Use of CD4-targeted viral vectors

Abstract

Provided herein are methods for transducing resting or non-activated T cells using CD4-targeted viral vectors.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 259,717, entitled "USE OF CD4-TARGETED VIRAL VECTORS," filed August 4, 2021; U.S. Provisional Application No. 63 / 298,213, entitled "USE OF CD4-TARGETED VIRAL VECTORS," filed January 10, 2022; U.S. Provisional Application No. 63 / 341,784, entitled "USE OF CD4-TARGETED VIRAL VECTORS," filed May 13, 2022; and U.S. Provisional Application No. 63 / 392,833, entitled "USE OF CD4-TARGETED VIRAL VECTORS," filed July 27, 2022, the contents of each of which are incorporated by reference in their entirety for all purposes.

[0002] Incorporation by reference of sequence listing This application has been filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 186152005940SeqList.XML, created on July 29, 2022, and measuring 342,794 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure relates to methods for transducing resting or non-activated T cells using CD4-targeted viral vectors. [Background technology]

[0004] Viral vectors, including lentiviral vectors, are commonly used to deliver exogenous substances to cells.However, the transduction of viral vectors into specific target cells can be problematic.There is a need for improved viral vectors, including lentiviral vectors, for use in methods for targeting and improving delivery to desired cells.The disclosure provided addresses this need. Summary of the Invention

[0005] The present application is based, inter alia, on the surprising finding that resting or non-activated T cells can be efficiently transduced both in vitro and in vivo using CD4-targeted viral vectors.

[0006] Provided herein are methods of transducing T cells, the methods comprising contacting resting T cells with a lentiviral vector comprising a CD4-binding agent, wherein the lentiviral vector transduces the resting T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the resting T cells are surface negative for one or more T cell activation markers selected from the group consisting of CD25, CD44, and CD69.

[0007] In some embodiments, the resting T cells have not been treated with an anti-CD3 antibody (e.g., OKT3). In some embodiments, the resting T cells have not been treated with an anti-CD28 antibody (e.g., CD28.2). In some embodiments, the resting T cells have not been treated with an anti-CD3 antibody (e.g., OKT3) or an anti-CD28 antibody (e.g., CD28.2). In some embodiments, the resting T cells have not been treated with beads coupled to an anti-CD3 antibody (e.g., OKT3) and an anti-CD28 antibody (e.g., CD28.2), optionally the beads are superparamagnetic beads. In some embodiments, the beads are superparamagnetic beads. In some embodiments, the resting T cells have not been treated with a T cell activating cytokine (e.g., recombinant IL-2, IL-7, IL-15, IL-21, or a combination thereof), optionally the T cell activating cytokine is a human cytokine. In some embodiments, the T cell activating cytokine is a human cytokine. In some embodiments, the resting T cells have not been treated with a soluble T cell costimulatory molecule (e.g., an anti-CD28 antibody or soluble CD80, soluble CD86, soluble CD137L, or soluble ICOS-L). In some of any provided embodiments, the lentiviral vector comprises a transgene encoding an engineered receptor that binds to or recognizes a protein or antigen expressed by or on cells associated with a disease or pathology (e.g., tumor cells).

