A, C153A, C158A, A182E, and L356A further comprises one or more additional mutations outside of amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153,158, 182, and 356 in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, L148A, C153A, C158A, A182E, and L356A does not comprise any other mutations outside the amino acid positions 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103.

[0124] In some embodiments, the reference viral glycoprotein is MARAV-G. Hence, in some embodiments, there is provided a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to a wildtype MARAV-G sequence of SEQ ID NO: 5, and wherein the MARAV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to wildtype MARAV-G. The MARAV-G variant may or may not comprise further mutation(s) in addition to the amino acid positions equivalent to 41, 46, 50, 51, and 182. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions 41, 46, 50, 51, and 182, wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. The MARAV-G variant may or may not comprise further mutation(s) in addition to the one or more substitutions at any of amino acid positions of 41, 46, 50, 51, and 182. The further mutation(s) can be at or in addition to the amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at amino acid positions L41, P46, K50, A51, and A182. The amino acid at any one of positions 41, 46, 50, 51, and 182 can be substituted for any other amino acid (preferably an amino acid with different side chain property), such as a hydrophilic amino acid (such as Cys, Ser, Thr, Asn, or Gin), an acidic amino acid (such as Asp or Glu), a basic amino acid (such as His, Lys, or Arg), an aromatic amino acid (such as Trp, Tyr, or Phe), an amino acid that influences chain orientation (such as Gly or Pro), or a hydrophobic amino acid (e.g., Norleucine, Met, Ala, Vai, He, Leu). In some embodiments, the amino acid of positions 41, 46,and / or 50 are substituted for a hydrophobic amino acid, such as an alanine (A). In some embodiments, the amino acid of position 50 is substituted for a hydrophobic amino acid, such as an isoleucine (I). In some embodiments, the amino acid of any of positions 50 and / or 182 are substituted for an acidic amino acid, such as a glutamic acid (E). In some embodiments, the amino acid of position 50 is substituted for a hydrophilic amino acid, such as a threonine (T). In some embodiments, the amino acid of position 50 is substituted for a hydrophilic amino acid, such as a glutamine (Q). Hence in some embodiments, there is provided a MARAV-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, and / or A182E, wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) a K50T mutation, wherein the amino acid position is in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, there is provided a MARAV-G variant comprising (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 60-63. In some embodiments, the MARAV-G variant that comprises one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, and / or A182E further comprises one or more additional mutations at one or more amino acid positions equivalent to 41, 46, 50, and 182 in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant that comprises one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, and / or A182E further comprises one or more additional mutations outside of amino acid positions 41, 46, 50, and 182 in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant that comprises one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, and / or A182E does not comprise any other mutations outside the amino acid positions 41, 46, 50, and 182 in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63.

[0125] In some embodiments, the reference viral glycoprotein is MARAV-G. Hence, in some embodiments, there is provided a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) a mutation (e.g., insertion, deletion, and / or substitution) at amino acid position 50 in reference to a wildtype MARAV-G sequence of SEQID NO: 5, and wherein the MARAV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to wildtype MARAV-G. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50, wherein the amino acid position is in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. The MARAV-G variant may or may not comprise further mutation(s) in addition to the substitution at amino acid position 50. The further mutation(s) can be in addition to the amino acid position 50 in reference to SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position K50. The amino acid at position 50 can be substituted for any other amino acid (preferably an amino acid with different side chain property), such as a hydrophilic amino acid (such as Cys, Ser, Thr, Asn, or Gin), an acidic amino acid (such as Asp or Glu), a basic amino acid (such as His, Lys, or Arg), an aromatic amino acid (such as Trp, Tyr, or Phe), an amino acid that influences chain orientation (such as Gly or Pro), or a hydrophobic amino acid (e.g., Norleucine, Met, Ala, Vai, lie, Leu). In some embodiments, the lysine at position 50 is substituted for a hydrophobic amino acid, such as an alanine (A). In some embodiments, the lysine at position 50 is substituted for a hydrophobic amino acid, such as an isoleucine (I). In some embodiments, the lysine at position 50 is substituted for an acidic amino acid, such as a glutamic acid (E). In some embodiments, the lysine at position 50 is substituted for a hydrophilic amino acid, such as a glutamine (Q). Hence in some embodiments, there is provided a MARAV-G variant comprising (or consisting essentially of, or consisting of) a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E, wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, there is provided a MARAV-G variant comprising (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, or a variant thereof having at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to any one of SEQ ID NOs: 60-63. In some embodiments, the MARAV-G variant that comprises a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E further comprises one or more additional mutations outside amino acid position 50 in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant that comprises a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E does not comprise any other mutations outside amino acid position 50 in reference to thewildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63.

[0126] In some embodiments, the viral glycoprotein variant has one single mutation with the substitution at amino acid position 50 or 51 selected from the group consisting of K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51D, and A51P, i.e., the variant does not comprise any other mutations.

[0127] In some embodiments, the viral glycoprotein variant has a combination of two mutations with the substitution at amino acid position 50 and 51 selected from the group consisting of K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51D, and A51P.

[0128] The binding of the viral glycoprotein variant to a viral glycoprotein receptor (e.g., LDL-R) can be measured by any known methods available in the art for detecting or measuring protein-protein binding, such as Western blot, dot blot, surface plasmon resonance (SPR) method, Bio-Layer Interferometry (BLI), FACS, and enzyme-linked immunosorbent assay (ELISA). In some embodiments, the dissociation constant (KD) between the viral glycoprotein variant and LDL-R is at least about 1.2-fold (e.g., such as at least about any of 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, or more) of the KD between the reference (e.g., wildtype) viral glycoprotein and LDL-R. In some embodiments, the binding between the viral glycoprotein variant and LDL-R is reduced by at least 20% (e.g., such as reduced by at least about any of 50%, 60%, 70%, 80%, 90%, 95%, or more, or abolished) as compared to the binding between the reference (e.g., wildtype) viral glycoprotein and LDL-R.

[0129] Fusogenic activity can be evaluated, for example, by assessing efficiency of the viral glycoprotein variant to induce membrane fusion and CAR transduction. In some embodiments, the fusogenic activity of the viral glycoprotein variant is retained by at least 20% (e.g., such as retained by at least about any of 50%, 60%, 70%, 80%, 90%, 95%, or more) as compared to the fusogenic activity of the reference (e.g., wildtype) viral glycoprotein.

[0130] In some embodiments, the infectivity of the virus pseudotyped with the viral glycoprotein variant can be restored (e.g., such as restored by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, or by at least about any of 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, or more) with an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell (e.g., an anti-CD3 scFv).

[0131] In some embodiments, the infectivity of the virus pseudotyped with the viral glycoprotein variant cannot be restored (e.g., such as restored by no more than about 20%,15%, 10%, 5%, 2%, or 0%) with an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell (e.g., an anti-CD3 scFv).

[0132] Also provided are isolated nucleic acids (e.g., a first nucleic acid) encoding any of the viral glycoprotein variants described herein (e.g., any of the viral glycoprotein variants with reduced infectivity of its pseudotyped virus and comprising one or more substitutions at any of amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356, or any of the viral glycoprotein variants with reduced binding to LDL-R and optionally retained fusogenic activity and comprising one or more substitutions at any of amino acid positions equivalent to 41, 46, 50, 51, and 182 (e.g., equivalent to 50), in reference to any of SEQ ID NOs: 1-5), and vectors (e.g., an envelope vector) comprising any of the isolated nucleic acids or encoding any of the viral glycoprotein variants described herein. In some embodiments, the vector (e.g., envelope vector) further comprises a promoter (e.g., CMV promoter) upstream of the nucleic acid (e.g., first nucleic acid) encoding any of the viral glycoprotein variants described herein.III. Recombinant viruses

[0133] A number of viral based systems have been developed for gene transfer into target cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acid can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the target cell in vitro or ex vivo. Vectors derived from retroviruses such as lentivirus are suitable tools to achieve long-term gene transfer, because they allow long-term, stable integration of a transgene and its propagation in progeny cells. Lentiviral vectors also have low immunogenicity, and can transduce non-proliferating cells.

[0134] Provided herein are recombinant viruses that are enveloped (e.g., pseudotyped) with any of the viral glycoprotein variants described herein (e.g., any of the viral glycoprotein variants with reduced infectivity of its pseudotyped virus and comprising one or more substitutions at any of amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356, or any of the viral glycoprotein variants with reduced binding to LDL-R and optionally retained fusogenic activity and comprising one or more substitutions at any of amino acid positions equivalent to 41, 46, 50, 51, and 182 (e.g., equivalent to 50) in reference to any of SEQ ID NOs: 1-5, such as any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-63, 73, 74, 77, 80-86, 89, 91, 93, 94, 96-98, 100, and 103). Examples of recombinant viruses include, but are not limited to, adenovirus, adeno-associated virus (AAV), baculovirus,lentivirus, retrovirus, herpes simplex virus, vaccinia virus, and derivatives thereof. In some embodiments, the recombinant virus is lentivirus.

[0135] In some embodiments, the recombinant virus enveloped (e.g., pseudotyped) with any of the viral glycoprotein variants described herein is characterized by increased (e.g., increasing at least about any of 20%, 50%, 60%, 70%, 80%, 90%, 95%, 1.2-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, or more) stability, reduced (e.g., decreasing atleast about any of 20%, 50%, 60%, 70%, 80%, 90%, 95%, 1.2-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, or more) serum activation, and / or reduced (e.g., decreasing at least aboutany of 20%, 50%, 60%, 70%, 80%, 90%, 95%, 1.2-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, or more) transduction efficiency. Recombinant virus stability can be assessed by any method known in the art, such as thermostability, tolerability to varying pH or salt concentrations, resistance to serum, virus half-life, trypsin sensiti vity, and freeze-thaw tolerance. Serum sensitivity can be measured by any method known in the art, such as a serum neutralization assay. Transduction efficiency can be assessed by using a recombinant virus carrying a selectable marker, such as an antibiotic resistance gene, or an easily detectable marker, such as a transgene encoding a protein that is readily detectable on cell surfaces (e.g., Flag-tagged engineered receptor, see Example 1).

