Ligand discovery and gene delivery via retroviral surface display

Abstract

Disclosed herein are retroviral compositions and methods of using the same for gene delivery, wherein the retrovirus comprises a viral envelope protein that comprises at least one mutation that reduces its native function, a non-viral membrane-associated protein that comprises a membrane-associated domain and an extracellular targeting domain.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of the filing date of U.S. Provisional Application No. 62 / 851,889, entitled "LIGAND DISCOVERY AND GENE DELIVERY VIA RETROVIRAL SURFACE DISPLAY," filed May 23, 2019; the entire contents of which are incorporated herein by reference. [Background technology]

[0002] background It is well established that retroviruses (e.g. lentiviruses) can redirect their natural tropism to a desired target by pseudotyping the virus to express the envelope protein of another virus. This is most commonly achieved by pseudotyping with the VSV glycoprotein, which allows viral entry into a broad range of cells to target the LDL receptor. Recently, groups have shown that when lentiviruses are pseudotyped with envelope proteins from paramyxoviruses such as measles virus or Nipah virus, and mutations are made that abolish the native tropism, C-terminal fusions to these viruses allow receptor-mediated entry into the target cell of choice. Summary of the Invention

[0003] overview Herein, the inventors surprisingly demonstrate that the combination of overexpression of a second membrane protein with a mutation that abolishes a native function (e.g., tropism) allows the second protein to function as the basis for viral entry. These discoveries described herein enable new and innovative methodologies that allow, for example, screening of cells that are notoriously difficult to screen for specific antigens and functions (e.g., T cells) and delivery of nucleic acids to target cells in a target-specific manner. Some aspects of the disclosure provide retroviral (e.g., lentiviral) compositions that include: (i) a nucleic acid comprising a non-viral membrane-associated protein having the structure: S-ETD-MBD-IRES-R, where S encodes a signal sequence, ETD encodes an extracellular targeting domain; MBD encodes a membrane-associated domain, IRES encodes an internal ribosome entry site, and R encodes a reporter; and (ii) a mutated viral envelope protein that comprises at least one mutation that reduces its native function.

[0004] Some aspects of the present disclosure provide a retroviral (e.g., lentiviral) composition comprising: (i) a CD80 protein domain; and (ii) a mutated viral envelope protein comprising at least one mutation that reduces its native function. The CD80 protein domain can be an extracellular domain. In some embodiments, the CD80 extracellular domain binds to a receptor on a target cell. Some aspects of the present disclosure provide a method for screening a cell population, comprising: (i) providing a retrovirus comprising a viral envelope protein comprising at least one mutation that reduces its native function, a non-viral membrane-bound protein comprising a membrane-binding domain and an extracellular targeting domain, and a nucleic acid encoding a reporter; (ii) combining the retrovirus with a cell population; and (iii) sorting the cell population based on the presence or absence of the reporter. In some embodiments, the retrovirus (e.g., lentivirus) comprises a nucleic acid comprising a non-viral membrane-bound protein comprising the structure: S-ETD-MBD-IRES-R, where S encodes a signal sequence, ETD encodes an extracellular targeting domain; MBD encodes a membrane-binding domain, IRES encodes an internal ribosome entry site, and R encodes a reporter.

[0005] In some embodiments, the cells are somatic cells (e.g., antigen-specific cells, such as T cells or B cells). In some embodiments, the cells are isolated from a subject (e.g., a human subject). In some embodiments, the cells are isolated from the subject's blood or tumor. In some embodiments, the cells are maintained in liquid culture prior to combining with the retrovirus. In some embodiments, the viral envelope protein is VSV-G envelope protein, measles virus envelope protein, Nipah virus envelope protein, or coccus virus G protein. VSV-G envelope protein can be mutated at any one or more of H8, K47, Y209, and / or R354. Measles virus envelope protein can be mutated at any one or more of Y481, R533, S548, and / or F549. Nipah virus envelope protein can be mutated at any one or more of E501, W504, Q530, and / or E533. Coccus virus G protein can be mutated at K64 and / or R371.

[0006] In some embodiments, the non-viral membrane-bound protein comprises a major histocompatibility complex (MHC) protein. In some embodiments, the extracellular targeting domain is a protein (e.g., interleukin-13), a peptide, or an antibody (e.g., an anti-CD19 antibody, an anti-TCR antibody, or an anti-CD3 antibody). In some embodiments, the reporter is a fluorescent protein (e.g., green fluorescent protein, yellow fluorescent protein, red fluorescent protein) or an antibiotic resistance marker. In some embodiments, a linker is disposed between the membrane-bound domain and the extracellular targeting domain. The linker can be a rigid linker (e.g., a PDGFR stalk or a CD8α stalk), a flexible linker (e.g., an amino acid sequence comprising GAPGAS (SEQ ID NO: 5) or GGGGS (SEQ ID NO: 7)), or an oligomerization linker (e.g., an IgG4 hinge, or an amino acid sequence capable of forming a tetrameric coiled coil).

[0007] In some embodiments, the retrovirus is combined with the cell population in (ii) for 1 minute to 72 hours and at a temperature ranging from 4°C to 42°C. In some embodiments, the retrovirus and cell population are combined in (ii) in the presence of (a) cell culture medium, optionally RPMI or DMEM cell culture medium; (b) buffered saline, optionally phosphate buffered saline or HEPES buffered saline; and / or (c) a retroviral transduction enhancer, optionally heparin sulfate, polybrene, protamine sulfate, and / or dextran. In some embodiments, the extracellular targeting domain can bind to a cognate protein (e.g., a protein receptor) present on the cell surface of a subset of the cell population. In some embodiments, the cell population is washed between (ii) and (iii) (e.g., using phosphate buffered saline (PBS), e.g., to remove the retrovirus from the cell population). In some embodiments, sorting of the cell population is performed using fluorescence activated cell sorting, single cell next generation sequencing, or antibiotic selection.

[0008] In some embodiments, the method further comprises a second retrovirus, wherein the second retrovirus comprises a different extracellular targeting domain and / or a different reporter as compared to the first retrovirus. Another aspect of the disclosure provides a method of delivering a nucleic acid (e.g., encoding a gene of interest, e.g., a protein) to a cell, the method comprising: (i) providing a retrovirus comprising a nucleic acid, a viral envelope protein comprising at least one mutation that reduces its native function, and a non-viral membrane-associated protein comprising an extracellular targeting domain capable of binding to a cognate ligand of the cell; and (ii) contacting the retrovirus with the cell, thereby delivering the nucleic acid to the cell. In some embodiments, the retrovirus enters or infects the cell during (ii). Some aspects of the disclosure provide methods of delivering a nucleic acid to a cell, the methods including: (i) providing a retrovirus comprising a nucleic acid, a viral envelope protein comprising at least one mutation that reduces its native function, and a CD80 protein domain; and (ii) contacting the retrovirus with a cell, thereby delivering the nucleic acid to the cell.

[0009] Yet another aspect of the present disclosure provides a method for detecting an interaction between a retrovirus and a cell, the method comprising: (i) contacting a sample comprising a retrovirus and a cell with an antibody, where the retrovirus comprises a viral envelope protein that comprises at least one mutation that reduces its native function, a non-viral membrane-associated protein that comprises an extracellular targeting domain, and where the antibody binds to the extracellular targeting domain of the retrovirus; (ii) optionally removing unbound antibody from the sample; and (iii) imaging the sample to detect whether the antibody-retrovirus complex is bound to the cell. In some embodiments, the antibody further comprises a fluorescent label, optionally wherein the antibody is covalently attached to the fluorescent label. In some embodiments, the sample is imaged in (iii) using a confocal microscope or a fluorescent microscope. Some aspects of the disclosure provide a library of retroviruses comprising a plurality of unique retroviruses, wherein each unique retrovirus comprises a viral envelope protein comprising at least one mutation that reduces its native function, a non-viral membrane-associated protein comprising a membrane-binding domain and an extracellular targeting domain (e.g., comprising at least 5, at least 10, at least 15, at least 20, or at least 50 amino acids), and a nucleic acid encoding a reporter, and wherein each unique retrovirus comprises a different unique extracellular targeting domain.

[0010] In some embodiments, the library can be screened against a population of antigen-specific cells, optionally where the antigen-specific cells are B cells or T cells. The library comprises at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , or at least 10 10 In some embodiments, each different unique extracellular targeting domain is generated by site-directed mutagenesis. Some aspects of the present disclosure provide a cell population, wherein a subset of the cell population contains a retrovirus comprising a viral envelope protein that comprises at least one mutation that reduces its native function, a non-viral membrane-bound protein that comprises a membrane-binding domain and an extracellular targeting domain, and a nucleic acid that encodes a reporter. In some embodiments, a subset of the population of cells (e.g., antigen-specific cells, e.g., B cells or T cells) contains a retrovirus as described herein. In some embodiments, a subset of the cell population contains a retrovirus in each cell of the subset. The subset of the population that contains a retrovirus can be isolated and / or selected from the cells of the population that do not contain a retrovirus. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows an exemplary schematic of a retrovirus of the invention interacting with its cognate ligand on a target cell. [Diagram 2] FIG. 2 is an exemplary schematic diagram of a protein containing a signal sequence (SP), an extracellular targeting domain (peptide), a membrane-binding domain (MHC), an internal ribosome entry site (IRES), and a reporter (GFP); and the ability of a retrovirus containing said protein to interact with a target cell. [Diagram 3] Figures 3A-3B are graphs showing the entry of retroviruses into target cells. Figure 3A demonstrates that the binding interaction of the extracellular targeting domain present in the retrovirus to the cognate ligand of the target cell causes the entry of the retrovirus and the subsequent expression of the reporter (GFP). Figure 3B demonstrates that contacting the retrovirus with non-target cells does not cause the entry of the retrovirus. [Figure 4] FIG. 4 is a graph depicting the ability of a retrovirus to enter a target cell at various affinities between the extracellular targeting domain and its cognate ligand.

[0012] [Diagram 5] Figures 5A-5B are graphs showing that T cell signaling is active during retrovirus entry or infection. Figure 5A demonstrates that the binding interaction of the extracellular targeting domain present in retrovirus to the cognate ligand of the target cell causes retrovirus entry and T cell signaling activation. Figure 5B demonstrates that contacting retrovirus with non-target cells does not cause retrovirus entry or T cell signaling activation. [Figure 6] FIG. 6 shows an exemplary schematic of a pMHC retroviral library screening methodology. [Figure 7] Figures 7A-7B are graphs showing the entry of retroviruses into target cells. Figure 7A demonstrates that the binding interaction of the interleukin-13 (IL-13) protein domain present in the retrovirus to the IL-13 protein receptor of the target cell causes the entry of the retrovirus and the subsequent expression of a reporter (GFP). Figure 7B demonstrates that contacting a retrovirus containing the IL-13 protein domain with a non-target cell that does not express the IL-13 protein receptor does not cause the entry of the retrovirus. [Figure 8] FIG. 8 is a diagram showing three exemplary retroviruses, each containing a different viral envelope protein.

[0013] [Figure 9] FIG. 9 shows the ability of retroviruses containing Nipah virus envelope protein and anti-CD19 scFv antibody to infect and enter CD19+ Raji cells. [Figure 10] Figure 10 shows the ability of retroviruses containing mutated VSV-G viral envelope proteins and anti-CD19 scFv antibodies to infect and enter CD19+ Raji cells without infecting CD19- Jurkat cells, whereas retroviruses containing wild-type VSV-G viral envelope proteins and anti-CD19 scFv antibodies infect both CD19+ Raji cells and CD19- Jurkat cells. [Figure 11] FIG. 11 shows exemplary linkers for use with nucleic acids and proteins that contain a membrane binding domain and an extracellular targeting domain. [Figure 12] FIG. 12 is a graph showing the effect of short (PDGFR; GAPGAS (SEQ ID NO:5); CAR ECD; top, left to right) and long (CD8α; hinge domain; tetrameric coiled-coil; bottom, left to right) protein linkers on retroviral entry. [Figure 13] FIG. 13 is a graph showing that a retrovirus containing a mutated VSV-G viral envelope protein activates T cell signaling as determined by expression of CD69 (right panel), and that a retrovirus containing a wild-type VSV-G viral envelope protein does not activate T cell signaling (left panel).