[0008] In some embodiments, the engineered receptor is an engineered T cell receptor (eTCR). In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising intracellular components of a CD3 zeta signaling domain and a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is a CD28 costimulatory domain. In some embodiments, the CD28 costimulatory signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the costimulatory signaling domain is a 4-1BB signaling domain. In some embodiments, the 4-1BB signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 59. In some embodiments, the CD3 zeta signaling domain comprises the sequence set forth in SEQ ID NO: 61 or SEQ ID NO: 62. In some embodiments, the CD3 zeta signaling domain comprises the sequence set forth in SEQ ID NO: 61. In some embodiments, the CD3 zeta signaling domain comprises the sequence set forth in SEQ ID NO: 62. In some embodiments, the transmembrane domain comprises the sequence set forth in any one of SEQ ID NOs: 56, 57, and 58. In some embodiments, the transmembrane domain comprises the sequence set forth in SEQ ID NO: 56. In some embodiments, the transmembrane domain comprises the sequence set forth in SEQ ID NO: 57. In some embodiments, the transmembrane domain comprises the sequence set forth in SEQ ID NO: 58. In some embodiments, the CAR comprises a hinge domain. In some embodiments, the hinge domain comprises the sequence set forth in any one of SEQ ID NOs: 50, 51, 52, 53, 54, 55, and 142. In some embodiments, the hinge domain comprises the sequence set forth in SEQ ID NO: 51. In some embodiments, the hinge domain comprises the sequence set forth in SEQ ID NO: 52. In some embodiments, the hinge domain comprises the sequence set forth in SEQ ID NO: 53. In some embodiments, the hinge domain comprises the sequence set forth in SEQ ID NO: 54. In some embodiments, the hinge domain comprises the sequence set forth in SEQ ID NO: 55.In some embodiments, the hinge domain comprises the sequence set forth in SEQ ID NO:142.

[0009] In some embodiments, the antigen binding domain binds to an antigen selected from the group consisting of CD19, CD20, CD22, and BCMA.

[0010] In some embodiments, the antigen binding domain binds to CD19. In some embodiments, the antigen binding domain comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 70, 71, and 72, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 65, 66, and 67, respectively. In some embodiments, the antigen binding domain comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 69 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 64. In some embodiments, the antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO: 63 or 73. In some embodiments, the antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO: 63. In some embodiments, the antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO: 73. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 75, 77, 79, or 81. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 75. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 79. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the CAR comprises the amino acid sequence encoded by the polynucleotide sequence set forth in SEQ ID NO: 74, 76, 78, or 80. In some embodiments, the CAR comprises the amino acid sequence encoded by the polynucleotide sequence set forth in SEQ ID NO: 74. In some embodiments, the CAR comprises the amino acid sequence encoded by the polynucleotide sequence set forth in SEQ ID NO: 76. In some embodiments, the CAR comprises the amino acid sequence encoded by the polynucleotide sequence set forth in SEQ ID NO: 78. In some embodiments, the CAR comprises the amino acid sequence encoded by the polynucleotide sequence set forth in SEQ ID NO: 80.

[0011] In some embodiments, the antigen-binding domain binds to CD20. In some embodiments, the antigen-binding domain comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 88, 89, and 144, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 84, 85, and 86, respectively. A VH region comprising the amino acid sequence set forth in SEQ ID NO: 87, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 82.

[0012] In some embodiments, the antigen binding domain binds to CD22. In some embodiments, the antigen binding domain comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 92, 93, and 94, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 96, 97, and 98, respectively. In some embodiments, the antigen binding domain comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 101, 102, and 103, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 105, 106, and 107, respectively. In some embodiments, the antigen binding domain comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 91 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 95. In some embodiments, the antigen binding domain comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 100, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 104. In some embodiments, the antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO: 90 or 99. In some embodiments, the antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO: 90. In some embodiments, the antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO: 99.

[0013] In some embodiments, the antigen binding domain binds to BCMA. In some embodiments, the antigen binding domain comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 114, 115, and 116, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 110, 111, and 112, respectively. In some embodiments, the antigen binding domain comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 123, 124, and 125, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 119, 120, and 121, respectively. In some embodiments, the antigen binding domain comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 127, 128, and 129, respectively. In some embodiments, the antigen binding domain comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 136, 137, and 138, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 132, 133, and 134, respectively. In some embodiments, the antigen binding domain comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 113, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 109. In some embodiments, the antigen binding domain comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 122, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 118. In some embodiments, the antigen binding domain comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 135, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 131. In some embodiments, the antigen binding domain comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 126. In some embodiments, the antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO: 108, 117, or 130. In some embodiments, the antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO: 108. In some embodiments, the antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO:117.In some embodiments, the antigen binding domain comprises the amino acid sequence set forth in SEQ ID NO: 130. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 140. In some embodiments, the CAR comprises the amino acid sequence encoded by the polynucleotide sequence set forth in SEQ ID NO: 139.