[0136] In some embodiments, there is provided a recombinant virus (e.g., lentivirus), wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, and wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) infectivity of its pseudotyped virus compared to the reference viral glycoprotein. In some embodiments, the viral glycoprotein variant comprises one or more substitutions at any of amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the viral glycoprotein variant comprises any of 14A, 17A, 41 A, 44E, 46A, 46T, 46Q, 50T, 50Q, 50A, 501, 50E, 50D, 50H, 50S, 5 IE, 5 ID, 5 IP, 144A, 144E, 144Q, 145A, 146A, 148A, 153A, 158A, 182E, and / or 356A mutation, wherein the position is in reference to the wildtype COV-G sequence of SEQ ID NO: 1. The viral glycoprotein variant may or may not comprise further mutation(s) in addition to the one ormore substitutions at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to SEQ ID NO: 1. The further mutation(s) can be at or in addition to the amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to SEQ ID NO: 1.

[0137] In some embodiments, there is provided a recombinant virus (e.g., lentivirus), wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, and wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained atleast about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity, compared to the reference viral glycoprotein. In some embodiments, the viral glycoprotein variant comprises one or more substitutions at any of amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the viral glycoprotein variant comprises any of 41A, 46A, 50T, 50Q, 50A, 501, 50E, K50D, K50H, K50S, A51E, A51D, A51P, and / or 182E mutation, wherein the position is in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the viral glycoprotein variant a 50T mutation, wherein the position is in reference to the wildtype COV-G sequence of SEQ ID NO: 1. The viral glycoprotein variant may or may not comprise further mutation(s) in addition to the one or more substitutions at amino acid positions equivalent to 41, 46, 50, 51 and 182 in reference to SEQ ID NO: 1. The further mutation(s) can be at or in addition to the amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to SEQ ID NO: 1.

[0138] In some embodiments, there is provided a recombinant virus (e.g., lentivirus), wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) a mutation (e.g., insertion, deletion, and / or substitution) at amino acid position equivalent to 50 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, and wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99%, or 100%) fusogenic activity, compared to the reference viral glycoprotein. In some embodiments, the viral glycoprotein variant comprises a substitution at amino acid position equivalent to 50 in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the viral glycoprotein variant comprises any of 50Q, 50A, 501, or 50E mutation, wherein the position is in reference to the wildtype COV-G sequence of SEQ ID NO: 1. The viral glycoprotein variant may or may not comprise further mutation(s) in addition to the substitution at amino acid position equivalent to 50 in reference to SEQ ID NO: 1. The further mutation(s) can be in addition to the amino acid position equivalent to 50 in reference to SEQ ID NO: 1.

[0139] In some embodiments, the viral glycoprotein is COV-G, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the wildtype COV-G sequence of SEQ ID NO: 1, and wherein the COV-G variant has reduced (e.g., reduced at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) infectivity of its pseudotyped virus compared to wildtype COV-G. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356, and wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of W14A, V17A, M41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, F148A, C153A, C158A, V182E, and L356A in reference to SEQ ID NO: 1. In some embodiments, the COV-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, M41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, F148A, C153A, C158A, V182E, and L356A shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-47, 74, 77, and 80-83, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-47, 74, 77, and 80-83. In some embodiments,the COV-G variant consists of the amino acid sequence of any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-47, 74, 77, and 80-83.

[0140] In some embodiments, the viral glycoprotein is COV-G, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to the wildtype COV-G sequence of SEQ ID NO: 1, and wherein the COV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity, compared to wildtype COV-G. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 41, 46, 50, 51, and 182, and wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of M41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and V182E in reference to SEQ ID NO: 1. In some embodiments, the COV-G variant that comprises one or more mutations selected from the group consisting of M41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and V182E shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant consists of one or more mutations selected from the group consisting of M41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and V182E in reference to SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7, 17, 20, 24, 44-47, 74, 77, and 80-83, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 7, 17, 20, 24, 44-47, 74, 77, and 80-83. In some embodiments, the COV-G variant consists of the amino acid sequence of any of SEQ ID NOs: 7, 17, 20, 24, 44-47, 74, 77, and 80-83.

[0141] In some embodiments, the viral glycoprotein is COV-G, wherein the COV-G variant comprises (or consists essentially of, or consists of) a mutation (e.g., insertion, deletion, and / or substitution) at amino acid position 50 in reference to the wildtype COV-G sequence of SEQ ID NO: 1, and wherein the COV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to wildtype COV-G. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50, and wherein the amino acid position is in reference to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E in reference to SEQ ID NO: 1. In some embodiments, the COV-G variant that comprises one or more mutations selected from the group consisting of K50Q, K50A, K50I, and K50E shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the wildtype COV-G sequence of SEQ ID NO: 1. In some embodiments, the COV-G variant consists of one or more mutations selected from the group consisting of K50Q, K50A, K50I, and K50E in reference to SEQ ID NO: 1. In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 44-47, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 44-47. In some embodiments, the COV-G variant consists of the amino acid sequence of any of SEQ ID NOs: 44-47.

[0142] In some embodiments, the viral glycoprotein is VSV-G (e.g., VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4, and wherein the VSV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) infectivity of its pseudotyped virus compared to the corresponding reference VSV-G. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356, and wherein the amino acid positions are in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of 14A, 17A, 41A, 44E, 46A, 46T, 46Q, 50T, 50Q, 50A, 501, 50E, 50D, 50H, 50S, 5 IE, 5 ID, 5 IP, 144A, 144E, 144Q, 145 A, 146A, 148A, 153A, 158A, 182E, and 356A in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A,K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant that comprises one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant consists of one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A in reference to SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 52-55, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, the VSV Orsay-G variant consists of the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of W14A, V17A, I41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant that comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of W14A, V17A, I41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant consists of one or more mutations selected from the group consisting of W14A, V17A, 141 A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A in reference to SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 56-59, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 56-59. Insome embodiments, the VSV SJ-G variant consists of the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV-G variant is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A in reference to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, the VSV syn-G variant that comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, the VSV syn-G variant consists of one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, and L356A in reference to SEQ ID NO: 2. In some embodiments, the VSV syn-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93 or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93. In some embodiments, the VSV syn-G variant consists of the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93.

[0143] In some embodiments, the viral glycoprotein is VSV-G (e.g., VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 41, 46, 50, 51 and 182 in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4, and wherein the VSV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to the corresponding reference VSV-G. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 41, 46, 50, 51, and 182, and wherein the amino acid positions are in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of 41 A, 46A, 50T, 50Q, 50A, 501, 50E, 50D, 50H, 50S, 5 IE, 5 ID, 5 IP, and 182E in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant that comprises one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant consists of one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E in reference to SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 52-55, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, the VSV Orsay-G variant consists of the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of 141 A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant that comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of 141 A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant consists of one or more mutations selected from the group consisting of 141 A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E in reference to SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 56-59, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV SJ-G variant consists of the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV-G variantis a VS V syn-G variant comprising (or consisting essentially of, or consisting of) one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P and I182E in reference to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, the VSV syn-G variant that comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, the VSV syn-G variant consists of one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and I182E in reference to SEQ ID NO: 2. In some embodiments, the VSV syn-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 48-51 and 73, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93. In some embodiments, the VSV syn-G variant consists of the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93.

[0144] In some embodiments, the viral glycoprotein is VSV-G (e.g., VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) a mutation (e.g., insertion, deletion, and / or substitution) at amino acid position 50 in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4, and wherein the VSV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to the corresponding reference VSV-G. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50, and wherein the amino acid positions are in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4. In some embodiments, the VSV-G variant comprises (or consists essentially of, or consists of) a mutation selected from the group consisting of 50Q, 50A, 501, and 50E in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E in reference to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant that comprises a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E shares at leastabout 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the wildtype VSV Orsay-G sequence of SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant consists of a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E in reference to SEQ ID NO: 3. In some embodiments, the VSV Orsay-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 52-55, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, the VSV Orsay-G variant consists of the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E in reference to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant that comprises (or consists essentially of, or consists of) a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the wildtype VSV SJ-G sequence of SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant consists of a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E in reference to SEQ ID NO: 4. In some embodiments, the VSV SJ-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 56-59, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV SJ-G variant consists of the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV-G variant is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E in reference to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, the VSV syn-G variant that comprises (or consists essentially of, or consists of) a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the reference VSV syn-G sequence of SEQ ID NO: 2. In some embodiments, the VSV syn-G variant consists of a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E in reference to SEQ ID NO: 2. In some embodiments, the VSV syn-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 48-51, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 48-51. In some embodiments, the VSV syn-G variant consists of the amino acid sequence of any of SEQ ID NOs: 48-51.

[0145] In some embodiments, the viral glycoprotein is MARAV-G, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, and wherein the MARAV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) infectivity of its pseudotyped virus compared to wildtype MARAV-G. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356, and wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50D, K50H, K50S, A51E, A51D, A51P, K50Q, K50A, K50I, K50E, I144A, I144E, I144Q, D145A, S146A, L148A, C153A, C158A, A182E, and L356A in reference to SEQ ID NO: 5. In some embodiments, the MARAV-G variant that comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, L148A, C153A, C158A, A182E, and L356A shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant consists of one or more mutations selected from the group consisting of W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, L148A, C153A, C158A, A182E, and L356A in reference to SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103. In some embodiments, the MARAV-G variant consists of the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103.

[0146] In some embodiments, the viral glycoprotein is MARAV-G, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, and whereinthe MARAV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to wildtype MARAV-G. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 41, 46, 50, 51, and 182, and wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and A182E in reference to SEQ ID NO: 5. In some embodiments, the MARAV-G variant that comprises (or consists essentially of, or consists of) one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and A182E shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant consists of one or more mutations selected from the group consisting of L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, and A182E in reference to SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103. In some embodiments, the MARAV-G variant consists of the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103.

[0147] In some embodiments, the viral glycoprotein is MARAV-G, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) a mutation (e.g., insertion, deletion, and / or substitution) at amino acid position 50 in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, and wherein the MARAV-G variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to wildtype MARAV-G. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50, and wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) amutation selected from the group consisting of K50Q, K50A, K50I, and K50E in reference to SEQ ID NO: 5. In some embodiments, the MARAV-G variant that comprises (or consists essentially of, or consists of) a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E shares at least about 70% (e.g., at least about any of 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the wildtype MARAV-G sequence of SEQ ID NO: 5. In some embodiments, the MARAV-G variant consists of a mutation selected from the group consisting of K50Q, K50A, K50I, and K50E in reference to SEQ ID NO: 5. In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 60-63. In some embodiments, the MARAV-G variant consists of the amino acid sequence of any of SEQ ID NOs: 60-63.