[0014] [Figure 14] FIG. 14 is a graph showing that retroviruses with variable affinities between their extracellular targeting domains and T cell cognate ligands can activate T cell signaling. [Figure 15]FIG. 15 is a graph depicting the ability of retroviruses to enter target cells at various affinities, ranging from picomolar to micromolar binding affinities, between the extracellular targeting domain and its cognate ligand. [Figure 16] Figures 16A-16B are graphs showing the ability of retroviruses to enter target cells at various concentrations of virus. Figure 16A shows the ability of lentiviruses displaying NYESO-1 to transduce IG4-expressing T cells. Lentiviruses displaying SL-1 do not transduce IG4-expressing T cells. Figure 16B shows the mean fluorescence provided by the interaction between lentiviruses displaying NYESO-1 and IG4-expressing T cells. [Figure 17] FIG. 17 is a graph demonstrating the effect of protein linker oligomerization on retroviral transduction. [Figure 18A] Figures 18A-18B show the ability to detect binding interactions between retroviruses containing anti-CD19 scFv antibodies and target cells containing CD19 using fluorescently labeled antibodies that bind to the retrovirus. Figure 18A shows an exemplary binding schematic. [Figure 18B] FIG. 18B is a graph depicting the ability of retroviral constructs to interact with target cells.

[0015] [Figure 19] FIG. 19 shows the ability of lentiviruses displaying SL9 peptides to transduce T cells expressing the 868 TCR ("868") and lentiviruses displaying CMV NLV peptides to transduce T cells expressing the C7 TCR ("CMV C7"). [Figure 20]Figure 20 shows the ability of lentivirus displaying NYESO-1 to selectively transduce T cells expressing the IG4 T cell receptor (TCR) variant (target cells) (binding affinity of approximately 26 pM or approximately 32 μM for NYESO-1) compared to cells that do not express the IG4 TCR (off-target) at off-target to target cell ratios as high as 100,000:1. [Figure 21] FIG. 21 shows the ability of lentiviruses displaying various target peptides (CMV NLV; EBV; SL9; GL9; NYESO-1) to selectively transduce T cells expressing a target peptide-specific TCR (on-target) compared to T cells not expressing a target peptide-specific TCR (off-target). [Figure 22] Figures 22A-22B show the ability of various lentiviruses (GL9 peptide targeting; NYESO-1 peptide targeting; wild type VSV) to transduce primary T cells expressing GL9-specific TCR and T cells expressing IG4 TCR variant. Figure 22A shows the transduction rate of each lentivirus. Figure 22B is a graph showing FACS data of GL9 virus and NYESO-1 virus.

[0016] [Figure 23] FIG. 23 is a graph showing that NYESO-1-responsive primary cells are specifically infected. [Figure 24] FIG. 24 is a graph showing that viruses targeting NYESO-1 specifically infect grown primary cells. [Diagram 25] FIG. 25 is a graph depicting the ability of retroviruses containing mutated VSV-G viral envelope proteins and CD80 domains to specifically infect Jurkat T cells compared to B cell lines. [Figure 26]FIG. 26 shows the ability of retroviruses containing mutated VSV-G viral envelope proteins and either anti-TCR-alphabeta antibody (H57) or anti-CD3 antibody (C11) to infect TCR-transduced 58− / − mouse cell lines. [Figure 27] Figure 27 shows the ability of retroviruses containing either mutated VSV-G viral envelope protein or mutated coccus viral envelope protein; and anti-CD19 scFv antibody to infect and enter CD19+ Raji cells without infecting CD19- Jurkat cells. Retroviruses containing wild-type VSV-G viral envelope protein and anti-CD19 scFv antibody infect both CD19+ Raji cells and CD19- Jurkat cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description of the Invention Provided herein are new and innovative methods for, for example, screening cells (e.g., T cells) that are notoriously difficult to screen for specific antigens and functions, and for delivering nucleic acids to target cells in a target-specific manner. In some embodiments, described herein are systems that allow, for example, repertoire-scale analysis of T cell receptor (TCR)-peptide-major histocompatibility complex (pMHC) specificity, which was a difficult bottleneck before, because previously described methods required significant efforts to determine what a single T cell clone can recognize (e.g., as in a typical immune response). In some embodiments, described herein are retrovirus-based systems that repurpose viral tropism as a method of molecular interaction selection, replacing the binding functions of wild-type viral surface proteins with those of protein variants of interest, for example, by encoding these protein variants on the corresponding transfer plasmids used to generate the virus, thereby ensuring that the resulting viruses display the protein variants on their surface and package the corresponding gene sequences. Thus, once the virus enters a target cell (e.g., one that has a receptor that binds to the extracellular targeting domain of the displayed protein variant), cell entry results in integration of the genetic sequence of the displayed protein into the genome of the target cell.

[0018] Previous approaches to study T cell specificity required the combination of generated T cell lines, recombinant expression of T cell receptors, and / or individual validation of T cell binding or activity through candidate antigen-based approaches. Each of these elements posed inherent limitations in the throughput of screened T cells or antigens. For example, yeast display-based methods for deorphanizing T cell receptors alleviated the bottleneck in the number of antigens to be investigated (>10 8The current strategy of the present invention described herein has been developed to screen for a wide variety of ligands, but is still severely limited by the need to recombinantly express the TCR. 8 This represents a major advance in the study of T cell specificity and in T cell screening by allowing the screening of more than 100 ligands and eliminating the need for recombinant TCR expression.

[0019] Retroviruses Described herein is a retrovirus, comprising a viral envelope protein that comprises at least one mutation that reduces its native function, a non-viral membrane-bound protein that comprises a membrane-binding domain and an extracellular targeting domain, and a nucleic acid that codes a reporter.In some embodiments, the retrovirus comprises a viral envelope protein that comprises at least one mutation that reduces its native function, and a non-viral membrane-bound protein that comprises a membrane-binding domain and an extracellular targeting domain. The retroviruses disclosed herein contain one or more elements derived from the retroviral genome (naturally occurring or modified) of an appropriate species. Retroviruses include seven families: alpharetroviruses (avian leukosis viruses), betaretroviruses (mouse mammary tumor viruses), gammaretroviruses (murine leukemia viruses), deltaretroviruses (bovine leukemia viruses), epsilonretroviruses (walleye cutaneous sarcoma viruses), lentiviruses (human immunodeficiency virus 1), and spumaviruses (human spumaviruses). Six additional examples of retroviruses are provided in U.S. Patent No. 7,901,671.

[0020] In some embodiments, retrovirus is lentivirus.Lentivirus is a genus of retrovirus, which typically causes slowly progressive disease due to its ability to integrate into host genome.Modified lentivirus genome is useful as a viral vector for delivering nucleic acid to host cell.Host cell can be transfected with lentivirus vector and optionally additional vector for expressing lentivirus packaging protein (e.g., VSV-G, Rev, and Gag / Pol) to produce lentivirus particles in culture medium. Retroviral and lentiviral constructs are well known in the art, and any suitable retrovirus can be used to construct the retrovirus (or a plurality of retroviruses or libraries thereof) described herein. Non-limiting examples of retroviral constructs include lentiviral vectors, human immunodeficiency virus (HIV) vectors, avian leukemia virus (ALV) vectors, murine leukemia virus (MLV) vectors, mouse mammary tumor virus (MMTV) vectors, murine stem cell virus, and human T-cell leukemia virus (HTLV) vectors. These retroviral constructs contain proviral sequences from the corresponding retrovirus.

[0021] The retroviruses described herein may include the viral elements described herein from one or more suitable retroviruses that are RNA viruses with single-stranded positive-stranded RNA molecules. Retroviruses include reverse transcriptase and integrase enzymes. Upon entering a target cell, retroviruses utilize reverse transcriptase to transcribe their RNA molecules into DNA molecules. The integrase enzyme is then used to integrate the DNA molecule into the host cell genome. Once integrated into the host cell genome, the sequence from the retrovirus is called a provirus (e.g., proviral sequence or provirus sequence). The retroviral vectors described herein may further include additional functional elements known in the art to address safety concerns and / or improve vector functions such as packaging efficiency and / or viral titer. Additional information can be found in US20150316511 and WO2015 / 117027, the relevant disclosures of each of which are incorporated herein by reference for the purposes and subject matter referenced herein. Additional information regarding lentiviruses can be found, for example, in WO2019 / 056015, the relevant disclosure of which is incorporated herein by reference for this particular purpose. In some embodiments, lentivirus can target target-specific cells via pMHC-TCR interaction or any other protein-protein, cell-cell interaction.In some embodiments, T cells with known related specificity can be enhanced (in the case of cancer or infection) or ablated (in the case of autoimmunity) without affecting other T cells, dramatically limiting the risk of off-target effects.In some embodiments, lentivirus can code extracellular domain to target any other surface-expressed molecule on target cells.

[0022] Viral envelope proteins The retroviruses described herein comprise a viral envelope protein that comprises at least one mutation that reduces its native function (e.g., the wild-type function of non-mutated viral envelope protein). In some embodiments, the viral envelope protein is any viral envelope protein of any retrovirus (e.g., lentivirus). The viral envelope protein can be VSV-G envelope protein, measles virus envelope protein, Nipah virus envelope protein, or coccus virus G protein. In some embodiments, the native function that is reduced by the mutation of the viral envelope protein is the tropism of the virus (e.g., the ability to infect cells, the ability to bind to cells, etc.). In some embodiments, the viral envelope protein comprising at least one mutation that reduces its native function is a mutated VSV-G envelope protein. In some embodiments, the viral envelope protein comprising at least one mutation that reduces its native function is a mutated measles virus envelope protein. In some embodiments, the viral envelope protein comprising at least one mutation that reduces its native function is a mutated Nipah virus envelope protein. In some embodiments, the viral envelope protein comprising at least one mutation that reduces its native function is a mutated coccus virus G protein.

[0023] In some embodiments, the mutated VSV-G envelope protein comprises mutations at H8, K47, Y209, and / or R354. In some embodiments, the mutated VSV-G envelope protein comprises H8A, K47A, K47Q, Y209A, R354A, and / or R354Q mutations. In some embodiments, the mutated VSV-G envelope protein is as described in Nikolic et al., "Structural basis for the recognition of LDL-receptor family members by VSV glycoprotein." Nature Comm., 2018, 9:1029, the relevant disclosure of which is incorporated herein by reference for this particular purpose. In some embodiments, the mutated measles virus envelope protein comprises a mutation at Y481, R533, S548, and / or F549. In some embodiments, the mutated measles virus envelope protein comprises a Y481A, R533A, S548L, and / or F549S mutation. In some embodiments, the mutated Nipah virus envelope protein comprises a mutation at E501, W504, Q530, and / or E533. In some embodiments, the mutated Measles virus envelope protein comprises an E501A, W504A, Q530A, and / or E533A mutation. In some embodiments, the mutated coccal virus G protein comprises a mutation at K64 and / or R371. In some embodiments, the mutated coccal virus G protein comprises a mutation at K64Q and / or R371A.

[0024] In some embodiments, the mutated envelope protein is derived from any other enveloped virus, including, but not limited to, baculovirus, herpes simplex virus (HSV), cytomegalovirus (CMV), lymphocytic choriomeningitis virus (LCMV), Epstein-Barr virus (EBV), vaccinia virus, hepatitis A, B, or C virus, vaccinia virus, alphavirus, dengue virus, yellow fever virus, Zika virus, influenza virus, hantavirus, Ebola virus, rabies virus, human immunodeficiency virus (HIV), coronavirus, other members of the Rhabdoviridae family. In some embodiments, the viral envelope protein comprising at least one mutation comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations. In some embodiments, the viral envelope protein comprising at least one mutation comprises a nucleotide sequence and / or amino acid sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, or 97% identical to the wild-type viral envelope protein. In some embodiments, the viral envelope protein comprising at least one mutation that reduces its native function retains less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the function of the wild-type viral envelope protein. In some embodiments, the viral envelope protein comprising at least one mutation lacks all of its native functions. In some embodiments, a retrovirus comprising a viral envelope protein that includes at least one mutation that reduces its native function comprises less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of the cell infectivity of a retrovirus comprising a wild-type viral envelope protein.