[0014] In some embodiments, the CAR comprises (i) an antigen-binding domain comprising a VL region set forth in SEQ ID NO:64, a linker comprising the amino acid sequence set forth in SEQ ID NO:68, and a VH region set forth in SEQ ID NO:69; and / or an scFv set forth in SEQ ID NO:63; (ii) a hinge comprising the amino acid sequence set forth in SEQ ID NO:50; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:56; (iv) a 4-1BB signaling domain comprising the amino acid sequence set forth in SEQ ID NO:59; and (v) a CD3 zeta signaling domain comprising the amino acid sequence set forth in SEQ ID NO:61. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:75. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO:74.

[0015] In some embodiments, the resting T cells are human T cells.

[0016] In some embodiments, the resting T cells are in a subject. In some embodiments, the resting T cells are in vitro. In some embodiments, the resting T cells are ex vivo from a subject. In some embodiments of the provided methods, prior to the contacting, the subject has not been subjected to a T cell activation treatment.

[0017] In some embodiments, any of the methods provided herein are performed in vivo. In some embodiments, any of the methods provided herein are not ex vivo or in vitro.

[0018] In some embodiments of any of the provided methods, the subject has a disease or condition, e.g., cancer. In some embodiments, the lentiviral vector comprises a transgene encoding an engineered receptor that binds to or recognizes a protein or antigen expressed by or on a cell (e.g., a tumor cell) associated with the disease or condition, and optionally the engineered receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR). In some embodiments, the engineered receptor is an engineered T cell receptor (TCR).

[0019] In some embodiments, the T cell is edited to inactivate the B2M, CIITA, TRAC, and TRB genes. In some embodiments, the T cell is edited to inactivate the B2M, CIITA, and TRAC ...

[0020] Also provided herein are transduced T cells produced by any of the provided methods. In some embodiments, the T cells are inactivated at both alleles of one or more genes. Also provided herein are compositions comprising the provided transduced T cells. In some embodiments, the composition is a pharmaceutical composition.

[0021] Provided herein are methods of transducing a population of T cells, the method comprising contacting a population of resting T cells with a composition comprising a lentiviral vector comprising a CD4 binding agent, wherein the population of resting T cells is transduced with an efficiency of at least 1%. In some embodiments, the population of resting T cells is transduced with an efficiency of at least 5%. In some embodiments, the population of resting T cells is transduced with an efficiency of at least 10%. In some embodiments, the population of resting T cells is transduced with an efficiency of at least 15%. In some embodiments, the population of resting T cells is transduced with an efficiency of at least 20%. In some embodiments, the population of resting T cells is transduced with an efficiency of at least 25%. In some embodiments, the population of resting T cells is transduced with an efficiency of at least 30%. In some embodiments, the population of resting T cells is transduced with an efficiency of at least 35%. In some embodiments, the population of resting T cells is transduced with an efficiency of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0022] In some embodiments, at least 75% of the T cells in a population of resting T cells are surface negative for one or more T cell activation markers selected from the group consisting of CD25, CD44, and CD69 (e.g., at least 80%, at least 85%, at least 90%, at least 95% of the T cells in the population are surface negative for a T cell activation marker). In some embodiments, the population of resting T cells comprises CD4+ T cells (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the population of resting T cells are CD4+ T cells). In some embodiments, at least 75% of the CD4+ T cells are surface negative for one or more T cell activation markers selected from the group consisting of CD25, CD44, and CD69 (e.g., at least 80%, at least 85%, at least 90%, at least 95% of the CD4+ T cells in the population are surface negative for a T cell activation marker). In some embodiments, the one or more T cell activation markers is CD25. In some embodiments, the one or more T cell activation markers is CD44. In some embodiments, the one or more T cell activation markers is CD69. In some embodiments, the CD4+ T cells in the population of resting T cells are transduced with an efficiency of at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