[0148] In some embodiments, the viral glycoprotein variant is encoded by a first nucleic acid. In some embodiments, the first nucleic acid is on an envelope vector. In some embodiments, the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell (e.g., anti-CD3 scFv). In some embodiments, the envelope surface-bound targeting molecule is encoded by a second nucleic acid, such as carried on an envelope vector (either same or different from the envelope vector carrying the first nucleic acid). In some embodiments, the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a heterologous molecule (e.g., heterologous protein or RNA), such as an engineered receptor (e.g., CAR). In some embodiments, the third nucleic acid is on a transfer vector.

[0149] In some embodiments, there is provided a recombinant virus (e.g., lentivirus), wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) infectivity of its pseudotyped virus compared to the reference viral glycoprotein, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell). In some embodiments, there is provideda recombinant virus (e.g., lentivirus) comprising a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 (e.g., W14A, V17A, M41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, F148A, C153A, C158A, V182E, or L356A), wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1, wherein the COV-G variant has reduced infectivity of its pseudotyped virus compared to the wildtype COV-G, and wherein the recombinant virus further comprises an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell). In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-47, 74, 77 and 80-83. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 (e.g., W14A, V17A, L41A or I41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, or L356A), wherein the amino acid positions are in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4, wherein the VSV-G variant has reduced infectivity of its pseudotyped virus compared to the corresponding reference VSV-G, and wherein the recombinant virus further comprises an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell). In some embodiments, the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV-G variant is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 (e.g., W14A, V17A, L41A, K44E, P46A, P46T,P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, L148A, C153A, C158A, A182E, or L356A), wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, wherein the MARAV-G variant has reduced infeed vity of its pseudotyped virus compared to the wildtype MARAV-G, and wherein the recombinant virus further comprises an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell). In some embodiments, the MARAV-G comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103. In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain, e.g., a transmembrane domain derived from CD8. In some embodiments, the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb. In some embodiments, the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a co-stimulation molecule, e.g., CD86, CD80, CD137L, CD58, ICOSL, anti-CD28 antibody or antigen-binding fragment, anti-CD2 antibody or antigen-binding fragment, anti-ICOS antibody or antigenbinding fragment, or anti -4- IBB antibody or antigen-binding fragment. In some embodiments, the viral glycoprotein variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid. In some embodiments, the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a heterologous molecule, such as an engineered receptor (e.g., CAR). The first and second nucleic acids can be on the same vector or different vectors (e.g., envelope vector). In some embodiments, the first and second nucleic acids are on the same vector (e.g., envelope vector), and the third nucleic acid is on a different vector (e.g., transfer vector). The first and second nucleic acids, when on the same vector, can be under the control of a same or different promoters. When the first and second nucleic acids are under the control of a same promoter, the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A).

[0150] In some embodiments, there is provided a recombinant virus (e.g., lentivirus), wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positionsequivalent to 41, 46, 50, 51, and 182 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to the reference viral glycoprotein, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell). In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 41, 46, 50, 51, and 182 (e.g., M41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, or V182E), wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1, wherein the COV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype COV-G, and wherein the recombinant virus further comprises an envelope surfacebound targeting molecule (e.g., anti-CD3 scFv) that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell). In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7, 17, 20, 24, 44-47, 74, 77 and 80-83. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 41, 46, 50, 51 and 182 (e.g., L41A or I41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P or I182E), wherein the amino acid positions are in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4, wherein the VSV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the corresponding reference VSV-G, and wherein the recombinant virus further comprises an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell). In some embodiments, the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV-G variant is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (orconsisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 41, 46, 50, 51, and 182 (e.g., L41A, P46A, K50T, K50Q, K50A, K50I, K50E, or A182E), wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, wherein the MARAV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype MARAV-G, and wherein the recombinant virus further comprises an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell). In some embodiments, the MARAV-G comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103. In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain, e.g., a transmembrane domain derived from CD8. In some embodiments, the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb. In some embodiments, the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a costimulation molecule, e.g., CD86, CD80, CD137L, CD58, ICOSL, anti-CD28 antibody or antigen-binding fragment, anti-CD2 antibody or antigen-bi ndi ng fragment, anti-ICOS antibody or antigen-binding fragment, or anti -4- IBB antibody or antigen-binding fragment. In some embodiments, the viral glycoprotein variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid. In some embodiments, the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a heterologous molecule, such as an engineered receptor (e.g., CAR). The first and second nucleic acids can be on the same vector or different vectors (e.g., envelope vector). In some embodiments, the first and second nucleic acids are on the same vector (e.g., envelope vector), and the third nucleic acid is on a different vector (e.g., transfer vector). The first and second nucleic acids, when on the same vector, can be under the control of a same or different promoters. When the first and second nucleic acids are under the control of a same promoter, the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A).

[0151] In some embodiments, there is provided a recombinant virus (e.g., lentivirus), wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) a mutation (e.g., insertion, deletion, and / or substitution) at amino acid position equivalent to 50 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to the reference viral glycoprotein, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell). In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50 (e.g., K50Q, K50A, K50I, or K50E), wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1, wherein the COV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype COV-G, and wherein the recombinant virus further comprises an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell). In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 44-47. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50 (e.g., K50Q, K50A, K50I, or K50E), wherein the amino acid positions are in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4, wherein the VSV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the corresponding reference VSV-G, and wherein the recombinant virus further comprises an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell). In some embodiments, the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV-G variant is a VSV syn-G variant comprising (or consisting essentiallyof, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a recombinant virus (e.g., lent) virus) comprising a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50 (e.g., K50Q, K50A, K50I, or K50E), wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, wherein the MARAV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype MARAV-G, and wherein the recombinant virus further comprises an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell). In some embodiments, the MARAV-G comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63. In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain or membraneanchoring domain, e.g., a transmembrane domain derived from CD8. In some embodiments, the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb. In some embodiments, the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a co-stimulation molecule, e.g., CD86, CD80, CD137L, CD58, ICOSL, anti-CD28 antibody or antigen-binding fragment, anti-CD2 antibody or antigen-binding fragment, anti-ICOS antibody or antigen-binding fragment, or anti-4-lBB antibody or antigen-binding fragment. In some embodiments, the viral glycoprotein variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid. In some embodiments, the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a heterologous molecule, such as an engineered receptor (e.g., CAR). The first and second nucleic acids can be on the same vector or different vectors (e.g., envelope vector). In some embodiments, the first and second nucleic acids are on the same vector (e.g., envelope vector), and the third nucleic acid is on a different vector (e.g., transfer vector). The first and second nucleic acids, when on the same vector, can be under the control of a same or different promoters. When the first and second nucleic acids are under the control of a same promoter, the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A).

[0152] In some embodiments, there is provided a recombinant virus (e.g., lentivirus) wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) infectivity of its pseudotyped virus compared to the reference viral glycoprotein, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the viral glycoprotein variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the first and second nucleic acids are on the same vector (e.g., envelope vector). In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 (e.g., W14A, V17A, M41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, F148A, C153A, C158A, V182E, or L356A), wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1, wherein the COV-G variant has reduced infectivity of its pseudotyped virus compared to the wildtype COV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the COV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the first and second nucleic acids are on the same vector (e.g., envelope vector). In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7-17, 20, 24, 33, 36, and 41-47. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 (e.g., W14A, V17A, L41A or I41A, K44E, P46A, P46T,P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, or L356A), wherein the amino acid positions are in reference to a reference VSV-G sequence of any of SEQ ID NOs: 2-4, wherein the VSV-G variant has reduced infectivity of its pseudotyped virus compared to the corresponding reference VSV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the VSV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the first and second nucleic acids are on the same vector (e.g., envelope vector). In some embodiments, the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV-G variant is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 (e.g., W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, L148A, C153A, C158A, A182E, or L356A), wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, wherein the MARAV-G variant has reduced infectivity of its pseudotyped virus compared to the wildtype MARAV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the MARAV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the first and second nucleic acids are on the same vector (e.g., envelope vector). In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103. In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain, e.g., a transmembrane domain derived from CD8. In some embodiments, the envelope surface-bound targetingmolecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb. In some embodiments, the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a co-stimulation molecule, e.g., CD86, CD80, CD137L, CD58, ICOSL, anti-CD28 antibody or antigen-binding fragment, anti-CD2 antibody or antigen-binding fragment, anti-ICOS antibody or antigen-binding fragment, or anti-4-lBB antibody or antigenbinding fragment. In some embodiments, the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a heterologous molecule, such as an engineered receptor (e.g., CAR), wherein the third nucleic acid is on a different vector (e.g., transfer vector). The first and second nucleic acids can be under the control of a same or different promoters. In some embodiments, the first and second nucleic acids are under the control of a single / same promoter, and the first and second nucleic acids are connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A). In some embodiments, the first and second nucleic acids are under the control of a single / same promoter and connected via a linking nucleic acid encoding a P2A cleavable linker.

[0153] In some embodiments, there is provided a recombinant virus (e.g., lenti virus) wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to the reference viral glycoprotein, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the viral glycoprotein variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the first and second nucleic acids are on the same vector (e.g., envelope vector). In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positionsof 41, 46, 50, 51, and 182 (e.g., M41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, or V182E), wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1, wherein the COV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype COV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the COV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the first and second nucleic acids are on the same vector (e.g., envelope vector). In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7, 17, 20, 24, 44-47, 74, 77 and 80-83. In some embodiments, there is provided a recombinant virus (e.g., lenti virus) comprising a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 41, 46, 50, 51, and 182 (e.g., L41A or I41A, P46A, K50T, K50Q, K50A, K50I, K50E, or I182E), wherein the amino acid positions are in reference to a reference VSV-G sequence of any of SEQ ID NOs: 2-4, wherein the VSV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the corresponding reference VSV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the VSV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the first and second nucleic acids are on the same vector (e.g., envelope vector). In some embodiments, the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV-G variant is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51 and 73. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 41, 46, 50, and 182 (e.g., L41A, P46A, K50T, K50Q, K50A, K50I, K50E, or A182E), wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, wherein the MARAV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype MARAV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the MARAV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the first and second nucleic acids are on the same vector (e.g., envelope vector). In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63. In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain or membraneanchoring domain, e.g., a transmembrane domain derived from CD8. In some embodiments, the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb. In some embodiments, the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a co-stimulation molecule, e.g., CD86, CD80, CD137L, CD58, ICOSL, anti-CD28 antibody or antigen-binding fragment, anti-CD2 antibody or antigen-binding fragment, anti-ICOS antibody or antigen-binding fragment, or anti -4- IBB antibody or antigen-binding fragment. In some embodiments, the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a heterologous molecule, such as an engineered receptor (e.g., CAR), wherein the third nucleic acid is on a different vector (e.g., transfer vector). The first and second nucleic acids can be under the control of a same or different promoters. In some embodiments, the first and second nucleic acids are under the control of a single / same promoter, and the first and second nucleic acids are connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A). In some embodiments, the first and second nucleic acids are under the control of a single / same promoter and connected via a linking nucleic acid encoding a P2A cleavable linker.