[0025] Non-viral membrane-associated proteins The retrovirus described herein comprises a non-viral membrane-bound protein. The non-viral membrane-bound protein can comprise a membrane-bound domain and an extracellular targeting domain. In some embodiments, the non-viral membrane-bound protein is a chimeric protein that comprises sequences from at least two different proteins. In some embodiments, the non-viral membrane-bound protein is a full-length or truncated protein that comprises sequences from a single protein. A membrane binding domain is a protein or peptide that has an amino acid sequence that allows the protein or peptide to be fully or partially embedded in or associated with the membrane (e.g., envelope) of a retrovirus. In some embodiments, the membrane binding domain allows the presentation and delivery of the extracellular targeting domain to the extracellular environment. In some embodiments, the membrane binding domain comprises an intracellular domain, a transmembrane domain, and / or an extracellular domain. In some embodiments, the membrane binding domain comprises an intracellular domain and a transmembrane domain. In some embodiments, the membrane binding domain comprises a major histocompatibility complex (MHC) protein or a fragment thereof. The MHC protein can be a class I or class II MHC protein.

[0026] In some embodiments, the membrane binding domain comprises between 10-50, 10-100, 25-100, 50-200, 50-150, 100-500, 100-250, 250-500, or any reasonable number of total amino acids. In some embodiments, the retroviruses present in the retroviral library contain the same membrane-binding domain as some or all of the other retroviruses in the library, hi some embodiments, each retrovirus present in the retroviral library contains a different membrane-binding domain compared to some or all of the other retroviruses in the library. In some embodiments, the extracellular targeting domain is any protein or peptide that has an amino acid sequence and is a binding partner of a target molecule or ligand (e.g., a cognate protein) on the cell surface. When present in the extracellular environment beyond the interior of the retrovirus, the extracellular targeting domain can bind to the target cell. In some embodiments, the extracellular targeting domain binds to or targets a cognate protein or ligand (e.g., a protein receptor present on the target cell) present on the cell surface of a cell or a subset of a cell population. In some embodiments, the extracellular targeting domain binds to a cognate protein or ligand present on the cell surface of a single T cell or a subset of a T cell population. In some embodiments, the binding interaction between the extracellular targeting domain of the retrovirus and the cognate protein or ligand of the cell allows the retrovirus to enter the cell (e.g., an antigen-specific cell, e.g., a T cell).

[0027] In some embodiments, the extracellular targeting domain comprises between 10 and 50, between 10 and 100, between 25 and 100, between 50 and 200, between 50 and 150, between 100 and 500, between 100 and 250, between 250 and 500, or any reasonable number of total amino acids. In some embodiments, the extracellular targeting domain comprises at least 5, at least 10, at least 15, at least 20, or at least 50 amino acids. In some embodiments, the extracellular targeting domain is a protein, antibody, or peptide. In some embodiments, the antibody is a full-length antibody, an antibody fragment, a nanobody, or a single-chain antibody (scFv). In some embodiments, the extracellular targeting domain is an antibody that binds to a cognate protein on a target cell. In some embodiments, the extracellular targeting domain is an antibody that binds to a B cell or T cell antigen. In some embodiments, the extracellular targeting domain is an anti-CD19 antibody (e.g., an antibody that binds to CD19). In some embodiments, the extracellular targeting domain is an antibody that binds to any cell surface molecule. In some embodiments, the extracellular targeting domain is an antibody that binds to a lineage marker (e.g., CD3, CD20, integrin, or other receptor), a phenotypic marker (PD-1, CD25, CD45, or other). In some embodiments, the extracellular targeting domain is a protein or peptide that binds to a receptor (e.g., a receptor present on the surface of a target cell). In some embodiments, the extracellular targeting domain is a protein or peptide that binds to a cytokine receptor (e.g., an interleukin-13 (IL-13) receptor). In some embodiments, the extracellular targeting domain is a cytokine (e.g., IL-2, IL-6, IL-12, IL-13). In some embodiments, the extracellular targeting domain is a chemokine ligand (e.g., CXCL9, CXCL10, CXCL11, etc.). In some embodiments, the extracellular targeting domain is a cellular receptor, including cytokine receptors (e.g., IL-13Rα1, IL-13Rα2, IL-2 receptor, common gamma chain), GPCRs (including chemokine receptors such as CSCR3, CXCR4, etc.), and integrins. In some embodiments, the extracellular targeting domain is a peptide displayed by an MHC protein. In some embodiments, the non-viral membrane-bound protein comprises a membrane-binding domain comprising an MHC protein or fragment and an extracellular targeting domain comprising a peptide displayed by an MHC protein. In some embodiments, the extracellular domain binds to a T cell receptor and / or a B cell receptor.T cell receptors are naturally expressed on the surface of T cells, usually as alpha / beta and gamma / delta heterodimeric integral membrane proteins, with each subunit containing a short intracellular segment, a single transmembrane alpha helix and two globular extracellular Ig superfamily domains. The B cell receptor is a transmembrane receptor protein on the outer surface of B cells.

[0028] In some embodiments, the extracellular targeting domain is directed to a target cell or cell surface molecule. -9 ~10 -8 M, 10 -8 ~10 -7 M, 10 -7 ~10 -6 M, 10 -6 ~10 -5 M, 10 -5 ~10 -4 M, 10 -4 ~10 -3 M, or 10 -3 ~10 -2 In some embodiments, the extracellular targeting domain binds to a cognate protein or ligand on the target cell with a binding affinity of 10 -9 ~10 -8 M, 10 -8 ~10 -7 M, 10 -7 ~10 -6 M, 10 -6 ~10 -5 M, 10 -5 ~10 -4 M, 10 -4 ~10 -3 M, or 10 -3 ~10 -2 In some embodiments, the binding affinity between the extracellular targeting domain and its cognate protein or ligand is in the picomolar to nanomolar range (e.g., about 10 -12 ~about 10 -9 In some embodiments, the binding affinity between the extracellular targeting domain and the cognate protein or ligand is in the nanomolar to micromolar range (e.g., between about 10 -9 ~about 10 -6In some embodiments, the binding affinity between the extracellular targeting domain and the cognate protein or ligand is in the micromolar to millimolar range (e.g., between about 10 -6 ~about 10 -3 In some embodiments, the binding affinity between the extracellular targeting domain and the cognate protein or ligand is in the picomolar to micromolar range (e.g., between about 10 -12 ~about 10 -6 In some embodiments, the binding affinity between the extracellular targeting domain and the cognate protein or ligand is in the nanomolar to millimolar range (e.g., between about 10 -9 ~about 10 -3 (between 0.01 and 0.15 mm).

[0029] As used herein, the term antibody generally refers to a protein that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody may include a heavy (H) chain variable region (herein referred to as V H ), and / or a light (L) chain variable region (abbreviated herein as V L In another example, the antibody can include two heavy (H) chain variable regions and / or two light (L) chain variable regions. The antibody can have the structural characteristics of IgA, IgG, IgE, IgD, IgM (and subtypes thereof). H and V L The regions can be further subdivided into regions of hypervariability called "complementarity determining regions" ("CDRs"), separated by more conserved regions called "framework regions" ("FRs"). H and / or V L Typically, the V of an antibody is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. H or V LThe chains can further include a heavy or light chain constant region, thereby forming a heavy or light immunoglobulin chain, respectively. In some embodiments, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, where the heavy and light immunoglobulin chains are interconnected, for example, by disulfide bonds. In IgG, the heavy chain constant region includes three immunoglobulin domains, CH1, CH2, and CH3.

[0030] In some embodiments, a retrovirus present in a library of retroviruses comprises the same extracellular targeting domain as some or all of the other retroviruses in the library, hi some embodiments, each retrovirus present in a library of retroviruses comprises a different extracellular targeting domain compared to some or all of the other retroviruses in the library. In some embodiments, the non-viral membrane-associated protein further comprises a signal sequence (also referred to as a signal peptide of a localization sequence). In some embodiments, the signal sequence is at the N-terminus or C-terminus of the non-viral membrane-associated protein. The signal sequence functions to translocate the non-viral membrane-associated protein to the membrane (or envelope) of the retrovirus. In some embodiments, the signal sequence is 5-10, 5-15, 10-20, 15-20, 15-30, 20-30, or 25-30 amino acids. In some embodiments, the signal sequence is an Ig kappa leader sequence (e.g., a mouse Ig kappa leader sequence comprising METDTLLLWVLLLWVPGSTG (SEQ ID NO: 1)) or a B2M signal peptide sequence (e.g., a B2M signal peptide sequence comprising MSRSVALAVLALLSLSGLEA (SEQ ID NO: 2)). In some embodiments, the retroviruses present in the library of retroviruses comprise the same signal sequence as some or all of the other retroviruses in the library. In some embodiments, each retrovirus present in a library of retroviruses contains a different signal sequence as compared to some or all of the other retroviruses in the library.

[0031] In some embodiments, the nucleic acid encoding the non-viral membrane-associated protein further comprises an internal ribosome entry site (IRES). An IRES is an RNA sequence that allows initiation of translation during protein synthesis. In some embodiments, the IRES is located at or near the C-terminus. In some embodiments, the IRES is located C-terminal to the membrane-associated domain and the extracellular targeting domain. In some embodiments, the IRES is a viral IRES. In some embodiments, the IRES is an IRES unique to the retrovirus. In some embodiments, the IRES is a sequence derived from encephalomyocarditis virus (EMCV). In some embodiments, the retroviruses present in the retroviral library comprise the same IRES as some or all of the other retroviruses in the library. In some embodiments, each retrovirus present in the retroviral library comprises a different IRES compared to some or all of the other retroviruses in the library.

[0032] In some embodiments, the non-viral membrane-associated protein further comprises a linker disposed between the membrane-associated domain and the extracellular targeting domain. The linker is an amino acid linker and may be a rigid linker, a flexible linker, or an oligomerized linker. A rigid linker is an amino acid sequence that lacks flexibility (e.g., may contain at least one proline). In some embodiments, the rigid linker comprises a platelet-derived growth factor receptor (PDGFR) stalk or a CD8α stalk. In some embodiments, the PDGFR stalk comprises an amino acid sequence comprising AVGQDTQEVIVVPHSLPFK (SEQ ID NO: 3). In some embodiments, the PDGFR stalk comprises an amino acid sequence comprising ASAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAK (SEQ ID NO: 4). A flexible linker is an amino acid sequence that has many degrees of freedom (e.g., may contain multiple amino acids with small side chains, such as glycine or alanine). In some embodiments, the flexible linker comprises an amino acid sequence comprising GAPGAS (SEQ ID NO: 5). In some embodiments, the flexible linker comprises an amino acid sequence consisting of GAPGSGGGGSGGGGSAS (SEQ ID NO: 6). In some embodiments, the flexible linker comprises an amino acid sequence comprising GGGGS (SEQ ID NO: 7). In some embodiments, the flexible linker comprises an amino acid sequence consisting of (GAPGAS) N (SEQ ID NO: 29) or (G4S) N(SEQ ID NO:30), where N is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. An oligomerization linker is an amino acid capable of oligomerizing to another related amino acid. In some embodiments, an oligomerization linker is an amino acid sequence capable of forming a dimer, trimer, or tetramer. In some embodiments, an oligomerization linker comprises an IgG4 hinge domain (e.g., ASESKYGPPCPPCPAVGQDTQEVIVVPHSLPFK (SEQ ID NO:8)). In some embodiments, an oligomerization linker comprises an amino acid sequence capable of forming a tetrameric coiled coil (e.g., ASGGGGSGELAAIKQELAAIKKELAAIKWELAAIKQGAG (SEQ ID NO:9)). In some embodiments, an oligomerization linker comprises an amino acid sequence capable of forming a dimeric coiled coil (e.g., ASESKYGPPCPPCP (SEQ ID NO:10)).