[0023] In some embodiments, the population of resting T cells has not been treated with an anti-CD3 antibody (e.g., OKT3). In some embodiments, the population of resting T cells has not been treated with an anti-CD28 antibody (e.g., CD28.2). In some embodiments, the population of resting T cells has not been treated with an anti-CD3 antibody (e.g., OKT3) or an anti-CD28 antibody (e.g., CD28.2). In some embodiments, the population of resting T cells has not been treated with beads coupled to an anti-CD3 antibody (e.g., OKT3) and an anti-CD28 antibody (e.g., CD28.2), optionally wherein the beads are superparamagnetic beads. In some embodiments, the population of resting T cells has not been treated with beads coupled to an anti-CD3 antibody (e.g., OKT3) and an anti-CD28 antibody (e.g., CD28.2). In some embodiments, the beads are superparamagnetic beads. In some embodiments, the population of resting T cells has not been treated with a T cell activating cytokine (e.g., recombinant IL-2, IL-7, IL-15, IL-21, or a combination thereof), and optionally the T cell activating cytokine is a human cytokine. In some embodiments, the population of resting T cells has not been treated with a T cell activating cytokine (e.g., recombinant IL-2, IL-7, IL-15, IL-21, or a combination thereof). In some embodiments, the T cell activating cytokine is a human cytokine. In some embodiments, the population of resting T cells has not been treated with a soluble T cell costimulatory molecule (e.g., an anti-CD28 antibody or soluble CD80, soluble CD86, soluble CD137L, or soluble ICOS-L).

[0024] In some embodiments, the population of resting T cells are human cells.

[0025] In some embodiments, the population of resting T cells is in a subject. In some embodiments, the subject has not been subjected to a T cell activation treatment prior to the contacting. In some embodiments, the population of resting T cells is in vitro. In some embodiments, the population of resting T cells is ex vivo from the subject. In some embodiments, the population of resting T cells comprises peripheral blood mononuclear cells (PBMCs) or a subset thereof comprising CD4+ T cells. In some embodiments, the population of resting cells is an enriched population of T cells selected from a biological sample from the subject, optionally wherein the T cells are selected for T cell surface positivity for a T cell marker (e.g., CD3 or CD4). In some embodiments, the population of resting cells is an enriched population of T cells selected from a biological sample from the subject. In some embodiments, the T cells are selected for T cell surface positivity for a T cell marker (e.g., CD3 or CD4). In some embodiments, the T cell marker is CD3. In some embodiments, the T cell marker is CD4. In some embodiments, the biological sample is a whole blood sample, an apheresis sample, or a leukapheresis sample. In some embodiments, the biological sample is a whole blood sample. In some embodiments, the biological sample is an apheresis sample. In some embodiments, the biological sample is a leukapheresis sample.

[0026] In some embodiments, the subject has a disease or condition. In some embodiments, the lentiviral vector comprises a transgene encoding an engineered receptor that binds to or recognizes a protein or antigen expressed by or on cells associated with the disease or condition (e.g., tumor cells), and optionally the engineered receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In some embodiments, the lentiviral vector comprises a transgene encoding an engineered receptor that binds to or recognizes a protein or antigen expressed by or on cells associated with the disease or condition (e.g., tumor cells). In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR). In some embodiments, the engineered receptor is an engineered T cell receptor (TCR).

[0027] In some embodiments, the method further comprises editing a T cell or population of T cells to inactivate one or more of the B2M, CIITA, TRAC, and TRB genes. In some embodiments, the population of T cells is edited to inactivate the B2M, CIITA, and TRAC genes. In some embodiments, the T cells of the population of T cells are edited to inactivate the B2M, CIITA, and TRB genes. In some embodiments, the method further comprises inserting a gene encoding CD47 at a defined locus. In some embodiments, the defined locus is selected from the group consisting of the B2M locus, the CIITA locus, the TRAC locus, the TRB locus, or a safe harbor locus. In some embodiments, the safe harbor locus is selected from the group consisting of the AAVS1 locus, the CCR5 locus, and the ROSA26 locus.