[0154] In some embodiments, there is provided a recombinant virus (e.g., lenti virus) wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) a mutation (e.g., insertion, deletion, and / or substitution) at amino acid position equivalent to 50 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to the reference viral glycoprotein, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the viral glycoprotein variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the first and second nucleic acids are on the same vector (e.g., envelope vector). In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50 (e.g., K50Q, K50A, K50I, or K50E), wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1, wherein the COV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype COV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the COV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the first and second nucleic acids are on the same vector (e.g., envelope vector). In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 44-47. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid positions 50 (e.g., K50Q, K50A, K50I, or K50E), wherein the amino acid positions are in reference to a reference VSV-G sequence of any of SEQ ID NOs: 2-4, wherein the VSV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the corresponding reference VSV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the VSV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the first and second nucleic acids are on the same vector (e.g., envelope vector). In some embodiments, the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59. In someembodiments, the VSV-G variant is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a recombinant virus (e.g., lent) virus) comprising a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50 (e.g., K50Q, K50A, K50I, or K50E), wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, wherein the MARAV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype MARAV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the MARAV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the first and second nucleic acids are on the same vector (e.g., envelope vector). In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63. In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain, e.g., a transmembrane domain derived from CD8. In some embodiments, the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb. In some embodiments, the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a co- stimulation molecule, e.g., CD86, CD80, CD137L, CD58, ICOSL, anti-CD28 antibody or antigen-binding fragment, anti-CD2 antibody or antigen-binding fragment, anti-ICOS antibody or antigen-binding fragment, or anti-4-lBB antibody or antigen-binding fragment. In some embodiments, the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a heterologous molecule, such as an engineered receptor (e.g., CAR), wherein the third nucleic acid is on a different vector (e.g., transfer vector). The first and second nucleic acids can be under the control of a same or different promoters. In some embodiments, the first and second nucleic acids are under the control of a single / same promoter, and the first and second nucleic acids are connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A). In some embodiments, thefirst and second nucleic acids are under the control of a single / same promoter and connected via a linking nucleic acid encoding a P2A cleavable linker.

[0155] In some embodiments, there is provided a recombinant virus (e.g., lentivirus), wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) infectivity of its pseudotyped virus compared to the reference viral glycoprotein, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the viral glycoprotein variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., anti-CD20+CD19 CAR). In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 (e.g., W14A, V17A, M41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, I144A, I144E, I144Q, D145A, S146A, F148A, C153A, C158A, V182E, or L356A), wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1, wherein the COV-G variant has reduced infectivity of its pseudotyped virus compared to the wildtype COV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the COV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., anti-CD20+CD19 CAR). In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-47, 74, 77 and 80-83. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 (e.g., W14A, V17A, L41A or I41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, or L356A), wherein the amino acid positions are in reference to the reference VSV-G sequence of SEQ ID NO: 2-4, wherein the VSV-G variant has reduced infectivity of its pseudotyped virus compared to the corresponding reference VSV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the VSV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., anti-CD20+CD19 CAR). In some embodiments, the VSV-G is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV-G is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 74, 77 and 80-83. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 (e.g., W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, L148A, C153A, C158A, A182E, or L356A), wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, wherein the MARAV-G variant has reduced infectivity of its pseudotyped virus compared to the wildtype MARAV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the MARAV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., anti-CD20+CD19 CAR). In some embodiments, the MARAV-G variantcomprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103. In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain, e.g., a transmembrane domain derived from CD8. In some embodiments, the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb. In some embodiments, the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a co- stimulation molecule, e.g., CD86, CD80, CD137L, CD58, ICOSL, anti-CD28 antibody or antigen-binding fragment, anti-CD2 antibody or antigen-binding fragment, anti-ICOS antibody or antigen-binding fragment, or anti-4-1BB antibody or antigen-binding fragment. In some embodiments, the CAR comprises from N-terminus to C-terminus: (a) an extracellular antigen binding domain (e.g., an anti-CD19 antigen-binding fragment and / or an anti-CD20 antigen-binding fragment); (b) an optional hinge domain; (c) a transmembrane domain; and (d) an intracellular signaling domain. In some embodiments, the CAR further comprises an intracellular co-stimulatory signaling domain, either between the transmembrane domain and the intracellular signaling domain, or at the C-terminus of the intracellular signaling domain. In some embodiments, the extracellular antigen binding domain comprises two or more antibody moieties connected in tandem. The first and second nucleic acids can be on the same vector or different vectors (e.g., envelope vector). In some embodiments, the first and second nucleic acids are on the same vector (e.g., envelope vector), and the third nucleic acid is on a different vector (e.g., transfer vector). The first and second nucleic acids, when on the same vector, can be under the control of a same or different promoters. When the first and second nucleic acids are under the control of a single / same promoter, the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A). In some embodiments, the recombinant virus comprises two or more units of third nucleic acids encoding two or more CARs (e.g., one anti-CD19 CAR, one anti-CD20 CAR). The two or more units of third nucleic acids can be on the same vector or different vectors (e.g., transfer vector). The two or more units of third nucleic acids, when on the same vector, can be under the control of a same or different promoters. When the two or more units of third nucleic acids are under the control of a single / same promoter, the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A).

[0156] In some embodiments, there is provided a recombinant virus (e.g., lentivirus), wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to the reference viral glycoprotein, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the viral glycoprotein variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., anti-CD20+CD19 CAR). In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 41, 46, 50, 51, and 182 (e.g., M41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P or V182E), wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1, wherein the COV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype COV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the COV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., anti-CD20+CD19 CAR). In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7, 17, 20, 24, 44-47, 74, 77 and 80-83. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 41,46, 50, 51, and 182 (e.g., L41A or I41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P or I182E), wherein the amino acid positions are in reference to the reference VSV-G sequence of SEQ ID NO: 2-4, wherein the VSV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the corresponding reference VSV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the VSV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., anti-CD20+CD19 CAR). In some embodiments, the VSV-G is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV-G is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51 and 73. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a recombinant virus (e.g., lenti virus) comprising a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 41, 46, 50, 51, and 182 (e.g., L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, or A182E), wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, wherein the MARAV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype MARAV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the MARAV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., anti-CD20+CD19 CAR). In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103. In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain, e.g., a transmembrane domain derived from CD8. In some embodiments, the envelope surface-bound targeting molecule comprises an antibody orantigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb. In some embodiments, the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a co-stimulation molecule, e.g., CD86, CD80, CD137L, CD58, ICOSL, anti-CD28 antibody or antigen-binding fragment, anti-CD2 antibody or antigen-binding fragment, anti-ICOS antibody or antigenbinding fragment, or anti -4- IBB antibody or antigen-binding fragment. In some embodiments, the CAR comprises from N-terminus to C-terminus: (a) an extracellular antigen binding domain (e.g., an anti-CD19 antigen-binding fragment and / or an anti-CD20 antigen-binding fragment); (b) an optional hinge domain; (c) a transmembrane domain; and (d) an intracellular signaling domain. In some embodiments, the CAR further comprises an intracellular costimulatory signaling domain, either between the transmembrane domain and the intracellular signaling domain, or at the C-terminus of the intracellular signaling domain. In some embodiments, the extracellular antigen binding domain comprises two or more antibody moieties connected in tandem. The first and second nucleic acids can be on the same vector or different vectors (e.g., envelope vector). In some embodiments, the first and second nucleic acids are on the same vector (e.g., envelope vector), and the third nucleic acid is on a different vector (e.g., transfer vector). The first and second nucleic acids, when on the same vector, can be under the control of a same or different promoters. When the first and second nucleic acids are under the control of a single / same promoter, the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A). In some embodiments, the recombinant virus comprises two or more units of third nucleic acids encoding two or more CARs (e.g., one anti-CD19 CAR, one anti-CD20 CAR). The two or more units of third nucleic acids can be on the same vector or different vectors (e.g., transfer vector). The two or more units of third nucleic acids, when on the same vector, can be under the control of a same or different promoters. When the two or more units of third nucleic acids are under the control of a single / same promoter, the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A).

[0157] In some embodiments, there is provided a recombinant virus (e.g., lentivirus), wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) a mutation (e.g., insertion, deletion, and / or substitution) at amino acid position equivalent to 50 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein theviral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to the reference viral glycoprotein, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the viral glycoprotein variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., anti-CD20+CD19 CAR). In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50 (e.g., K50Q, K50A, K50I, or K50E), wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1, wherein the COV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype COV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the COV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., anti-CD20+CD19 CAR). In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 44-47. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50 (e.g., K50Q, K50A, K50I, or K50E), wherein the amino acid position is in reference to the reference VSV-G sequence of SEQ ID NO: 2-4, wherein the VSV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the corresponding reference VSV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the VSV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the recombinant virus further comprises a thirdnucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., anti-CD20+CD19 CAR). In some embodiments, the VSV-G is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV-G is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50 (e.g., K50Q, K50A, K50I, or K50E), wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, wherein the MARAV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype MARAV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the MARAV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, and wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., anti-CD20+CD19 CAR). In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63. In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain, e.g., a transmembrane domain derived from CD8. In some embodiments, the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb. In some embodiments, the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a co-stimulation molecule, e.g., CD86, CD80, CD137L, CD58, ICOSL, anti-CD28 antibody or antigen-binding fragment, anti-CD2 antibody or antigen-binding fragment, anti-ICOS antibody or antigen-binding fragment, or anti-4-lBB antibody or antigenbinding fragment. In some embodiments, the CAR comprises from N-terminus to C-terminus: (a) an extracellular antigen binding domain (e.g., an anti-CD19 antigen-binding fragment and / or an anti-CD20 antigen-binding fragment); (b) an optional hinge domain; (c) a transmembrane domain; and (d) an intracellular signaling domain. In some embodiments, the CAR further comprises an intracellular co-stimulatory signaling domain, either between thetransmembrane domain and the intracellular signaling domain, or at the C-terminus of the intracellular signaling domain. In some embodiments, the extracellular antigen binding domain comprises two or more antibody moieties connected in tandem. The first and second nucleic acids can be on the same vector or different vectors (e.g., envelope vector). In some embodiments, the first and second nucleic acids are on the same vector (e.g., envelope vector), and the third nucleic acid is on a different vector (e.g., transfer vector). The first and second nucleic acids, when on the same vector, can be under the control of a same or different promoters. When the first and second nucleic acids are under the control of a single / same promoter, the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A). In some embodiments, the recombinant virus comprises two or more units of third nucleic acids encoding two or more CARs (e.g., one anti-CD19 CAR, one anti-CD20 CAR). The two or more units of third nucleic acids can be on the same vector or different vectors (e.g., transfer vector). The two or more units of third nucleic acids, when on the same vector, can be under the control of a same or different promoters. When the two or more units of third nucleic acids are under the control of a single / same promoter, the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A).