[0033] Reporter In some embodiments, the retroviruses described herein may include a reporter (e.g., a reporter protein). In some embodiments, the retroviruses described herein include a nucleic acid that encodes a reporter (e.g., a reporter protein). As used herein, a reporter is generally a protein or gene that can be detected when expressed in a retrovirus and / or a target cell. In some embodiments, the presence or absence of a reporter in a target cell or a subset of target cells within a cell population allows the ability to sort the cells (e.g., using flow cytometry and / or fluorescence-activated cell sorting). In some embodiments, the reporter is a fluorescent protein. The fluorescent protein can be green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP). The fluorescent protein can be one described in U.S. Patent No. 7,060,869, entitled "Fluorescent protein sensors for detection of analytes." In some embodiments, the reporter is an antibiotic resistance marker. In some embodiments, the antibiotic resistance marker is a protein or gene that provides a competitive advantage to target cells that contain the marker. In some embodiments, the antibiotic resistance marker comprises a hygromycin resistance protein or gene, a kanamycin resistance protein or gene, an ampicillin resistance protein or gene, a streptomycin resistance protein or gene, or a neomycin resistance protein or gene.

[0034] cell The cells described herein can be any bacterial, mammalian, or yeast cell. In some embodiments, the cells are human, mouse, rat, or non-human primate cells. In some embodiments, the cells are somatic or germ cells. In some embodiments, the cells are epithelial cells, neuronal cells, hormone-secreting cells, immune cells, secretory cells, blood cells, stromal cells, or embryonic cells. In some embodiments, the cells are antigen-specific cells (e.g., cells that bind to a particular antigen). In some embodiments, the antigen-specific cells are immune cells. In some embodiments, the antigen-specific cells are B cells or T cells. In some embodiments, the cells are target cells (e.g., those that contain a cognate protein or ligand that can be targeted by the retroviruses described herein). The cell population described herein can be any bacterial, mammalian, or yeast cell population. In some embodiments, the cell population is a human, mouse, rat, or non-human primate cell population. In some embodiments, the cell population is a somatic cell population or a germ cell population. In some embodiments, the cell population comprises epithelial cells, neural cells, hormone-secreting cells, immune cells, secretory cells, blood cells, stromal cells, and / or germ cells. In some embodiments, the cell population comprises antigen-specific cells (e.g., cells that bind to a particular antigen). In some embodiments, the antigen-specific cell population comprises immune cells. In some embodiments, the antigen-specific cell population comprises B cells and / or T cells. In some embodiments, the cell population comprises a homogenous cell population. In some embodiments, the cell population comprises a heterogenous cell population.

[0035] In some embodiments, the cell population is a cell population isolated from a subject.The subject can be a human subject (e.g., a human subject suffering from a disease), a mouse subject, a rat subject, or a non-human primate subject.In some embodiments, the cell population is isolated from the blood or tumor of the subject. In some embodiments, the cell population has been previously frozen and thawed (e.g., 1, 2, 3, 4, 5, or more freeze / thaw cycles). In some embodiments, the cell population is maintained in liquid culture medium. In some embodiments, the cell population has been passaged 1, 2, 3, 4, 5, or more times using any known method. In some embodiments, the cell population is maintained in liquid culture medium before being combined with the retrovirus or multiple retroviruses. In some embodiments, the cell population is maintained in liquid culture medium after being combined with the retrovirus or multiple retroviruses. In some embodiments, the cell population is maintained in liquid culture medium prior to being combined with the retrovirus or multiple retroviruses.

[0036] In some embodiments, the cell population comprises any of the retroviruses described herein. In some embodiments, a subset of the cell population comprises any of the retroviruses described herein. In some embodiments, a subset of the cell population comprises a retrovirus in each cell of the subset (e.g., in the nucleus of each cell of the subset). In some embodiments, the cell population or a subset thereof expresses a reporter (e.g., a fluorescent protein or an antibiotic resistance marker). In some embodiments, the cell population or a subset thereof (e.g., containing a retrovirus) is isolated and / or sorted based on the presence or absence of a reporter. In some embodiments, a subset of the cell population containing a retrovirus as described herein is isolated and / or sorted based on the presence or absence of a reporter away from cells of the population that do not contain a retrovirus. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, or 95% of the cell population before cell sorting contains a retrovirus. In some embodiments, at least 70%, 80%, 90%, 95%, or 100% of the cell population contains a retrovirus after isolation and / or sorting based on the presence or absence of a reporter.

[0037] Screening Methods Described herein is a method for screening a cell population, comprising: (i) providing a retrovirus comprising a viral envelope protein that comprises at least one mutation that reduces its native function, a non-viral membrane-bound protein that comprises a membrane-binding domain and an extracellular targeting domain, and a nucleic acid that encodes a reporter; (ii) combining the retrovirus with a cell population; and (iii) sorting the cell population based on the presence or absence of the reporter. In some embodiments, the retrovirus of (i) comprises a nucleic acid that comprises the following structure: S-ETD-MBD-IRES-R, where S encodes a signal sequence, ETD encodes an extracellular targeting domain; MBD encodes a membrane-binding domain, IRES encodes an internal ribosome entry site, and R encodes a reporter; and comprises a mutated viral envelope protein that comprises at least one mutation that reduces its native function.

[0038] As used herein, the term "combining" (which in some embodiments is synonymous with the terms "providing" and "contacting") generally refers to the act of bringing a retrovirus into intimate physical contact with a cell population so that the extracellular targeting domain of the retrovirus can bind to a cognate ligand present on a subset of cells of the population. In some embodiments, combining the retrovirus with the cell population occurs when a solution containing the retrovirus is mixed with a solution containing the cell population. In some embodiments, combining the retrovirus with the cell population occurs when a solution containing a lyophilized retrovirus is mixed with a cell population. In some embodiments, combining the retrovirus with the cell population occurs when a lyophilized retrovirus is mixed with a lyophilized cell population and reconstituted with a solution. In some embodiments, the cells of the population are maintained in a monolayer of cells in cell culture medium and / or attached to a tissue culture plate or petri dish.

[0039] Generally, the retrovirus and cell population are combined (e.g., physically combined or contacted) for a defined period of time. In some embodiments, the period is measured in seconds, minutes, hours, or days. In some embodiments, the period is 0-30 seconds, 15-45 seconds, 30-60 seconds, 45-90 seconds, 60-90 seconds, or 60-120 seconds. In some embodiments, the retrovirus and cell population are combined and contacted for 0-30 seconds, 15-45 seconds, 30-60 seconds, 45-90 seconds, 60-90 seconds, or 60-120 seconds. In some embodiments, the time period is 1-2 minutes, 1-5 minutes, 1-10 minutes, 2-10 minutes, 5-10 minutes, 5-20 minutes, 10-20 minutes, 25-30 minutes, 25-60 minutes, 30-45 minutes, 30-40 minutes, 40-60 minutes, 50-70 minutes, or 60-120 minutes. In some embodiments, the retrovirus and cell population are contacted in combination for 1-2 minutes, 1-5 minutes, 1-10 minutes, 2-10 minutes, 5-10 minutes, 5-20 minutes, 10-20 minutes, 25-30 minutes, 25-60 minutes, 30-45 minutes, 30-40 minutes, 40-60 minutes, 50-70 minutes, or 60-120 minutes. In some embodiments, the period is 1-2 hours, 1-5 hours, 1-3 hours, 2-5 hours, 3-6 hours, 3-12 hours, 6-12 hours, 12-18 hours, 12-24 hours, 15-30 hours, 18-24 hours, 24-48 hours, 24-36 hours, or 36-50 hours. In some embodiments, the retrovirus and cell population are contacted in combination for 1-2 hours, 1-5 hours, 1-3 hours, 2-5 hours, 3-6 hours, 3-12 hours, 6-12 hours, 12-18 hours, 12-24 hours, 15-30 hours, 18-24 hours, 24-48 hours, 24-36 hours, or 36-50 hours. In some embodiments, the period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 5-15 days. In some embodiments, the retrovirus and the cell population are contacted in combination for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 5-15 days.

[0040] In some embodiments, cell populations are selected based on the presence or absence of reporter.In some embodiments, the subset of cell populations that contain reporters (e.g., express reporters) are selected from the remaining subset of cell populations that do not contain reporters.In some embodiments, the selection of cell populations is carried out using flow cytometry (e.g., fluorescence-activated cell sorting), next-generation genomic sequencing (e.g., single-cell next-generation sequencing), or antibiotic selection. In some embodiments, the condition of step (ii) that allows the retrovirus to have cell-cell interactions with a subset of the cell population includes combining the retrovirus with the cell population in a defined solution, composition, and at a specific temperature. In some embodiments, the retrovirus and the cell population are combined in the presence of cell culture medium (e.g., RPMI or DMEM cell culture medium). In some embodiments, the retrovirus and the cell population are combined in the presence of a buffered saline. In some embodiments, the buffered saline is phosphate buffered saline or HEPES buffered saline. In some embodiments, the buffered saline includes bovine serum albumin and / or EDTA. In some embodiments, the retrovirus and the cell population are combined in the presence of an enhancer of retroviral transduction (e.g., heparin sulfate, polybrene, protamine sulfate, or dextran). In some embodiments, the retrovirus and cell population are combined in (ii) at a temperature ranging from 4°C to 42°C, 4°C to 8°C, 4°C to 10°C, 8°C to 15°C, 10°C to 20°C, 18°C ​​to 23°C, 20°C to 30°C, 25°C to 35°C, 30°C to 40°C, or 37°C to 42°C.

[0041] In some embodiments, the screening method described herein further comprises washing the cell population with a washing solution between steps (ii) and (iii). In some embodiments, the washing solution is any liquid solution that allows for the maintenance of healthy cells (e.g., a solution with a neutral pH and a low to moderate level of ionic strength). In some embodiments, washing the cell population removes excess and / or residual retrovirus from the cell population. In some embodiments, the cell population is washed using cell culture medium (e.g., RPMI or DMEM cell culture medium). In some embodiments, the cell population is washed using a buffered saline. In some embodiments, the buffered saline is phosphate buffered saline or HEPES buffered saline. In some embodiments, the buffered saline comprises bovine serum albumin and / or EDTA. In some embodiments, the cell population is washed at a temperature ranging from 4°C to 42°C, 4°C to 8°C, 4°C to 10°C, 8°C to 15°C, 10°C to 20°C, 18°C ​​to 23°C, 20°C to 30°C, 25°C to 35°C, 30°C to 40°C, or 37°C to 42°C. In some embodiments, the cell population is maintained in liquid culture before being combined with the retrovirus. In some embodiments, the cell population is maintained in liquid culture after being combined with the retrovirus. In some embodiments, the cell population is maintained in liquid culture during the step of combining with the retrovirus. In some embodiments, the cell population is attached to a cell culture plate or a petri dish. In some embodiments, the cell population is maintained in a monolayer, an embryoid body, or any cell aggregate.

[0042] In some embodiments, the method of screening involves the use of a plurality of retroviruses. In some embodiments, the plurality of retroviruses comprises at least 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 1010 , 10 11 , or 10 12 In some embodiments, the plurality of retroviruses comprises at least 10 of each unique retrovirus. 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 There may be copies of In some embodiments, the method of screening comprises screening a cell population with at least two different unique retroviruses. In some embodiments, the different unique retroviruses comprise different extracellular targeting domains and / or different reporters. In some embodiments, the method of screening comprises a first retrovirus and a second retrovirus, where the first and second retroviruses comprise different extracellular targeting domains and / or different reporters. In some embodiments, the method of screening comprises screening a cell population with 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100 or more different retroviruses. In some embodiments, the method of screening comprises screening a cell population with a library of retroviruses. In some embodiments, the library of retroviruses comprises at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , or at least 10 10 Contains unique retroviruses.