[0028] In some embodiments, the method further comprises expanding the population of transduced T cells. In some embodiments, the expanding comprises incubating the transduced cells with one or more T cell activating cytokines (e.g., recombinant IL-2, IL-7, IL-15, IL-21, or a combination thereof), where optionally the T cell activating cytokines are human cytokines. In some embodiments, the expanding comprises incubating the transduced cells with one or more T cell activating cytokines (e.g., recombinant IL-2, IL-7, IL-15, IL-21, or a combination thereof). In some embodiments, the T cell activating cytokines are human cytokines. In some embodiments, the method further comprises incubating the transduced T cells with one or more T cell activating cytokines (e.g., recombinant IL-2, IL-7, IL-15, IL-21, or a combination thereof), where optionally the T cell activating cytokines are human cytokines. In some of the provided methods, the method further comprises incubating the transduced T cells with one or more T cell-activating cytokines (e.g., recombinant IL-2, IL-7, IL-15, IL-21, or a combination thereof). In some embodiments, the T cell-activating cytokine is a human cytokine.

[0029] Also provided herein are populations of transduced T cells generated by any of the provided methods. In some embodiments, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of the cells in the population of resting cells are inactivated with one or more genes. In some embodiments, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of the resting CD4+ T cells in the population are transduced and inactivated with one or more genes. In some embodiments, at least 1% of the resting CD4+ T cells in the population are transduced and inactivated with one or more genes. In some embodiments, at least 5% of the resting CD4+ T cells in the population are transduced and inactivated with one or more genes. In some embodiments, at least 10% of the resting CD4+ T cells in the population are transduced and inactivated with one or more genes. In some embodiments, at least 11% of the resting CD4+ T cells in the population are transduced and inactivated with one or more genes. In some embodiments, at least 15% of the resting CD4+ T cells in the population are transduced and inactivated with one or more genes. In some embodiments, at least 20% of the resting CD4+ T cells in the population are transduced and inactivated with one or more genes. In some embodiments, at least 25% of the resting CD4+ T cells in the population are transduced and inactivated with one or more genes. In some embodiments, at least 30% of the resting CD4+ T cells in the population are transduced and inactivated with one or more genes.In some embodiments, at least 35% of the resting CD4+ T cells in the population are transduced and inactivated with one or more genes, hi some embodiments, the cells in the population are inactivated with both alleles of one or more genes.

[0030] Also provided herein are compositions comprising a population of transduced T cells, optionally wherein the composition is a pharmaceutical composition. Also provided herein are compositions comprising a population of transduced T cells. In some embodiments, the composition is a pharmaceutical composition. Also provided herein are pharmaceutical compositions comprising a population of transduced T cells. Also provided herein are methods of treating a subject having a disease or condition, the method comprising administering to the subject any of the provided compositions comprising a population of transduced T cells. In some embodiments, the composition is not administered subcutaneously (SC). In some embodiments, the composition is not administered intramuscularly (IM). In some embodiments, the composition is administered intravenously (IV).

[0031] In some of the provided compositions, the composition further comprises a cryoprotectant. In some embodiments, the cryoprotectant is DMSO.

[0032] Provided herein are methods for in vivo transduction of T cells, the method comprising administering to a subject a composition comprising a lentiviral vector comprising a CD4 binding agent, wherein the lentiviral vector transduces T cells in the subject, and the subject is not subjected to a T cell activation treatment in conjunction with (e.g., before, after, or simultaneously with) administration of the composition. Also provided herein are methods for in vivo transduction of T cells, the method comprising administering to a subject any of the provided compositions, wherein the lentiviral vector transduces T cells in the subject, and the subject is not subjected to a T cell activation treatment in conjunction with (e.g., before, after, or simultaneously with) administration of the composition. In some embodiments, the subject has a disease or condition.