[0158] In some embodiments, there is provided a recombinant virus (e.g., lentivirus), wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) infectivity of its pseudotyped virus compared to the reference viral glycoprotein, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the viral glycoprotein variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., an anti-CD20+CD19 CAR), wherein the first and second nucleic acids are on the samevector (e.g., envelope vector) and the third nucleic acid comprising a nucleic acid sequence encoding a CAR is on a different vector (e.g., transfer vector). In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 (e.g., W14A, V17A, M41 A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, F148A, C153A, C158A, V182E, or L356A), wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1, wherein the COV-G variant has reduced infectivity of its pseudotyped virus compared to the wildtype COV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the COV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., an anti-CD20+CD19 CAR), wherein the first and second nucleic acids are on the same vector (e.g., envelope vector) and the third nucleic acid comprising a nucleic acid sequence encoding a CAR is on a different vector (e.g., transfer vector). In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-47, 74, 77 and 80-83. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 (e.g., W14A, V17A, L41A or I41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, V144A, V144E, V144Q, D145A, S146A, F148A, C153A, C158A, I182E, or L356A), wherein the amino acid positions are in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4, wherein the VSV-G variant has reduced infectivity of its pseudotyped virus compared to the corresponding reference VSV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the VSV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acidsequence encoding a CAR (e.g., an anti-CD20+CD19 CAR), wherein the first and second nucleic acids are on the same vector (e.g., envelope vector) and the third nucleic acid encoding a CAR is on a different vector (e.g., transfer vector). In some embodiments, the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV-G variant is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 (e.g., W14A, V17A, L41A, K44E, P46A, P46T, P46Q, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, I144A, I144E, I144Q, D145A, S146A, L148A, C153A, C158A, A182E, or L356A), wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, wherein the MARAV-G variant has reduced infectivity of its pseudotyped virus compared to the wildtype MARAV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the MARAV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., an anti-CD20+CD19 CAR), wherein the first and second nucleic acids are on the same vector (e.g., envelope vector) and the third nucleic acid comprising a nucleic acid sequence encoding a CAR is on a different vector (e.g., transfer vector). In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103. In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain, e.g., a transmembrane domain derived from CD8. In some embodiments, the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb. In some embodiments, the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a costimulation molecule, e.g., CD86, CD80, CD137L, CD58, ICOSL, anti-CD28 antibody orantigen-binding fragment, anti-CD2 antibody or antigen-binding fragment, anti-ICOS antibody or antigen-binding fragment, or anti -4- IBB antibody or antigen-binding fragment. In some embodiments, the CAR comprises from N-terminus to C -terminus: (a) an extracellular antigen binding domain (e.g., an anti-CD19 antigen-binding fragment and / or an anti-CD20 antigenbinding fragment); (b) an optional hinge domain; (c) a transmembrane domain; and (d) an intracellular signaling domain. In some embodiments, the CAR further comprises an intracellular co-stimulatory signaling domain, either between the transmembrane domain and the intracellular signaling domain, or at the C-terminus of the intracellular signaling domain. In some embodiments, the extracellular antigen binding domain comprises two or more antibody moieties connected in tandem. The first and second nucleic acids can be under the control of a same or different promoters. In some embodiments, the first and second nucleic acids are under the control of a single / same promoter and connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A). In some embodiments, the first and second nucleic acids are under the control of a single / same promoter and connected via a linking nucleic acid encoding a P2A cleavable linker. In some embodiments, the recombinant virus comprises two or more units of third nucleic acids encoding two or more CARs (e.g., one anti-CD19 CAR, one anti-CD20 CAR). The two or more units of third nucleic acids can be on the same vector or different vectors (e.g., transfer vector). The two or more units of third nucleic acids, when on the same vector, can be under the control of a same or different promoters. When the two or more units of third nucleic acids are under the control of a single / same promoter, the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A).

[0159] In some embodiments, there is provided a recombinant virus (e.g., lentivirus), wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) one or more mutations (e.g., insertion(s), deletion(s), and / or substitution(s)) at amino acid positions equivalent to 41, 46, 50, 51, and 182 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) fusogenic activity compared to the reference viral glycoprotein, wherein the recombinant virus further comprises an envelope surface-boundtargeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the viral glycoprotein variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., an anti-CD20+CD19 CAR), wherein the first and second nucleic acids are on the same vector (e.g., envelope vector) and the third nucleic acid comprising a nucleic acid sequence encoding a CAR is on a different vector (e.g., transfer vector). In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 41, 46, 50, 51, and 182 (e.g., M41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, or V182E), wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1, wherein the COV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype COV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the COV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., an anti-CD20+CD19 CAR), wherein the first and second nucleic acids are on the same vector (e.g., envelope vector) and the third nucleic acid comprising a nucleic acid sequence encoding a CAR is on a different vector (e.g., transfer vector). In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7, 17, 20, 24, 44-47, 74, 77 and 80-83. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 41, 46, 50, 51, and 182 (e.g., L41A or I41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, or I182E), wherein the amino acid positions are in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4, wherein the VSV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the corresponding reference VSV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell suchas T cell), wherein the VSV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., an anti-CD20+CD19 CAR), wherein the first and second nucleic acids are on the same vector (e.g., envelope vector) and the third nucleic acid comprising a nucleic acid sequence encoding a CAR is on a different vector (e.g., transfer vector). In some embodiments, the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV-G variant is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51, 73, 84-86, 89, 91 and 93. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) one or more substitutions at any of amino acid positions of 41, 46, 50, 51, and 182 (e.g., L41A, P46A, K50T, K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, A51P, or A182E), wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, wherein the MARAV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype MARAV-G, wherein the recombinant virus further comprises an envelope surfacebound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the MARAV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., an anti-CD20+CD19 CAR), wherein the first and second nucleic acids are on the same vector (e.g., envelope vector) and the third nucleic acid comprising a nucleic acid sequence encoding a CAR is on a different vector (e.g., transfer vector). In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103. In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain, e.g., a transmembrane domain derived from CD8. In some embodiments, the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb. Insome embodiments, the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a co-stimulation molecule, e.g., CD86, CD80, CD137L, CD58, ICOSL, anti-CD28 antibody or antigen-binding fragment, anti-CD2 antibody or antigen-binding fragment, anti-ICOS antibody or antigen-binding fragment, or anti-4-lBB antibody or antigenbinding fragment. In some embodiments, the CAR comprises from N-terminus to C-terminus: (a) an extracellular antigen binding domain (e.g., an anti-CD19 antigen-binding fragment and / or an anti-CD20 antigen-binding fragment); (b) an optional hinge domain; (c) a transmembrane domain; and (d) an intracellular signaling domain. In some embodiments, the CAR further comprises an intracellular co-stimulatory signaling domain, either between the transmembrane domain and the intracellular signaling domain, or at the C-terminus of the intracellular signaling domain. In some embodiments, the extracellular antigen binding domain comprises two or more antibody moieties connected in tandem. The first and second nucleic acids can be under the control of a same or different promoters. In some embodiments, the first and second nucleic acids are under the control of a single / same promoter and connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A). In some embodiments, the first and second nucleic acids are under the control of a single / same promoter and connected via a linking nucleic acid encoding a P2A cleavable linker. In some embodiments, the recombinant virus comprises two or more units of third nucleic acids encoding two or more CARs (e.g., one anti-CD19 CAR, one anti-CD20 CAR). The two or more units of third nucleic acids can be on the same vector or different vectors (e.g., transfer vector). The two or more units of third nucleic acids, when on the same vector, can be under the control of a same or different promoters. When the two or more units of third nucleic acids are under the control of a single / same promoter, the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A).