[0043] Retroviral Libraries Described herein is a library of retroviruses, wherein the library comprises a plurality of unique retroviruses, wherein each unique retrovirus comprises a viral envelope protein comprising at least one mutation that reduces its native function, a non-viral membrane-associated protein comprising a membrane-associated domain and an extracellular targeting domain, and a nucleic acid encoding a reporter, wherein each unique retrovirus comprises a different unique extracellular targeting domain. Also described herein is a library of cells comprising retroviruses, wherein the library comprises a plurality of unique cells, wherein each unique cell comprises a unique retrovirus. In some embodiments, the library comprises a pMHC-encoded (peptide / MHC-encoded) retroviral (e.g., lentiviral) library for use in screening T cell populations. In such libraries, the pMHC displayed on the viral surface will allow T cell infection in a TCR-specific manner. Infected T cells can be collected and sequenced, allowing for the identification of pMHC ligands that can infect a subset of the T cell population of interest and the ability to simultaneously track TCR sequences and reactive pMHC ligands. In some embodiments, the pMHC retroviral library minimally comprises a randomized transfer vector containing a randomized pMHC targeting element. In some embodiments, the randomly derived library is generated using degenerate oligonucleotide primers. In some embodiments, a target library specific to a unique set of antigens (e.g., all possible viral or bacterial antigens for a particular target of interest - human immunodeficiency virus, tuberculosis TB, etc.; or all possible neoantigens for a particular target) is generated.

[0044] In some embodiments, the library can be screened against a population of antigen-specific cells (e.g., B cells or T cells). In some embodiments, the library contains at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , or at least 10 10 In some embodiments, the library containing unique retroviruses contains an extracellular targeting domain that is at least 5, at least 10, at least 15, at least 20, or at least 50 amino acids in length. In some embodiments, each different unique extracellular targeting domain is generated by site-directed mutagenesis.

[0045] The size of a retrovirus or cell library can vary from hundreds to hundreds of thousands, millions, or more unique retroviruses or unique cells. In some embodiments, the library of the present disclosure contains at least 500,000 unique retroviruses or unique cells. The libraries of the present invention include retrovirus libraries and cell libraries. Libraries are synthetic (i.e., isolated, synthetically produced, no components naturally found together in a cell, purified before entering the library) collections of members that have a common element and at least one different element. The library contains 1,000 or more (e.g., at least 1,000; 2,000; 3,000; 4,000; 5,000; 10,000; 50,000; 100,000; 500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 2,000,000; 3,000,000; 4,000,000; or more) members. The upper limit of the library size is defined by the combinatorial theory of the domain or module, providing clarity or diversity among the members. For example, the upper limit can be 4,000,000 members. Thus, in some embodiments, the library is highly diverse and contains at least 500,000 distinct members. A highly diverse library is one that is 10 6 In some embodiments, the retroviral library is generated using site-directed mutagenesis of nucleic acid as described herein. In some embodiments, the site-directed mutagenesis includes the use of primers and low-fidelity RNA polymerase to allow randomized mutagenesis of common nucleic acid as described herein.

[0046] Methods for delivering nucleic acids into cells Described herein is a method of delivering a nucleic acid to a cell, the method comprising: (i) providing a retrovirus comprising a nucleic acid, a viral envelope protein comprising at least one mutation that reduces its native function, and a non-viral membrane-associated protein comprising an extracellular targeting domain capable of binding to a cognate ligand of the cell, as described herein; and (ii) contacting the retrovirus with a cell, so that the retrovirus enters or infects the cell. In some embodiments, the nucleic acid encodes an mRNA molecule, where optionally, the mRNA is a gene of interest. In some embodiments, the nucleic acid encodes a double-stranded RNA, an antisense RNA, a microRNA, or any other RNA molecule. In some embodiments, the gene of interest encodes a protein. In some embodiments, the gene of interest encodes a therapeutic protein (e.g., a protein for compensating for a disease state of a subject). In some embodiments, the nucleic acid is delivered to the cell when the retrovirus enters or infects the cell during step (ii). In some embodiments, the methods of delivering nucleic acid described herein do not require a transfection agent (e.g., a lipophilic transfection agent such as lipofectin).

[0047] Detection Method Described herein is a method for detecting an interaction between a retrovirus and a cell, the method comprising: (i) contacting a sample containing a retrovirus and a cell with an antibody, where the retrovirus comprises a viral envelope protein that comprises at least one mutation that reduces its native function, a non-viral membrane-associated protein that comprises an extracellular targeting domain, and where the antibody binds to the extracellular targeting domain of the retrovirus; (ii) optionally removing unbound antibody from the sample; and (iii) imaging the sample to detect whether the antibody-retrovirus complex is bound to the cell. In some embodiments, the antibody further comprises at least one fluorescent label. In some embodiments, the fluorescent label is a xanthene derivative (e.g., fluorescein, rhodamine, Oregon Green, eosin, and Texas Red), a cyanine derivative (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine), a naphthalene derivative (e.g., dansyl and prodan derivatives), a coumarin derivative, an oxadiazole derivative (e.g., pyridyloxazole, nitrobenzoxadiazole, and benzoxadiazole), a pyrene derivative (e.g., cascade blue), an oxazine derivative (e.g., Nile Red, Nile Blue, cresyl violet, and oxazine 170), an acridine derivative (e.g., proflavine, acridine orange, and acridine yellow), an arylmethine derivative (e.g., auramine, crystal violet, and malachite green), or a tetrapyrrole derivative (e.g., porphine, phthalocyanine, and bilirubin). The fluorescent label may be non-covalently bound to the antibody or covalently bound to the antibody. In some embodiments, the sample is imaged in step (iii) using a confocal microscope or a fluorescent microscope. In some embodiments, the detection method can be carried out using standard microscope equipment (e.g., a confocal microscope or a fluorescent microscope). In some embodiments, the sample is detected in a supermultiplexed format while being imaged using a standard confocal microscope or an epifluorescence microscope.

[0048] nucleic acid As used herein, the term "nucleic acid" generally refers to a molecule that includes a plurality of linked nucleotides (i.e., a sugar (e.g., ribose or deoxyribose) linked to an exchangeable organic base, which is either a pyrimidine (e.g., cytosine (C), thymidine (T) or uracil (U)) or a purine (e.g., adenine (A) or guanine (G)). Nucleic acids include DNA and RNA, such as D- and L-DNA, and various modifications. Modifications include base modifications, sugar modifications, and backbone modifications. It should be understood that the nucleic acids used in the retroviruses and methods of the present invention can be homogeneous or heterogeneous in nature. As an example, they can be completely DNA in nature, or they can be composed of DNA and non-DNA (e.g., LNA) monomers or sequences. Thus, any combination of nucleic acid elements can be used. Modifications can make nucleic acids more stable and / or less susceptible to degradation under certain conditions. For example, in some instances, the nucleic acids are nuclease-resistant. Methods for synthesizing nucleic acids, including automated nucleic acid synthesis, are also known in the art.

[0049] Nucleic acids may contain modifications to their bases. Modified bases include modified cytosines (e.g., 5-substituted cytosines (e.g., 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5-iodo-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstituted or substituted 5-alkynyl-cytosines), 6-substituted cytosines, N4-substituted cytosines (e.g., N4-ethyl-cytosine), 5-aza-cytosine, 2-methyl-cytosine ...methyl-cytosine, 5-fluoro-cytosine, 5-methyl-cytosine, 5-fluoro-cytosine, 5-bromo-cytosine, 5-iodo-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstituted or substituted 5-alkynyl-cytosines). leuceptocytosine, isocytosine, pseudo-isocytosine, cytosine analogues with fused ring systems (e.g., N,N'-propylenecytosine or phenoxazine), and uracil and its derivatives (e.g., 5-fluoro-uracil, 5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil, 5-propynyl-uracil), modified guanines, such as 7-deazaguanine, 7-deaza-7-substituted guanines (7-deaza-7(C2C 6) alkynylguanine), 7 deaza 8 substituted guanine, hypoxanthine, N2-substituted guanine (e.g. N2-methyl-guanine), 5-amino-3-methyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione, 2,6 diaminopurine, 2 aminopurine, purine, indole, adenine, substituted adenines (e.g. N6-methyl-adenine, 8-oxo-adenine), 8 substituted guanines (e.g. 8 hydroxyguanine and 8 bromoguanine), and 6 Thioguanine is included. Nucleic acid can contain universal bases (e.g. 3-nitropyrrole, P-base, 4-methyl-indole, 5-nitro-indole, and K-base) and / or aromatic ring systems (e.g. fluorobenzene, difluorobenzene, benzimidazole or dichloro-benzimidazole, 1-methyl-1H-[1,2,4]triazole-3-carboxylic acid amide). Particular base pairs that can be incorporated into the oligonucleotide of the present invention are dZ and dP nonstandard nucleobase pairs reported in Yang et al. NAR, 2006, 34(21):6095-6101.The pyrimidine analog, dZ, is 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and its Watson-Crick complement, dP, the purine analog, is 2-amino-8-(1'-β-D-1'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one.

[0050] Amino acid substitutions In some embodiments, the amino acid residue modification is a conservative amino acid residue substitution. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, for example, according to methods found in references that compile such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made between amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0051] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. EXAMPLES

[0052] Example 1. Generation of retroviruses that target antigen-specific T cells Targeted lentiviruses were generated by polyethyleneimine (PEI) transfection of HEK293T cells with the following plasmids: an envelope plasmid encoding a mutated VSV-G envelope protein containing the K47Q and R354A mutations, at least one helper plasmid (pRRE, pRev, or psPAX2.1), and a transfer plasmid (Figure 1). The transfer plasmid used encoded a nucleic acid comprising the structure: S-ETD-MBD-IRES-R, where S encodes the B2M signal sequence (provided by SEQ ID NO:2), ETD encodes a variable extracellular targeting domain (e.g., the cancer-testis antigen NYESO-1); MBD encodes a membrane binding domain (e.g., the MHC HLA-A2 domain), IRES encodes an internal ribosome entry site derived from encephalomyocarditis virus (EMCV), and R encodes a green fluorescent protein GFP reporter (Figure 2). The resulting virus was harvested and purified by standard centrifugation techniques before being mixed (using pipette mixing) with a T cell line (e.g., Jurkat T cells) expressing a T cell receptor (TCR) specific for a known pMHC in the presence of hexadimethrine bromide. When the virus displaying HLA-A2-NYESO-1 pMHC was mixed with Jurkat T cells expressing the IG4 T cell receptor (TCR) variant (SEQ ID NO: 27), which efficiently recognizes the displayed NYESO-1 with a binding affinity of about 26 pM, efficient infection was observed, with 41.9% of the T cells expressing the GFP reporter after mixing (Figure 3A). This result indicates that 41.9% of the T cells in this cell population are infected with the virus. Conversely, when the virus displaying HLA-A2-NYESO-1 pMHC was mixed with cells not expressing the IG4 TCR, minimal infection was observed, with only 1.1% of the T cells expressing the GFP reporter after mixing (Figure 3B).

[0053] Mixing the virus displaying HLA-A2-NYESO-1 pMHC with T cells expressing different versions of the IG4 TCR (where the different versions of the IG4 TCR show reduced binding to the NYESO-1 antigen compared to the IG4 TCR variant containing SEQ ID NO:27) demonstrated that the virus was able to infect T cells even when the binding affinity between the NYESO-1 antigen and the IG4 TCR was reduced to 32 μM (FIG. 4). When measuring GFP expression, a binding affinity of 32 μM between the NYESO-1 antigen and the IG4 TCR variant (SEQ ID NO:24) provided 3.6% transduction; a binding affinity of 84 nM between the NYESO-1 antigen and the IG4 TCR variant (SEQ ID NO:25) provided 17.6% transduction; a binding affinity of 5 nM between the NYESO-1 antigen and the IG4 TCR variant (SEQ ID NO:26) provided 18.1% transduction. 1G4-expressing T cells exhibited T cell activation when transduced / infected with a virus displaying HLA-A2-NYESO-1, as evidenced by upregulation of CD69 (Fig. 5A). Conversely, a virus displaying HLA-A2-SL9 did not transduce / infect or activate 1G4-expressing T cells (Fig. 5B).