[0033] Also provided herein are methods of treating a subject having a disease or condition, the method comprising administering to the subject a composition comprising a lentiviral vector comprising a CD4 binding agent, wherein the subject is not administered a T cell activation treatment in conjunction with (e.g., before, after, or simultaneously with) administration of the composition. Also provided herein are methods of treating a subject having a disease or condition, the method comprising administering to the subject any of the provided compositions, wherein the subject is not administered a T cell activation treatment in conjunction with (e.g., before, after, or simultaneously with) administration of the composition. In some embodiments, the disease or condition is cancer.

[0034] Also provided herein is a method for expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof, the method comprising administering to the subject a composition comprising a lentiviral vector comprising a CD4 binding agent, wherein the subject is not administered a T cell activation treatment in conjunction with (e.g., before, after, or simultaneously with) administration of the composition. Also provided herein is a method for expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof, the method comprising administering to the subject a composition provided herein, wherein the subject is not administered a T cell activation treatment in conjunction with (e.g., before, after, or simultaneously with) administration of the composition. Also provided herein is a method for expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof, the method comprising administering to the subject a composition provided herein. In some embodiments, the composition is not administered subcutaneously (SC). In some embodiments, the composition is not administered intramuscularly (IM). In some embodiments, the composition is administered intravenously (IV).

[0035] Also provided herein is the use of a composition comprising a lentiviral vector comprising a CD4 binding agent for treating a subject having a disease or condition, optionally cancer. Also provided herein is the use of a composition provided herein for formulating a medicament for treating a subject having a disease or condition, optionally cancer. Also provided herein is the use of a composition comprising a lentiviral vector comprising a CD4 binding agent for treating a subject having a disease or condition. Also provided herein is the use of a composition provided herein for formulating a medicament for treating a subject having a disease or condition. In some embodiments, the disease or condition is cancer.

[0036] Also provided herein are compositions comprising a lentiviral vector comprising a CD4 binding agent for use in treating a subject having a disease or condition, optionally cancer. Also provided herein are compositions provided herein for use in treating a subject having a disease or condition, optionally cancer. Also provided herein are compositions comprising a lentiviral vector comprising a CD4 binding agent for use in treating a subject having a disease or condition. Also provided herein are any of the compositions provided herein for use in treating a subject having a disease or condition, optionally cancer. In some embodiments, the disease or condition is cancer.

[0037] Also provided herein is the use of a composition comprising a lentiviral vector containing a CD4 binding agent for the formulation of a medicament for expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof. Also provided herein is the use of a composition provided herein for the formulation of a medicament for expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof.

[0038] Provided herein is a composition comprising a lentiviral vector containing a CD4 binding agent for use in expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof. Also provided herein is a composition provided herein for use in expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof.

[0039] In some of any of the provided embodiments, the uses or compositions for use provided herein are for use in a subject who does not or should not receive a T cell activation treatment in conjunction with (e.g., before, after, or simultaneously with) administration of the composition.

[0040] In some embodiments of any of the methods, uses, or compositions for use provided herein, the disease or condition is cancer. In some embodiments, the lentiviral vector comprises a transgene encoding an engineered receptor that binds to or recognizes a protein or antigen expressed by or on cells associated with the disease or condition (e.g., tumor cells). In some embodiments, the lentiviral vector comprises a transgene encoding an engineered receptor that binds to or recognizes a protein expressed on tumor cells. In some embodiments, the lentiviral vector comprises a transgene encoding an engineered receptor that binds to or recognizes a protein or antigen expressed by or on cells associated with the disease or condition (e.g., tumor cells), optionally the engineered receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In some embodiments, the lentiviral vector comprises a transgene encoding an engineered receptor that binds to or recognizes a protein expressed on tumor cells, optionally the engineered receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