[0160] In some embodiments, there is provided a recombinant virus (e.g., lentivirus), wherein the recombinant virus comprises a viral glycoprotein variant of a reference viral glycoprotein (e.g., COV-G, VSV-G (such as VSV Orsay-G, VSV SJ-G, or VSV syn-G), or MARAV-G), wherein the viral glycoprotein variant comprises (or consists essentially of, or consists of) a mutation (e.g., insertion, deletion, and / or substitution) at amino acid position equivalent to 50 in reference to a wildtype COV-G sequence of SEQ ID NO: 1, wherein the viral glycoprotein variant has reduced (e.g., reduced by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, or abolished) binding to LDL-R and optionally has retained (e.g., retained at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%,or 100%) fusogenic activity compared to the reference viral glycoprotein, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the viral glycoprotein variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., an anti-CD20+CD19 CAR), wherein the first and second nucleic acids are on the same vector (e.g., envelope vector) and the third nucleic acid comprising a nucleic acid sequence encoding a CAR is on a different vector (e.g., transfer vector). In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a COV-G variant, wherein the COV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50 (e.g., K50Q, K50A, K50I, or K50E), wherein the amino acid positions are in reference to the wildtype COV-G sequence of SEQ ID NO: 1, wherein the COV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype COV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the COV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., an anti-CD20+CD19 CAR), wherein the first and second nucleic acids are on the same vector (e.g., envelope vector) and the third nucleic acid comprising a nucleic acid sequence encoding a CAR is on a different vector (e.g., transfer vector). In some embodiments, the COV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 7, 17, 20, 24, and 44-47. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a VSV-G variant (e.g., variant of VSV Orsay-G, VSV SJ-G, or VSV syn-G), wherein the VSV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50 (e.g., K50Q, K50A, K50I, or K50E), wherein the amino acid positions are in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4, wherein the VSV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the corresponding reference VSV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the VSV-G variant is encoded by a first nucleic acid, and the envelopesurface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., an anti-CD20+CD19 CAR), wherein the first and second nucleic acids are on the same vector (e.g., envelope vector) and the third nucleic acid comprising a nucleic acid sequence encoding a CAR is on a different vector (e.g., transfer vector). In some embodiments, the VSV-G variant is a VSV SJ-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 56-59. In some embodiments, the VSV-G variant is a VSV syn-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 48-51. In some embodiments, the VSV-G variant is a VSV Orsay-G variant comprising (or consisting essentially of, or consisting of) the amino acid sequence of any of SEQ ID NOs: 52-55. In some embodiments, there is provided a recombinant virus (e.g., lentivirus) comprising a MARAV-G variant, wherein the MARAV-G variant comprises (or consists essentially of, or consists of) a substitution at amino acid position 50 (e.g., K50Q, K50A, K50I, or K50E), wherein the amino acid positions are in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5, wherein the MARAV-G variant has reduced binding to LDL-R and optionally has retained fusogenic activity compared to the wildtype MARAV-G, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein (e.g., CD3) on a target cell (e.g., immune cell such as T cell), wherein the MARAV-G variant is encoded by a first nucleic acid, and the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) is encoded by a second nucleic acid, wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., an anti-CD20+CD19 CAR), wherein the first and second nucleic acids are on the same vector (e.g., envelope vector) and the third nucleic acid comprising a nucleic acid sequence encoding a CAR is on a different vector (e.g., transfer vector). In some embodiments, the MARAV-G variant comprises (or consists essentially of, or consists of) the amino acid sequence of any of SEQ ID NOs: 60-63. In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain, e.g., a transmembrane domain derived from CD8. In some embodiments, the envelope surface-bound targeting molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes the cell surface protein (e.g., CD3) on the target cell, such as Fab, scFv, or sdAb. In some embodiments, the envelope surface-bound targeting molecule comprises a T-cell activation molecule or a co- stimulation molecule, e.g., CD86, CD80, CD137L, CD58, ICOSL, anti-CD28 antibody or antigen-binding fragment, anti-CD2 antibodyor antigen-binding fragment, anti-ICOS antibody or antigen-binding fragment, or anti-4-lBB antibody or antigen-binding fragment. In some embodiments, the CAR comprises from N-terminus to C-terminus: (a) an extracellular antigen binding domain (e.g., an anti-CD19 antigen-binding fragment and / or an anti-CD20 antigen-binding fragment); (b) an optional hinge domain; (c) a transmembrane domain; and (d) an intracellular signaling domain. In some embodiments, the CAR further comprises an intracellular co-stimulatory signaling domain, either between the transmembrane domain and the intracellular signaling domain, or at the C-terminus of the intracellular signaling domain. In some embodiments, the extracellular antigen binding domain comprises two or more antibody moieties connected in tandem. The first and second nucleic acids can be under the control of a same or different promoters. In some embodiments, the first and second nucleic acids are under the control of a single / same promoter and connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A). In some embodiments, the first and second nucleic acids are under the control of a single / same promoter and connected via a linking nucleic acid encoding a P2A cleavable linker. In some embodiments, the recombinant virus comprises two or more units of third nucleic acids encoding two or more CARs (e.g., one anti-CD19 CAR, one anti-CD20 CAR). The two or more units of third nucleic acids can be on the same vector or different vectors (e.g., transfer vector). The two or more units of third nucleic acids, when on the same vector, can be under the control of a same or different promoters. When the two or more units of third nucleic acids are under the control of a single / same promoter, the nucleic acids can be connected via a linking nucleic acid sequence, such as IRES, or a linking nucleic acid encoding a cleavable linker (e.g., 2A peptide, such as P2A, T2A).A. Envelope surface-bound targeting molecule

[0161] Recombinant viruses enveloped (e.g., pseudotyped) with any of the viral glycoprotein variants described herein (e.g., any of the viral glycoprotein variants with reduced infectivity of its pseudotyped virus and comprising one or more substitutions at any of amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to any of SEQ ID NOs: 1-5, such as any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-63, 73, 74, 77, 80-86, 89, 91, 93, 94, 96-98, 100 and 103, or any of the viral glycoprotein variants with reduced binding to LDL-R and optionally retained fusogenic activity, and comprising one or more substitutions at any of amino acid positions equivalent to 41, 46, 50, 51, and 182 (e.g., equivalent to 50 in reference to any of SEQ ID NOs: 1-5, such as any ofSEQ ID NOs: 7, 17, 20, 24, 44-63, 73, 74, 77, 80-86, 89, 91, 93, 94, 96-98, 100 and 103) can have reduced binding affinity between the viral glycoprotein variant and LDL-R. To increase specificity to target cells, recombinant viruses can be engineered to further comprise an envelope surface-bound targeting molecule. In some embodiments, the envelope surfacebound targeting molecule specifically recognizes a cell surface protein on a target cell, such as anti-CD3 scFv that can target T cells. In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain (e.g., CD8-derived transmembrane domain) or membrane-anchoring domain. In some embodiments, the envelope surface-bound targeting molecule is encoded by a second nucleic acid.

[0162] The envelope surface-bound targeting molecule may comprise an antibody or antigenbinding fragment thereof which can enhance recombinant virus targeting to any target cell of interest. The target cell can be dividing or non-dividing, such as hematopoietic cells, lymphoid cells, myeloid cells, epithelial cells, fibroblasts, muscle cells, and neuronal cells. In some embodiments, the target cell is a disease cell (e.g., cancer cell) to be treated. In some embodiments, the target cell is an immune cell, such as an immune effector cell. In some embodiments, the immune cell is selected from the group consisting of a monocyte, a dendritic cell, a macrophage, a B cell, a T cell, a natural killer (NK) cell, or a combination thereof. In some embodiments, the immune cell is a T cell, such as a killer T cell (Tc), cytotoxic T lymphocyte (CTL), a helper T cell (Th), a regulatory T cell (Treg), an af> T cell, a y5 T cell, and a natural killer T (NKT) cell.

[0163] Cell surface protein on a target cell can be any antigen displayed on the surface of the target cell. To increase target cell transduction specificity, a cell surface protein specific for the target cell can be chosen to be targeted by the envelope surface-bound targeting molecule.

[0164] The cell surface protein of a T cell can be any antigen displayed on the surface of a T cell. To increase T-cell transduction specificity, a T-cell specific antigen can be used. In some embodiments, the T cell surface protein is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD25, CD27, CD28, CD31, CD38, CD45, CD56, CD58, CD69, CD95, CD103, CD122, CD127, CD161, CCR4, CCR5, CCR6, CCR7, CCR10, CTLA4, CXCR3, CDlla, CD40L, and TCR. In some embodiments, the envelope surface-bound antibody or antigen-binding fragment thereof specifically recognizes CD3. Exemplary anti-CD3 antibodies or antigen-binding fragments include but are not limited to, clones OKT3 (e.g., Invitrogen), UCHT1 (e.g., Invitrogen), SP7 (e.g., Invitrogen), SK7 (e.g., Invitrogen), F7.2.38 (e.g., Invitrogen), HIT3A (e.g., Invitrogen), 7D6 (e.g., Invitrogen), S4.1 (e.g., Invitrogen), RIV9 (e.g., Invitrogen), MEM-57 (e.g., Invitrogen), 3F3A1 (e.g., Proteintech), UMAB54 (e.g.,OriGene), GT0013 (e.g., Invitrogen), APA1 / 1 (e.g., Invitrogen), 4E2 (e.g., Invitrogen), 4D10A6 (e.g., Invitrogen), OTI3E10 (e.g., OriGene), B-B12 (e.g., NeoBiotechnologies), C3e / 1308 (e.g., NeoBiotechnologies), C3e / 2479 (e.g., NeoBiotechnologies), CRIS-7 (e.g., NeoBiotechnologies), 145-2C11 (e.g., AbboMax), 4C1 (e.g., Abnova), 3E12 (e.g., Abnova), ICO-90 (e.g., Invitrogen), 3H5 (e.g., Abnova), M2AB (e.g., Exalpha), ZM45 (e.g., Zeta).

[0165] The antibody or antigen-binding fragment thereof comprised by the envelope surfacebound targeting molecule can be of any format. In some embodiments, the envelope surfacebound antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab, a Fab’, a (Fab’)2, an Fv, an scFv, and an sdAb. In some embodiments, the antibody or antigen-binding fragment thereof is an scFv.

[0166] Traditional transduction and activation of target immune cells typically requires separate steps, for example, activation of immune cells followed by transduction. In some embodiments, recombinant viruses expressing the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) can transduce and activate immune cells (e.g., T cells) at the same time. In some embodiments, the target cell is a T cell, and the activation of the T cell comprises upregulation (e.g., upregulating at least about 20%, 50%, 90%, 1-fold, 2-fold, 10-fold, or more) of a marker selected from one or more of CD25, CD38 CD69, CD71, GITR (glucocorticoid-induced TNFR-related receptor), CTLA-4, NTB-A, and HLA-DR. In some embodiments, the target cell is an NK cell, and the activation of the NK cell comprises upregulation (e.g., upregulating at least about 20%, 50%, 90%, 1-fold, 2-fold, 10-fold, or more) of a marker selected from one or more of CD16, CD27, CD56, CD69, Sca-1, NKG2D, and KLRG1 (killer cell lectin-like receptor Gl).

[0167] In some embodiments, the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain. In some embodiments, the membrane-anchoring domain is a glycosylphosphatidylinositol (GPI) anchor. In some embodiments, the transmembrane domain is derived from a transmembrane protein selected from the group consisting of CD8, CD4, CD28, 4-1BB, CD80, CD86, CD152, and PD-1. In some embodiments, the transmembrane domain is derived from CD8. In some embodiments, the envelope surface-bound targeting molecule further comprises a domain C-terminal to the transmembrane domain. The domain C-terminal to the transmembrane domain of the envelope surface-bound targeting molecule can derive from an intracellular portion of any transmembrane protein, which can be a primary intracellular signaling domain, intracellular co-stimulatory signaling domain, or a cytoplasmic portion without signaling function. For example, the domain C-terminal to the transmembrane domain of the envelope surface-boundtargeting molecule can derive from any of CD3^, FcRy, FcR, CD3y, CD35, CD3a, TCRa, TCR, TCRy, TCR5, CD5, CD22, CD79a, CD79b, CD66d, and 4-1BB. In some embodiments, the envelope surface-bound targeting molecule thereof comprises an intracellular domain derived from 4-1BB C-terminal to the transmembrane domain. In some embodiments, the envelope surface-bound targeting molecule comprises from N’ to C’: an antibody or antigenbinding fragment thereof that specifically recognizes a cell surface protein on a target cell (e.g., anti-CD3 scFv), a transmembrane domain or membrane-anchoring domain (e.g., CD8-derived transmembrane domain), and optionally a domain C-terminal to the transmembrane domain or membrane-anchoring domain (e.g., intracellular domain of 4-1BB or fragment thereof).