[0054] Example 2. Generation of a retrovirus targeting the IL-13 receptor A lentivirus containing an interleukin-13 (IL-13) extracellular targeting domain was generated as described in Example 1. The IL-13 extracellular targeting domain consists of a full-length IL-13 protein connected to an IgG4 hinge linker protein linker and a PDGFR transmembrane domain containing a truncated extracellular tail (PDGFR transmembrane domain: VVVISAILALVVLTIISLIILIMLWQKKPR (SEQ ID NO: 28)). When the resulting virus was mixed with Jurkat cells expressing the IL-13Rα1 receptor, efficient transduction was observed, with 85.2% of the cells expressing the GFP reporter after mixing (Figure 7A). When the same virus was mixed with cells not expressing any IL-13 receptor, minimal infection was observed (Figure 7B). Additional IL-13-expressing lentiviruses were generated using alternative protein linkers to connect the IL-13 extracellular targeting domain to the PDGFR transmembrane domain. Equal amounts of all lentiviruses tested were mixed with Jurkat cells bearing the IL13Rα1 receptor, and transduction rates were assessed by the GFP reporter (Figure 12).

[0055] Short and long protein linkers were tested for their ability to allow cell-cell interaction between the extracellular targeting domain (IL-13) and the cognate ligand (IL13Rα1 receptor). Lentiviruses containing short linkers were poorly able to infect Jurkat cells, with the PDGFR linker (AVGQDTQEVIVVPHSLPFK (SEQ ID NO: 3)) allowing infection of 6.1% of the cells; the GAPGAS linker (GAPGAS (SEQ ID NO: 5)) allowing infection of 1.0% of the cells; and the CAR ECD linker (ASESKYGPPCPPCP (SEQ ID NO: 10)) allowing infection of 1.6% of the cells. Lentiviruses containing long linkers were highly successful in infecting Jurkat cells, with the CD8a stalk linker (ASAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAK (SEQ ID NO: 4)) allowing infection of 88.5% of the cells; the IgG4 hinge linker (ASESKYGPPCPPCPAVGQDTQEVIVVPHSLPFK (SEQ ID NO: 8)) allowing infection of 85.2% of the cells; and an oligomerization linker containing an amino acid sequence capable of forming a tetrameric coiled-coil (e.g., ASGGGGSGELAAIKQELAAIKKELAAIKWELAAIKQGAG (SEQ ID NO: 9)) allowing infection of 38.5% of the cells.

[0056] Example 3. Generation of CD19-targeting retroviruses Lentiviruses pseudotyped with Nipah virus F protein and Nipah virus G protein fused to anti-CD19 scFv express CD19 +RajiB cells (cells expressing CD19) could be transduced and 14.5% of the cells expressed the GFP reporter after mixing (Figure 9). Lentiviruses pseudotyped with VSV-mut (mutated VSV-G) and an anti-CD19 scFv fused to an IgG hinge domain, a truncated PDGFR stalk, and the PDGFR transmembrane domain inhibited CD19 + RajiB cells were transduced with 1 μL of VSV-mut lentivirus (200-fold concentrated). + When applied to RajiB cells, it resulted in 10.0% infected cells (based on expression of the GFP reporter). 10 μL of VSV-mut provided 10.7% infected cells (Figure 10). The same VSV-mut lentiviral construct did not transduce / infect CD19-Jurkat cells (cells that do not express CD19), indicating that the lentivirus is specific for cells expressing the cognate ligand of its extracellular targeting domain (anti-CD19 scFv). Lentiviruses pseudotyped with VSV-wt (wild-type VSV-G) and an anti-CD19 scFv fused to an IgG hinge domain, a truncated PDGFR stalk, and the PDGFR transmembrane domain inhibited CD19 + Raji B cells and CD19 - Jurkat cells. This comparative result demonstrates that lentiviruses containing mutated VSV-G, where the mutation reduces the native function of the VSV-G protein, are selective for transduction / infection of cells expressing the cognate ligand targeted by the lentiviral extracellular targeting domain.

[0057] Example 4. Ability of targeted lentiviruses to induce T cell signaling Viruses were evaluated to determine whether infection via TCR targeting provided evidence of TCR signaling. Non-targeted VSV-wt virus efficiently infected T cells but did not activate them compared to controls as assessed by CD69 expression levels (Figure 13, left panel). However, when HLA-A2-NYESO-1-displaying virus was used to target Jurkat T cells expressing high affinity wild-type 1G4 TCR, upregulation of CD69 was observed in transduced cells, indicating that TCR signaling occurred during infection (Figure 13, right panel). These results were replicated when HLA-A2-NYESO-1-displaying virus was mixed with T cells expressing the low affinity mutant 1G4 TCR construct described in Example 1 (Figure 14).

[0058] Example 5. Lentiviral transduction with physiological tropism via TCR Jurkat T cell populations were purified based on the expression of IG4 TCR before contacting with the virus displaying HLA-A2-NYESO-1. The virus efficiently transduced the purified population of Jurkat T cells expressing each of the affinity variants of IG4 TCR based on the expression of the GFP reporter (Figure 15). A binding affinity of 32 μM between the NYESO-1 antigen and the IG4 TCR variant provided 51.3% transduction; a binding affinity of 84 nM between the NYESO-1 antigen and the IG4 TCR variant provided 84.8% transduction; a binding affinity of 5 nM between the NYESO-1 antigen and the IG4 TCR variant provided 82.3% transduction; and a binding affinity of 26 nM between the NYESO-1 antigen and the IG4 TCR variant provided 73.2% transduction. Differences between the different IG4 TCR affinity variants were further investigated by performing dose-responses in which cells were incubated with different amounts (0.5-10 μL of virus) of HLA-A2-NYESO-1-displaying viruses. NYESO-1-displaying lentiviruses transduce IG4-expressing T cells even at high viral doses (Figure 16A). Conversely, SL-1-displaying lentiviruses (negative control) do not transduce IG4-expressing T cells (SL-1 and IG4 TCR are not cognate binding partners).

[0059] The protein structure of membrane-bound NYESO-1 targeting protein was changed to test the ability of constructs displaying oligomerized NYESO-1 to transduce T cells. Lentiviruses displaying and expressing NYESO-1 were generated using alternative protein linkers to connect the NYESO-1 extracellular targeting domain to the transmembrane domain (protein linker containing IgG4 hinge (dimer); and oligomerization linker containing amino acid sequence capable of forming tetrameric coiled coil (tetramer)). As demonstrated in Figure 17, lentiviruses expressing these alternative structures were able to transduce T cells expressing 26pM and 32μM IG4 TCR variants.

[0060] Example 6. Viral binding assay Viruses were pseudotyped with either: (1) wild-type VSV-G (VSV-G); (2) measles virus F protein and receptor-blind H protein fused to anti-CD19 scFv and FLAG tag (MV-αCD19); (3) Nipah virus F protein and receptor-blind G protein fused to anti-CD19 scFv and FLAG tag (NiV-αCD19); or (4) Nipah virus F and receptor-blind G proteins with FLAG tag but no scFv (NiV-dead). These viruses, respectively, are CD19 +The viruses were mixed with Raji B cells and then exposed to a fluorescently labeled antibody against the FLAG tag. As shown in Figures 18A-18B, the viruses that expressed anti-CD19 scFv (MV-αCD19 and NiV-αCD19) bound to Raji B cells. The viruses that did not express anti-CD19 scFv (VSV-G, NiV-dead) did not bind to Raji B cells.

[0061] Example 7. Screening retroviral libraries against T cell populations A retrovirus (10) containing a mutated VSV-G envelope protein, a non-viral membrane-associated protein containing an MHC membrane-binding domain, a variable IL-13 extracellular targeting domain, and a GFP reporter was 7 ~10 10 A library of unique retroviruses. First, a library of nucleic acids encoding unique non-viral membrane-bound proteins containing an MHC membrane-binding domain and a variable IL-13 extracellular targeting domain is prepared using PCR primers that contain degenerate codons at positions known to interact with the IL-13 receptor and induce random mutagenesis in the IL-13 extracellular targeting domain. The resulting nucleic acid constructs are assembled into viral transfer plasmids with fluorescent reporters by standard molecular cloning techniques. These plasmids ("transfer plasmids"), together with a helper plasmid and an envelope plasmid encoding a mutated VSV-G envelope protein (or an equivalent pseudotype), are transfected into retroviruses to generate a library of retroviruses. The virus is purified and mixed with a cell population (T cell line, HEK293 cell line, etc.) expressing the IL-13 receptor of interest. The virus is incubated with the cell population at an appropriate temperature (e.g., 37°C) for an appropriate time (e.g., 1-48 hours). Mixing is performed with hexadimethrine bromide in standard cell culture medium. After 24-48 hours, cells are sorted based on expression of the GFP reporter. High expressing cells are retained and the sorting process is repeated as needed. After these selection rounds of sorting, the retained cells are lysed and cellular RNA and DNA are isolated for analysis using next generation sequencing methods to determine which IL-13 variants were able to mediate viral entry.

[0062] Example 8. Generation of additional retroviruses targeting antigen-specific T cells Additional targeted lentiviruses were generated using the protocol described in Example 1. As shown in Figure 19, mixing (i) lentiviruses containing mutated VSV-G envelope proteins and pMHC displaying SL9 peptides, and (ii) 868 TCR expressing T cells demonstrated that the viruses were capable of transducing and infecting T cells with small amounts of added virus (~70% transduction with 1 μL of virus). Conversely, mixing these viruses with off-target Jurkat cells resulted in low levels of transduction (less than 5% up to 10 μL of added virus). Similar experiments As also shown in Figure 19, mixing (i) lentiviruses containing mutated VSV-G envelope proteins and pMHC displaying cytomegalovirus (CMV) NLV peptides, and (ii) C7 TCR-expressing T cells demonstrated that the viruses were capable of transducing and infecting T cells (~25% transduction with 10 μL of virus). Conversely, mixing these viruses with off-target Jurkat cells resulted in low levels of transduction (less than 5% up to 10 μL of added virus).

[0063] Example 9. Generation of retroviruses containing targeted pMHC stabilized by disulfides Additional targeted lentiviruses were generated using the protocol described in Example 1. These additional lentiviruses further contained disulfides located within the pMHC to stabilize the structure of the pMHC. As shown in Figure 21, mixing (i) lentiviruses containing mutated VSV-G envelope proteins and disulfide-stabilized pMHC displaying targeting peptides, and (ii) target cells expressing the cognate receptor demonstrated that the targeted viruses were able to transduce and infect the target cells. Conversely, mixing these targeted viruses with off-target cells resulted in no or very limited (0.1%) transduction. Specifically, lentivirus containing disulfide-stabilized CMV NLV pMHC transduced on-target C7 TCR-expressing T cells at a transduction rate of 21.8%; lentivirus containing disulfide-stabilized EBV pMHC transduced on-target AS01 cells at a transduction rate of 4.8%; lentivirus containing disulfide-stabilized SL9 pMHC transduced on-target 868 TCR-expressing T cells at a transduction rate of 22.1%; and lentivirus containing disulfide-stabilized NYESO-1 pMHC transduced on-target IG4 TCR-expressing T cells at a transduction rate of 19.0%.

[0064] Example 10. Targeted viruses transduce primary cells Primary T cell lines specific for GL9 (presented by HLA-A2) were specifically and efficiently transduced by viruses displaying GL9 with mutated VSV-G envelope proteins (Figures 22A-22B). Approximately 50% of primary T cells were transduced by these GL9-targeting viruses. Conversely, these GL9-targeting viruses transduced only 0.3% of T cells expressing IG4 TCR. T cells expressing IG4 TCR were specifically and efficiently transduced by viruses displaying NYESO-1 with mutated VSV-G envelope proteins (55.9% transduction). Furthermore, the targeted viruses infect primary cells more efficiently than wild-type VSV-G viruses. These results indicate that the targeted viruses can infect even unstimulated primary cells. Similarly, primary T cell lines specific for NYESO (presented by HLA-A2) were specifically and efficiently transduced by viruses bearing mutated VSV-G envelope proteins and displaying NYESO-1 (Figures 23-24). Conversely, viruses targeting GL9 were unable to efficiently transduce primary T cell lines specific for NYESO. Taken together, these data demonstrate that primary NYESO-1-reactive cells, including expanded primary cells, can be specifically infected by viruses targeting NYESO.

[0065] Example 11. Generation of retroviruses containing CD80 domains Virus pseudotyped with mutated VSV-G envelope proteins and containing the CD80 ectodomain was generated using the protocol described in Example 1. This virus was able to specifically and efficiently infect Jurkat T cells (25.5% transduction) compared to B cells (0.0% transduction) (Figure 25). Similarly, a virus pseudotyped with mutated VSV-G envelope proteins and containing NYESO-1 pMHC and CD80 extracellular domains was generated using the protocol described in Example 1. This virus was able to specifically and efficiently infect Jurkat T cells (13.5% transduction) compared to B cells (0.2% transduction) (Figure 25). The presence of the CD80 domain allowed the virus to transduce Jurkat T cells; this was demonstrated by the inability of a virus pseudotyped with mutated VSV-G envelope proteins and containing only NYESO-1 pMHC to infect these Jurkat T cells (0.9% transduction). Taken together, these data indicate that the presence of CD80 on the viral surface mediates specific infection of T cells and demonstrate that CD80 can be used to generally target viruses to T cells.

[0066] Example 12. Mouse anti-CD3 antibodies mediate infection of TCR-transduced 58- / - cells Displaying antibodies specific for mouse TCR constant region (H57 antibody) or mouse CD3 (2C11 antibody) on the surface of the virus enabled the virus to infect mouse T cell lines. Viruses containing mutated VSV-G viral envelope proteins and anti-TCR antibodies were able to infect mouse T cell lines. - β - provided 11.0% transduction of mouse T hybridoma cells; the virus containing the mutated VSV-G viral envelope protein and anti-CD3 antibodies was - β - It provided 12.8% transduction of mouse T hybridoma cells.

[0067] Example 13. Generation of viruses containing killed coccal virus G proteins The lentivirus was pseudotyped with the coccoccus virus G protein (Cocal-dead; amino acid sequence comprising SEQ ID NO: 53) containing mutations to reduce its infectivity. These mutations at K64Q and R371A in the coccoccus virus G protein were similar to the mutations used in the VSV-dead variant described in Example 1. The Cocal-dead virus further contained a displayed anti-CD19 scFv antibody. As shown in FIG. 27, the Cocal-dead virus expressed CD19 in a manner similar to that of the lentivirus containing the VSV-dead protein and displaying the anti-CD19 scFv antibody. + Raji B cells could be transduced (8.6% transduction). These data demonstrate that the viral targeting strategy is highly robust and can effectively use any viral envelope protein that can be mutated to reduce infectivity (e.g., mutant VSV-G, mutant Nipah envelope, mutant measles envelope, mutant Staphylococcus virus envelope).

[0068] Other Aspects All features disclosed herein can be combined in any combination. Each feature disclosed herein can be replaced with an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, those skilled in the art can easily ascertain the essential characteristics of the present invention, and can make various changes and modifications to adapt the present invention to various usages and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are also within the scope of the claims.

[0069] Equivalent While several inventive embodiments have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more of the described advantages, and each such variation and / or modification is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application or applications for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that, within the scope of the appended claims and their equivalents, the inventive embodiments may be practiced otherwise than as specifically described and claimed. The inventive aspects of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, any combination of two or more of such features, systems, articles, materials, kits, and / or methods is included within the inventive scope of the present disclosure, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0070] All definitions defined and used herein should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms. All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which may, in some cases, include the entire document. The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one." The phrase "and / or" as used in the present specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner; i.e., "one or more" of the elements so conjoined. Other elements other than the elements specifically identified by the "and / or" clause may optionally be present, whether related to the elements specifically identified or not. Thus, as a non-limiting example, a reference to "A and / or B", when used with open-ended language such as "comprising", may in one embodiment refer only to A (optionally including elements other than B); in another embodiment, it may refer only to B (optionally including elements other than A); in yet another embodiment, it may refer to both A and B (optionally including other elements), etc.

[0071] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive; i.e., including at least one of a number or list of elements, but also including a plurality, optionally including additional unlisted items. Terms clearly indicated to the contrary, such as "only one" or "exactly one," or "consisting of," when used in the claims, refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein should be interpreted as an exclusive alternative (i.e., "either one, but not both") when preceded by a term of exclusivity, such as "any," "one of," "only one," or "exactly one." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0072] As used in this specification and claims, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows that elements other than those specifically identified in the list of elements to which the phrase "at least one" refers may optionally be present, whether or not related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently, "at least one of A and / or B") can refer to: in one embodiment, at least one, optionally including two or more As, and no B (and optionally including elements other than B); in another embodiment, at least one, optionally including two or more Bs, and no A (and optionally including elements other than A); in yet another embodiment, at least one, optionally including two or more As, and at least one, optionally including two or more Bs (and optionally including other elements), etc. It should also be understood that, unless expressly stated to the contrary, in any method described herein that includes two or more steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described.

[0073] array >Kappa leader sequence, amino acid (SEQ ID NO:1): METDTLLLWVLLLWVPGSTG >B2M signal peptide sequence, amino acid (SEQ ID NO:2): MSRSVALAVLALLSLSGLEA >PDGFR short stalk, amino acid (SEQ ID NO: 3): MSRSVALAVLALLSLSGLEA >PDGFR long stalk, amino acid (SEQ ID NO: 4): ASAKPTTTPAPRPPTPAPTIASQPLSLRPEAARPAAGGAVHTRGLDFAK >Short flexible linker, amino acid (SEQ ID NO:5): GAPGAS Long flexible linker, amino acid (SEQ ID NO: 6): GAPGSGGGSGGGGSAS >Short flexible linker, amino acid (SEQ ID NO: 7): GGGGS >IgG4 hinge domain, amino acid (SEQ ID NO: 8): ASESKYGPPCPPCPAVGQDTQEVIVVPHSLPFK >Tetrameric coiled coil, amino acid (SEQ ID NO: 9): ASGGGGSGELAAIKQELAAIKKELAAIKWELAAIKQGAG > Dimeric coiled coil, amino acid (SEQ ID NO: 10): ASESKYGPPCPPCP >Wild type VSV-G envelope protein (with leader sequence), DNA sequence (SEQ ID NO: 11):

[0074] Wild-type VSV-G envelope protein (with leader sequence), amino acid sequence (SEQ ID NO: 12): MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPP QSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEM ADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVE IGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0075] Wild-type VSV-G envelope protein, amino acid sequence (SEQ ID NO: 13): KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAE AVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAA ARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERELWDDWAPYEDVEIGPN GVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0076] >VSV-G envelope protein (with leader sequence), DNA sequence (SEQ ID NO: 14):

[0077] >VSV-G envelope protein (with leader sequence), amino acid sequence (SEQ ID NO: 15): MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPQSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPP QSCGYATVTDAEAVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEM ADKDLFAAARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEAELWDDWAPYEDVE IGPNGVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0078] >I41L / K47Q / R354A VSV-G envelope protein, amino acid sequence (SEQ ID NO: 16): KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTALQVKMPQSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAE AVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAA ARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEAELWDDWAPYEDVEIGPN GVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0079] >K47Q / R354A VSV-G envelope protein, amino acid sequence (SEQ ID NO: 17): KFTIVFPHNQKGNWKNVPSNYHYCPSSSDLNWHNDLIGTAIQVKMPQSHKAIQADGWMCHASKWVTTCDFRWYGPKYITQSIRSFTPSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAE AVIVQVTPHHVLVDEYTGEWVDSQFINGKCSNYICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKEGTGFRSNYFAYETGGKACKMQYCKHWGVRLPSGVWFEMADKDLFAA ARFPECPEGSSISAPSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRVDIAAPILSRMVGMISGTTTEAELWDDWAPYEDVEIGPN GVLRTSSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDDESLFFGDTGLSKNPIELVEGWFSSWKSSIASFFFIIGLIIGLFLVLRVGIHLCIKLKHTKKRQIYTDIEMNRLGK

[0080] Exemplary wild-type measles envelope protein (with leader sequence), DNA sequence (SEQ ID NO: 18):

[0081] Exemplary wild-type measles envelope protein, amino acid sequence (SEQ ID NO: 19): MGSRIVINREHLMIDRPYVLLAVLFVMFLSLIGLLAIAGIRLHRAAIYTAEIHKSLSTNLDVTNSIEHQVKDVLTPLFKIIGDEVGLRTPQRFTDLVKFISDKIKFLNPDREYDFRDLTWCINPPERIKLDYDQYCADVAAELMNALVNS TLLETRTTNQFLAVSKGNCSGPTTIRGQFSNMSLSLLDLYLGRGYNVSSIVTMTSQGMYGGTYLVEKPNLSSKRSELSQLSMYRVFEVGVIRNPGLGAPVFHMTNYLEQPVSNDLSNCMVALGELKLAALCHGEDSITIPYQGSGKGVSFQ LVKLGVWKSPTDMQSWVPLSTDDPVIDRLYLSSHRGVIADNQAKWAVPTTRTDDKLRMETCFQQACKGKIQALCENPEWAPLKDNRIPSYGVLSVDLSLTVELKIKIASGFGPLITHGSGMDLYKSNHNNVYWLTIPPMKNLALGVINTLE WIPRFKVSPYLFTVPIKEAGGDCHAPTYLPAEVDGDVKLSSNLVILPGQDLQYVLATYDTSRVEHAVVYYVYSPSRSFSYFYPFRLPIKGVPIELQVECFTWDQKLWCRHFCVLADSESGGHITHSGMVGMGVSCTVTREDGTNDYKDDDDK

[0082] Exemplary mutant measles envelope protein, DNA sequence (SEQ ID NO:20):

[0083] Exemplary mutant measles envelope protein, amino acid sequence (SEQ ID NO:21): MGSRIVINREHLMIDRPYVLLAVLFVMFLSLIGLLAIAGIRLHRAAIYTAEIHKSLSTNLDVTNSIEHQVKDVLTPLFKIIGDEVGLRTPQRFTDLVKFISDKIKFLNPDREYDFRDLTWCINPPERIKLDYDQYCADVAAELMNALVNS TLLETRTTNQFLAVSKGNCSGPTTIRGQFSNMSLSLLDLYLGRGYNVSSIVTMTSQGMYGGTYLVEKPNLSSKRSELSQLSMYRVFEVGVIRNPGLGAPVFHMTNYLEQPVSNDLSNCMVALGELKLAALCHGEDSITIPYQGSGKGVSFQ LVKLGVWKSPTDMQSWVPLSTDDPVIDRLYLSSHRGVIADNQAKWAVPTTRTDDKLRMETCFQQACKGKIQALCENPEWAPLKDNRIPSYGVLSVDLSLTVELKIKIASGFGPLITHGSGMDLYKSNHNNVYWLTIPPMKNLALGVINTLE WIPRFKVSPALFNVPIKEAGGDCHAPTYLPAEVDGDVKLSSNLVILPGQDLQYVLATYDTSAVEHAVVYYVYSPSRSFSYFYPFRLPIKGVPIELQVECFTWDQKLWCRHFCVLADSESGGHITHSGMVGMGVSCTVTREDGTNDYKDDDDK

[0084] Exemplary mutant Nipah envelope protein, DNA sequence (SEQ ID NO:22):

[0085] Exemplary mutant Nipah envelope protein, amino acid sequence (SEQ ID NO:23): MKKINEGLLDSKILSAFNTVIALLGSIVIIVMNIMIIQNYTRSTDNQAVIKDALQGIQQQIKGLADKIGTEIGPKVSLIDTSSTITIPANIGLLGSKISQSTASINENVNEKCKFTLPPLKIHECNISCPNPLPFREYRPQTEGVSNL VGLPNNICLQKTSNQILKPKLISYTLPVVGQSGTCITDPLLAMDEGYFAYSHLERIGSCSRGVSKQRIIGVGEVLDRGDEVPSLFMTNVWTPPNPNTVYHCSAVYNNEFYYVLCAVSTVGDPILNSTYWSGSLMMTRLAVKPKSNGGGY NQHQLALRSIEKGRYDKVMPYGPSGIKQGDTLYFPAVGFLVRTEFKYNDSNCPITKCQYSKPENCRLSMGIRPNSHYILRSGLLKYNLSDGENPKVVFIEISDQRLSIGPSKIYDSLGQPVFYQASFSWDTMIKFGDVLTVNPLVVN WRNNTVISRPGQSQCPRFNTCPAICAEGVYNDAFLIDRINWISAGVFLDSNATAANPVFTVFKDNEILYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDNVIRPKLFAVKIPEQCTGGGGSGGGGSGGGGSASDYKDDDDK