[0168] In some embodiments, the envelope surface-bound targeting molecule (e.g., envelope surface-bound antibody or antigen-binding fragment thereof) binds to the cell surface protein on target cell with a KD of < lOpM, < IpM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., about 10’5M or less, such as any of from about 10’5M to about 10’13M, from about 10’8M to about 1013M, from about 10’9M to about 1013M, from about IO10M to about 1013M, or from about 1011M to about 1013M). A variety of methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure, including by RIA, for example, performed with the an antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81); by biolayer interferometry (BLI) or surface plasmon resonance (SPR) assays by Octet®, using, for example, an Octet®Red96 system, or by Biacore®, using, for example, a Biacore®TM-2000 or a Biacore®TM-3000. An “on-rate” or “rate of association” or “association rate” or “kon” may also be determined with the same BLI or SPR techniques described above using, for example, the Octet®Red96, the Biacore®TM-2000, or the Biacore®TM-3000 system.

[0169] In some embodiments, the envelope surface-bound targeting molecule comprises an immune cell (e.g., T-cell) activation molecule or co-stimulation molecule. In some embodiments, the immune cell activation molecule or co-stimulation molecule activates and / or co-stimulates T cells. Examples of T cell activation or co-stimulation molecules include, but are not limited to, CD3, CD8a, CD27, CD28, CD80, CD86, FCRy, 0X40, ICOS, CD40 ligand (CD40L), 4-1BB, and CD137 ligand (CD137L). In some embodiments, the T cell activation or co-stimulation molecule is selected from the group consisting of an anti-CD28 antibody or antigen-binding fragment thereof, anti-CD137 antibody or antigen-binding fragment thereof, anti-CD2 antibody or antigen-binding fragment, anti-ICOS antibody or antigen-binding fragment, an anti-4-lBB antibody or antigen-bindi ng fragment thereof, CD86, CD80, CD137L, CD58, and ICOSL. In some embodiments, the T cell activation or co-stimulation moleculecomprises a transmembrane domain (e.g., derived from CD8) or membrane-anchoring domain. In some embodiments, the envelope surface-bound targeting molecule comprises from N’ to C’: an immune cell activation molecule or co- stimulation molecule (e.g., anti-CD28 or anti-4-1BB antigen-binding fragment), a transmembrane domain or membrane-anchoring domain (e.g., CD8-derived transmembrane domain), and optionally a domain C-terminal to the transmembrane domain or membrane-anchoring domain (e.g., intracellular domain of 4-1BB or fragment thereof). In some embodiments, the envelope surface-bound targeting molecule is a full length CD86, CD80, or CD137L.

[0170] In some embodiments, the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) improves transduction efficiency of the recombinant virus comprising any of the viral glycoprotein variants disclosed herein (e.g., any of the viral glycoprotein variants with reduced infectivity of its pseudotyped virus and comprising one or more substitutions at any of amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to any of SEQ ID NOs: 1-5, such as any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-63, 73, 74, 77, 80-86, 89, 91, 93, 94, 96-98, 100, and 103, or any of the viral glycoprotein variants with reduced binding to LDL-R and optionally retained fusogenic activity, and comprising one or more substitutions at any of amino acid positions equivalent to 41, 46, 50, 51, and 182 (e.g., equivalent to 50) in reference to any of SEQ ID NOs: 1-5, such as any of SEQ ID NOs: 7, 17, 20, 24, 44-63, 73, 74, 77, 80-86, 89, 91, 93, 94, 96-98, 100, and 103) by specifically targeting the recombinant virus to the target cell of interest (e.g., T cell), such as improving at least about 5% (e.g., at least about any of 10%, 20%, 50%, 90%, 1-fold, 1.5-fold, 2-fold, 5 -fold, 10-fold, 20- fold, or more) transduction efficiency of the recombinant virus compared to: i) a corresponding recombinant virus comprising a same viral glycoprotein variant but does not comprise the envelope surface-bound targeting molecule; and / or ii) a recombinant virus comprising a corresponding reference viral glycoprotein with or without the envelope surface-bound targeting molecule. In some embodiments, a recombinant virus comprising any of the viral glycoprotein variants disclosed herein (e.g., any of the viral glycoprotein variants with reduced infectivity of its pseudotyped virus and comprising one or more substitutions at any of amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to any of SEQ ID NOs: 1-5, such as any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-63, 73, 74, 77, 80-86, 89, 91, 93, 94, 96-98, 100, 103, or any of the viral glycoprotein variants with reduced binding to LDL-R and optionally retained fusogenic activity, and comprising one or more substitutions at any of amino acid positions equivalent to 41, 46, 50, 51, and 182 (e.g., equivalent to 50) in reference to any of SEQ IDNOs: 1-5, such as any of SEQ ID NOs: 7, 17, 20, 24, 44-63, 73, 74, 77, 80-86, 89, 91, 93, 94, 96-98, 100, and 103) and an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) has reduced transduction of non-target cells (e.g., non-T cell), such as reducing at least about 5% (e.g., at least about any of 10%, 20%, 50%, 90%, 95%, 99%, or 100%) transduction efficiency of non-target cells compared to: i) a corresponding recombinant virus comprising a same viral glycoprotein variant but does not comprise the envelope surface-bound targeting molecule; and / or ii) a recombinant virus comprising a corresponding reference viral glycoprotein with or without the envelope surface-bound targeting molecule. In some embodiments, the transduction efficiency is reflected by the expression of a heterologous nucleic acid (e.g., encoding a CAR) comprised in the recombinant virus by the target cell. In some embodiments, the transduction efficiency is reflected by the expression and / or function of a heterologous nucleic acid (e.g., encoding a CAR or siRNA) comprised in the recombinant virus in the target cell, such as CAR-T mediated cytotoxicity, or gene knock-down in the target cell. In some embodiments, the recombinant virus comprises a heterologous nucleic acid comprising a nucleic acid sequence encoding a CAR (e.g., anti-CD20+CD19 CAR), and any of the viral glycoprotein variants disclosed herein (e.g., any of the viral glycoprotein variants with reduced infectivity of its pseudotyped virus and comprising one or more substitutions at any of amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to any of SEQ ID NOs: 1-5, such as any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-63, 73, 74, 77, 80-86, 89, 91, 93, 94, 96-98, 100, and 103, or any of the viral glycoprotein variants with reduced binding to LDL-R and optionally retained fusogenic activity, and comprising one or more substitutions at any of amino acid positions equivalent to 41, 46, 50, 51, and 182 (e.g., equivalent to 50) in reference to any of SEQ ID NOs: 1-5, such as any of SEQ ID NOs: 7, 17, 20, 24, 44-63, 73, 74, 77, 80-86, 89, 91, 93, 94, 96-98, 100, and 103) and an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv), wherein the target cell (e.g., T cell) transduced with the recombinant virus has comparable (e.g., within about 5% difference) or increased CAR-mediated cytotoxicity (e.g., against CD19+and / or CD20+B cell), such as increasing at least about 5% (e.g., at least about any of 10%, 20%, 50%, 90%, 1-fold, 1.5 -fold, 2-fold, 5 -fold, 10-fold, 20-fold, or more) CAR-mediated cytotoxicity compared to a target cell (e.g., T cell) transduced with: i) a corresponding recombinant virus comprising a same viral glycoprotein variant but does not comprise the envelope surface-bound targeting molecule; and / or ii) a recombinant virus comprising a corresponding reference viral glycoprotein but does not comprise the envelope surface-bound targeting molecule.

[0171] In some embodiments, the envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) of the recombinant virus can activate the transduced target cell (e.g., T cell) simultaneously with transduction. In some embodiments, at least about 20% (such as at least about any of 50%, 80%, 90%, 95%, 99%, or 100%) of transduced target cells are activated (e.g., upregulating CD25 expression) simultaneously with transduction. In some embodiments, a recombinant virus comprising any of the viral glycoprotein variants disclosed herein (e.g., any of the viral glycoprotein variants with reduced infectivity of its pseudotyped virus and comprising one or more substitutions at any of amino acid positions equivalent to 14, 17, 41, 44, 46, 50, 51, 144, 145, 146, 148, 153, 158, 182, and 356 in reference to any of SEQ ID NOs: 1-5, such as any of SEQ ID NOs: 7-17, 20, 24, 33, 36, 41-63, 73, 74, 77, 80-86, 89, 91, 93, 94, 96-98, 100, and 103, or any of the viral glycoprotein variants with reduced binding to LDL-R and optionally retained fusogenic activity, and comprising one or more substitutions at any of amino acid positions equivalent to 41, 46, 50, 51, and 182 (e.g., equivalent to 50) in reference to any of SEQ ID NOs: 1-5, such as any of SEQ ID NOs: 7, 17, 20, 24, 44-63, 73, 74, 77, 80-86, 89, 91, 93, 94, 96-98, 100, and 103) and an envelope surface-bound targeting molecule (e.g., anti-CD3 scFv) increases activation of target cells (e.g., T cell), such as increasing at least about 10% (e.g., at least about any of 20%, 50%, 90%, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, or more) activation of target cells compared to: i) a corresponding recombinant virus comprising a same viral glycoprotein variant but does not comprise the envelope surface-bound targeting molecule; and / or ii) a recombinant virus comprising a corresponding reference viral glycoprotein but does not comprise the envelope surface-bound targeting molecule. In some embodiments, the target cell is a T cell, and the activation of T cell comprises upregulation (e.g., upregulating at least about 20%, 50%, 90%, 1-fold, 2-fold, 10-fold, or more) of CD25 on the cell surface.B. Heterologous nucleic acid

[0172] In some embodiments, the recombinant virus further comprises a nucleic acid (e.g., a third nucleic acid) that encodes a heterologous molecule (e.g., heterologous protein or RNA), such as a therapeutic protein or RNA (e.g., siRNA). “Heterologous” nucleic acids or molecules encoded thereof refer to a nucleic acid or a molecule to be introduced to the target cell by the recombinant viruses described herein. The target cell may or may not have an endogenous version of this to-be-introduced nucleic acid or molecule. For example, in some embodiments, the recombinant virus further comprises a nucleic acid (e.g., a third nucleic acid) that encodes a functional or wildtype version of an RNA or protein, which can compensate for the expressionand / or function of a corresponding RNA or protein (e.g., mutant protein) in the target cell to be transduced. In some embodiments, the heterologous molecule is a therapeutic protein. In some embodiments, the therapeutic protein is an immunomodulator, such as immunostimulatory or immunosuppressant protein. Immunomodulators include but are not limited to, immune checkpoint inhibitors, immune checkpoint stimulators, cytokines, and immunocytokines. In some embodiments, the therapeutic protein (or immune che...