[0086] >IG4 TCR (variant that binds to the NYESO-1 antigen with a binding affinity of 32 μM), amino acid sequence (SEQ ID NO: 24): MSIGLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAE ISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV KRKDSRGGSATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGSSTLYIAASQPGDSATYLCAVRPTSGGSYIPTFG RGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSAAA

[0087] >IG4 TCR (variant that binds to the NYESO-1 antigen with a binding affinity of 84 nM), amino acid sequence (SEQ ID NO: 25): MSIGLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAQTTDQGEVPNGYNVSRSTIEDFPLRLLSAAPSQTSVYFCASSYLGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAE ISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV KRKDSRGGSATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGSSTLYIAASQPGDSATYLCAVRPMIGGTYIPTFG RGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSAAA

[0088] >IG4 TCR (variant that binds to the NYESO-1 antigen with a binding affinity of 5 nM), amino acid sequence (SEQ ID NO:26): MSIGLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGTTDRGEVPNGYNVSRSTIEDFPLRLLSAAPSQTSVYFCASSYVGDTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAE ISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV KRKDSRGGSATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGSSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFG RGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSAAA

[0089] >IG4 TCR (variant that binds to the NYESO-1 antigen with a binding affinity of 26 pM), amino acid sequence (SEQ ID NO: 27): MSIGLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVAIQTTDQGEVPNGYNVSRSTIEDFPLRLLSAAPSQTSVYFCASSYLGNTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAE ISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV KRKDSRGGSATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLITPWQREQTSGRLNASLDKSSGSSTLYIAASQPGDSATYLCAVRPLLDGTYIPTFG RGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSAAA

[0090] >868 TCR (beta chain-P2A-alpha chain), amino acid sequence (SEQ ID NO:31): MSIGLLCCAALSLLWAGPVNADAGVTQSPTHLIKTRGQQVTLRCSPKQGHDTVSWYQQALGQGPQFIFQYYEEERQRGNFPDRFSGHQFPNYSSELNVNALLLGDSALYLCASSDTVSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPS EAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPANLDSRYCLSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVL MAVMKRKDSRGGSATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSSKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYISLLIRDSKLSDSATYLCAVRTNSGYALNF GKGTSLLVTPHIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0091] >CMV C7 TCR、amino acid sequence(sequence number 33): MGTRLLFWVAFCLLGADHTGAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQRLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGESVSTLTIQRTQQEDSAVYLCASSQTQLWETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEA EISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAM VKRKDSRGGSATNFSLLKQAGDVEENPGPMEKNPLAAPLLILWFHLDCVSSILNVEQSPQSLHVQEGDSTNFTCFSPSSNFYALHWYRWETAKSPEALFVMTLNGDEKKKGRISATLNTKEGYSYLYIKGSQPEDSATYLCAFITGNQFYFG TGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSSAAA

[0092] >SL9 pMHC (SL9 peptide portion in bold), amino acid sequence (SEQ ID NO:35): [Table 1]

[0093] >CMV pMHC (CMV NLV peptide portion in bold), amino acid sequence (SEQ ID NO:37): [Table 2]

[0094] Disulfide-trapped MHC containing a >Y84C mutation (bold) and a C at position 2 of the linker (bold); amino acid sequence (SEQ ID NO:39) shown with the HGH signal peptide (underlined) and the GL9 peptide (italics): [Table 3]

[0095] >GL9 pMHC (GL9 peptide portion in bold), amino acid sequence (SEQ ID NO: 41): [Table 4]

[0096] >CD80, amino acid sequence (SEQ ID NO:43): MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTL SVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLPSWAITLISVNGIFVICCLTYCFAPRCRE

[0097] >CD86, amino acid sequence (SEQ ID NO:45): MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFS QPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVLPTVIICVMVFCLILWKWKKKKR

[0098] >Mouse anti-TCR beta clone H57-597 Fab antibody (light chain-P2A-heavy chain, PDGFR transmembrane domain), amino acid sequence: (SEQ ID NO: 47): METDTLLLWVLLLWVPGSTGADYKDDDDKDIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIKR ADAKPTVSIFPPSSEQLGTGSATLVCFVNNFYPKDINVKWKVDGSEKRDGVLQSVTDQDSKDSTYSLSSTLSLTKADYERHNLYTCEVTHKTSTAAIVKTLNRNECGSGATNFSLLKQAGDVEENPGPMVPCTLLLLL AAALAPTQTRAEVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSAKTTAPSVYPL APACDSTTSTTNTVTLGCLVKGYFPEVTVIWNSGALTSGVHTFPSVLHSGLYSLSSSVTVPSSTWPSQTVTCNVAHPASSTTVDLKIEAVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQKKPR

[0099] >Mouse anti-CD3 epsilon clone 145-2C11 Fab antibody (light chain-P2A-heavy chain, PDGFR transmembrane domain), amino acid sequence (SEQ ID NO: 49): *

[0100] > Coccus virus glycoprotein, amino acid sequence: (SEQ ID NO: 51): MNFLLLTFIVLPLCSHAKFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHKAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPP QNCGYATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEF VDQDVYAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVE IGPNGILKTPTGYKFPLFMIGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK*

[0101] > Coccus virus glycoprotein, DNA sequence: (SEQ ID NO: 52):

[0102] >Cocal-dead (mutations to remove native tropism are shown in bold in the protein sequence; these are K64Q and R371A counting from the start codon), amino acid sequence: (SEQ ID NO:53): [Table 5]

[0103] >Cocal-dead (mutation to remove native tropism), DNA sequence: (SEQ ID NO:54):

Claims

1. 1. A composition for use in delivering a nucleic acid to a cell, comprising: a retrovirus comprising a nucleic acid, a VSV-G envelope protein comprising at least one mutation that reduces its native function, and a non-viral membrane-associated protein comprising a membrane-associated domain and an extracellular targeting domain capable of binding to a cognate cellular ligand, wherein the at least one mutation is at amino acid position K47 or R354; wherein delivering the nucleic acid to the cell comprises contacting a retrovirus with the cell, thereby delivering the nucleic acid to the cell.

2. 2. The composition of claim 1, wherein (a) the nucleic acid encodes a gene of interest, optionally wherein the gene of interest encodes a protein; (b) the cognate ligand is a protein, optionally a protein receptor; (c) the extracellular targeting domain is capable of binding to a cognate ligand present on the cell surface of a T cell or a B cell; or (d) the retrovirus is a lentivirus.

3. The composition of claim 1 or 2, wherein the VSV-G envelope protein comprises mutations at amino acid positions K47 and R354.

4. 4. The composition of any one of claims 1 to 3, wherein (a) the non-viral membrane-associated protein comprises a major histocompatibility complex (MHC) protein, (b) the non-viral membrane-associated protein comprises a class I or class II MHC protein, (c) the non-viral membrane-associated protein is an HLA-A2 protein, or (d) the non-viral membrane-associated protein is a disulfide-stabilized MHC protein or a disulfide-stabilized MHC protein comprising a Y84C mutation.

5. The composition of any one of claims 1 to 4, wherein the extracellular targeting domain is a protein, peptide, or antibody.

6. 6. The composition of claim 5, wherein (a) the extracellular targeting domain is an interleukin-13 or CD80 protein domain, (b) the extracellular targeting domain is a full-length antibody, an antibody fragment, a nanobody, or a single-chain antibody (scFv), or (c) the extracellular targeting domain is an anti-CD19 antibody, an anti-TCR antibody, or an anti-CD3 antibody.

7. 7. The composition of any one of claims 1 to 6, wherein the cognate ligand is (a) a T cell receptor (TCR), (b) a cytokine receptor, (c) a cytokine, (d) a cell surface marker, or (e) a cell surface marker selected from the group consisting of CD3, CD19, and CD20.

8. (a) a nucleic acid encoding a gene of interest; (b) a VSV-G envelope protein comprising at least one mutation that reduces its native function, wherein the at least one mutation is at amino acid position K47 or R354; and (c) a non-viral membrane-associated protein comprising a membrane-associated domain and an extracellular targeting domain capable of binding to a cognate cellular ligand; including retroviruses.

9. The retrovirus of claim 8, wherein the VSV-G envelope protein contains mutations at amino acid positions K47 and R354.

10. 10. The retrovirus of claim 8 or 9, wherein (a) the gene of interest encodes a protein; (b) the cognate ligand is a protein, optionally a protein receptor; (c) the extracellular targeting domain is capable of binding to a cognate ligand present on the cell surface of a T cell or a B cell; or (d) the retrovirus is a lentivirus.

11. 11. The retrovirus of any one of claims 8 to 10, wherein (a) the non-viral membrane-associated protein comprises a major histocompatibility complex (MHC) protein, (b) the non-viral membrane-associated protein comprises a class I or class II MHC protein, (c) the non-viral membrane-associated protein is an HLA-A2 protein, or (d) the non-viral membrane-associated protein is a disulfide-stabilized MHC protein or a disulfide-stabilized MHC protein comprising a Y84C mutation.

12. The retrovirus according to any one of claims 8 to 11, wherein the extracellular targeting domain is a protein, peptide, or antibody.

13. 13. The retrovirus of claim 12, wherein (a) the extracellular targeting domain is an interleukin-13 or CD80 protein domain, (b) the extracellular targeting domain is a full-length antibody, an antibody fragment, a nanobody, or a single-chain antibody (scFv), or (c) the extracellular targeting domain is an anti-CD19 antibody, an anti-TCR antibody, or an anti-CD3 antibody.

14. 14. The retrovirus of any one of claims 8 to 13, wherein the cognate ligand is (a) a T cell receptor (TCR), (b) a cytokine receptor, (c) a cytokine, (d) a cell surface marker, or (e) a cell surface marker selected from the group consisting of CD3, CD19, and CD20.

15. below: (a) a CD80 protein domain; and (b) a mutated VSV-G envelope protein comprising at least one mutation that reduces its native function, wherein the at least one mutation is at amino acid position K47 or R354; including retroviruses.

16. The retrovirus of claim 15, wherein the VSV-G envelope protein contains mutations at amino acid positions K47 and R354.

17. The retrovirus of claim 15 or 16, wherein the CD80 protein domain binds to a receptor on a target cell.

18. The retrovirus according to any one of claims 15 to 17, further comprising a non-viral membrane-associated protein comprising an extracellular targeting domain capable of binding to a cellular cognate ligand.

19. The retrovirus according to any one of claims 15 to 18, wherein the retrovirus is a lentivirus.

20. (a) a non-viral membrane-associated protein comprising a membrane-associated domain and an extracellular targeting domain comprising an anti-CD3 antibody; and (b) a mutated VSV-G envelope protein comprising at least one mutation that reduces its native function, wherein the at least one mutation is at amino acid position K47 or R354; Retroviruses including.

21. The retrovirus of claim 20, wherein the VSV-G envelope protein contains mutations at amino acid positions K47 and R354.

22. 22. The retrovirus of claim 20 or 21, wherein the retrovirus further comprises a nucleic acid encoding a gene of interest, optionally wherein the gene of interest encodes a protein.

23. The retrovirus according to any one of claims 20 to 22, wherein the retrovirus is a lentivirus.

24. A composition for delivering a nucleic acid to a cell, comprising the retrovirus of any one of claims 8, 15 and 20, wherein: The composition, wherein delivering the nucleic acid to the cell comprises contacting a retrovirus with the cell, thereby delivering the nucleic acid to the cell.

Images