Claims

1. CLAIMS1. A viral glycoprotein variant of a reference viral glycoprotein, wherein the viral glycoprotein variant comprises a mutation at amino acid position equivalent to 50 or 51 in reference to a wildtype cocal virus glycoprotein (COV-G) sequence of SEQ ID NO: 1, and wherein the viral glycoprotein variant has reduced binding to low-density lipoprotein receptor (LDL-R) compared to the reference viral glycoprotein.

2. The viral glycoprotein variant of claim 1, wherein the reference viral glycoprotein is COV-G, vesicular stomatitis virus glycoprotein (VSV-G), or Maraba virus glycoprotein (MARAV-G).

3. The viral glycoprotein variant of claim 2, wherein the viral glycoprotein variant is a variant of COV-G, wherein the COV-G variant comprises a substitution at amino acid position 50 or 51, and wherein the amino acid position is in reference to the wildtype COV-G sequence of SEQ ID NO: 1.

4. The viral glycoprotein variant of claim 2 or 3, wherein the COV-G variant comprises a substitution selected from the group consisti ng of K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, and A51P.

5. The viral glycoprotein variant of claim 2 or 3, wherein the COV-G variant comprises a substitution selected from the group consisti ng of K50Q, K50A, K50I, and K50E.

6. The viral glycoprotein variant of any one of claims 3-5, wherein the COV-G variant comprises the amino acid sequence of any of SEQ ID NOs: 44-47, 74, 77, and 80-83, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 44-47, 74, 77, and 80-83.

7. The viral glycoprotein variant of any one of claims 3-5, wherein the COV-G variant comprises the amino acid sequence of any of SEQ ID NOs: 44-47, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 44-47.

8. The viral glycoprotein variant of claim 2, wherein the viral glycoprotein variant is a variant of VSV-G, wherein the VSV-G variant comprises a substitution at amino acidposition 50 or 51, and wherein the amino acid position is in reference to the reference VSV-G sequence of any of SEQ ID NOs: 2-4.

9. The viral glycoprotein variant of claim 2 or 8, wherein the VSV-G variant comprises a substitution selected from the group consisti ng of K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, and A51P.

10. The viral glycoprotein variant of claim 2 or 8, wherein the VSV-G variant comprises a substitution selected from the group consisting of K50Q, K50A, K50I, and K50E.

11. The viral glycoprotein variant of any one of claims 8-10, wherein the VSV-G variant comprises the amino acid sequence of any of SEQ ID NOs: 48-59, 84-86, 89, 91 and 93, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 48-59, 84-86, 89, 91 and 93.

12. The viral glycoprotein variant of any one of claims 8-10, wherein the VSV-G variant comprises the amino acid sequence of any of SEQ ID NOs: 48-59.

13. The viral glycoprotein variant of claim 2, wherein the viral glycoprotein variant is a variant of MARAV-G, wherein the MARAV-G variant comprises a substitution at amino acid position 50 or 51, and wherein the amino acid position is in reference to the wildtype MARAV-G sequence of SEQ ID NO: 5.

14. The viral glycoprotein variant of claim 2 or 13, wherein the MARAV-G variant comprises a substitution selected from the group consisting of K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, and A51P.

15. The viral glycoprotein variant of claim 2 or 13, wherein the MARAV-G variant comprises a substitution selected from the group consisting of K50Q, K50A, K50I, and K50E.

16. The viral glycoprotein variant of any one of claims 13-15, wherein the MARAV-G variant comprises the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and103, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 60-63, 94, 96-98, 100 and 103.

17. The viral glycoprotein variant of any one of claims 13-15, wherein the MARAV-G variant comprises the amino acid sequence of any of SEQ ID NOs: 60-63, or having at least about 70% sequence identity to the amino acid sequence of any of SEQ ID NOs: 60-63.

18. The viral glycoprotein variant of any one of claims 1-17, wherein the viral glycoprotein variant has a combi nation of two mutations with the substitution at amino acid position 50 and 51 selected from the group consisting of K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, and A51P.

19. The viral glycoprotein variant of any one of claims 1-17, wherein the viral glycoprotein variant has one single mutation with the substitution at amino acid position 50 or 51 selected from the group consisting of K50Q, K50A, K50I, K50E, K50D, K50H, K50S, A51E, A51D, and A51P.

20. A first nucleic acid comprising a nucleic acid sequence encoding the viral glycoprotein variant according to any one of claims 1-19.

21. A vector comprising the first nucleic acid of claim 20.

22. A recombinant virus comprising the viral glycoprotein variant of any one of claims 1-19.

23. The recombinant virus of claim 22, wherein the recombinant virus is a lentivirus.

24. The recombinant virus of claim 22 or 23, wherein the recombinant virus further comprises an envelope surface-bound targeting molecule that specifically recognizes a cell surface protein on a target cell, optionally wherein the envelope surface-bound targeting molecule is encoded by a second nucleic acid.

25. The recombinant virus of claim 24, wherein the envelope surface-bound targeting molecule further comprises a transmembrane domain or membrane-anchoring domain.

26. The recombinant virus of claim 25, wherein the transmembrane domain is derived from the group consisting of CD8a, CD4, CD28, 4-1BB, CD80, CD86, CD152, and PD-1; optionally wherein the transmembrane domain is derived from CD8a.

27. The recombinant virus of any one of claims 24-26, wherein the target cell is an immune cell.

28. The recombinant virus of claim 27, wherein the immune cell is selected from the group consisting of a monocyte, a dendritic cell, a macrophage, a B cell, a T cell, a natural killer (NK) cell, or a combination thereof.

29. The recombinant virus of claim 28, wherein the immune cell is a T cell.

30. The recombinant virus of any one of claims 27-29, wherein the cell surface protein is selected from the group consisting of CD3, CD5, CD2, TCR, CD4, CD8 and CD7.

31. The recombinant virus of claim 30, wherein the cell surface protein is CD3.

32. The recombinant virus of any one of claims 24-31, wherein the envelope surfacebound targeting molecule comprises an antibody or antigen-bi ndi ng fragment thereof that specifically recognizes the cell surface protein on the target cell, and optionally wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a full-length antibody, a Fab, a Fab’, a (Fab’, an Fv, an scFv, and an sdAb.

33. The recombinant virus of claim 32, wherein the antibody or antigen-binding fragment thereof is an scFv.

34. The recombinant virus of claim 27-29, wherein the envelope surface-bound targeting molecule is a T-cell activation molecule or a co-stimulation molecule.

35. The recombinant virus of claim 34, wherein the T-cell activation or co-stimulation molecule comprises CD86, CD80, CD137L, an anti-CD28 antibody or antigen-binding fragment, or an anti-4- 1 BB antibody or antigen-binding fragment.

36. The recombinant virus of any one of claims 22-35, wherein the recombinant virus further comprises a third nucleic acid comprising a nucleic acid sequence encoding a heterologous molecule.

37. The recombinant virus of claim 36, wherein the heterologous molecule is an engineered receptor.

38. The recombinant virus of claim 37, wherein the engineered receptor is a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), a chimeric TCR (cTCR), a T cell antigen coupler (TAC), or a TAC-like engineered receptor.

39. The recombinant virus of claim 38, wherein the engineered receptor is a CAR, and wherein the CAR comprises an extracellular binding domain specifically recognizing a cancer-associated antigen, a cancer-specific antigen, or an autoimmune disease-associated antigen.

40. The recombinant virus of claim 39, wherein extracellular binding domain specifically recognizes a cancer-associated antigen or a cancer-specific antigen selected from the group consisting of CD19, CD20, CD22, BCMA, DLL3, B7H3, PD-L1, PD-L2, CLL1, GPC3, GU2CYC, CD7, CD38, CD41, CD123, Claudin 18.2, Claudin 6, NKG2D, GPRC5D, CD70, and any combi nation thereof.

41. The recombinant virus of any one of claims 24-40, wherein the first nucleic acid and the second nucleic acid are on the same vector.

42. The recombinant virus of claim 41, wherein the first nucleic acid and the second nucleic acid are under the control of a same promoter.

43. The recombinant virus of claim 42, wherein the first nucleic acid and the second nucleic acid are connected via a linking nucleic acid encoding a cleavable linker.

44. The recombinant virus of claim 43, wherein the cleavable linker is a 2 A peptide.

45. The recombinant virus of any one of claims 24-40, wherein the first nucleic acid and the second nucleic acids are on two separate vectors.

46. The recombinant virus of any one of claims 36-40, wherein the first nucleic acid, the second nucleic acid, and the third nucleic acid are on different vectors.

47. A method of specifically delivering a heterologous nucleic acid into a target cell, comprising contacting the target cell with a recombinant virus, wherein the recombinant virus comprises the heterologous nucleic acid to be delivered into the target cell and the viral glycoprotein variant of any one of claims 1-19.

48. The method of claim 47, wherein the heterologous nucleic acid encodes a heterologous molecule.

49. The method of claim 48, wherein the heterologous molecule is a CAR, an engineered TCR, a cTCR, a TAC, or a TAC-like engineered receptor.

50. A method of producing a recombinant virus comprising the viral glycoprotein variant of any one of claims 1-19, wherein the method comprises introducing a first nucleic acid comprising a nucleic acid sequence encoding the viral glycoprotein variant and a packaging vector comprising one or more nucleic acids encoding one or more packaging proteins into a producer cell, thereby producing the recombinant virus.

51. The method of claim 50, further comprising introducing a second nucleic acid comprising a nucleic acid sequence encoding an envelope surface-bound targeti ng molecule into the producer cell, wherein the envelope surface-bound targeting molecule specifically recognizes a cell surface protein on a target cell.

52. The method of claim 50 or 51, further comprising introducing a third nucleic acid comprising a nucleic acid sequence encoding a heterologous molecule into the producer cell.

53. A pharmaceutical composition comprising the recombinant virus of any one of claims 36-46, and a pharmaceutically acceptable excipient.

54. A method of treating a disease or disorder in an individual, comprising administering to the individual a therapeutically effecti ve amount of the pharmaceutical composition of claim 53.

55. The method of claim 54, wherein the disease or disorder is a cancer or an autoimmune disease.

56. The method of claim 54 or 55, wherein the individual is a human.

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