[0041] In some embodiments, the T cell activation treatment comprises administration of an anti-CD3 antibody (e.g., OKT3). In some embodiments, the T cell activation treatment comprises administration of a soluble T cell costimulatory molecule (e.g., an anti-CD28 antibody, or recombinant CD80, CD86, CD137L, ICOS-L). In some embodiments, the T cell activation treatment comprises administration of a T cell activating cytokine (e.g., recombinant IL-2, IL-7, IL-15, IL-21). In some embodiments, the T cell activating cytokine is a human cytokine. In some embodiments, the T cell activation treatment comprises administration of a T cell activating cytokine (e.g., recombinant IL-2, IL-7, IL-15, IL-21), optionally where the T cell activating cytokine is a human cytokine. In some embodiments, the T cell activation treatment comprises administration of recombinant IL-7, optionally human IL-7. In some embodiments, the T cell activation treatment comprises administration of recombinant IL-7. In some embodiments, the T cell activation treatment comprises administration of recombinant human IL-7. In some of any of the embodiments, the T cell activation treatment comprises administration of lymphodepleting therapy, optionally administration of cyclophosphamide and / or fludarabine. In some of any of the embodiments, the T cell activation treatment comprises administration of lymphodepleting therapy. In some embodiments, the T cell activation treatment comprises administration of cyclophosphamide and / or fludarabine. In some embodiments, the T cell activation treatment comprises administration of cyclophosphamide or fludarabine. In some embodiments, the T cell activation treatment comprises administration of cyclophosphamide. In some embodiments, the T cell activation treatment comprises administration of fludarabine. In some embodiments, the T cell activation treatment comprises administration of cyclophosphamide and fludarabine.

[0042] In some of the provided embodiments, the subject is not administered a T cell activation treatment simultaneously with the lentiviral vector. In some of the provided embodiments, the subject is not administered a T cell activation treatment within one month prior to contact with the lentiviral vector or administration of a composition comprising the lentiviral vector. In some of the provided embodiments, the subject is not administered a T cell activation treatment within one week, two weeks, three weeks, or four weeks, or at or about one week, two weeks, three weeks, or four weeks, optionally at or about one week, two weeks, three weeks, or seven days, prior to contact with the lentiviral vector or administration of a composition comprising the lentiviral vector. In some of the provided embodiments, the subject is not administered a T cell activation treatment within one week, two weeks, three weeks, four weeks, five days, or about one week, two weeks, three weeks, or seven days prior to contact with the lentiviral vector or administration of a composition comprising the lentiviral vector. In some of the provided embodiments, the subject is not administered a T cell activation treatment within one month after contact with the lentiviral vector or administration of a composition comprising the lentiviral vector. In some of the provided embodiments, the subject is not administered a T cell activation treatment within one week, two weeks, three weeks, or four weeks, or at or about one week, two weeks, three weeks, or four weeks, optionally at or about one week, two weeks, three weeks, or seven days, after contact with the lentiviral vector or administration of a composition comprising the lentiviral vector. In some of the provided embodiments, the subject is not administered a T cell activation treatment within one week, two weeks, three weeks, four weeks, five days, six days, or seven days, after contact with the lentiviral vector or administration of a composition comprising the lentiviral vector.

[0043] In some of the provided embodiments, the lentiviral vector does not comprise or encode a T cell activator. In some of the provided embodiments, the lentiviral vector does not comprise or encode a membrane-bound T cell activator. In some of the provided embodiments, the lentiviral vector does not comprise or encode a surface-displayed T cell activator. In some of the provided embodiments, the lentiviral vector does not comprise a surface-displayed T cell activator, for example, the T cell activator is selected from the group consisting of a CD3 antibody (e.g., anti-CD3 scFv); a T cell activating cytokine (e.g., IL-2, IL-7, IL-15, or IL-21); or a T cell costimulatory molecule (e.g., an anti-CD28 antibody, CD80, CD86, CD137L, or ICOS-L). In some embodiments, the T cell activator is selected from the group consisting of a CD3 antibody (e.g., an anti-CD3 scFv); a T cell activating cytokine (e.g., IL-2, IL-7, IL-15, or IL-21); and a T cell costimulatory molecule (e.g., an anti-CD28 antibody, CD80, CD86, CD137L, or ICOS-L). In some embodiments, the T cell activator is a polypeptide capable of binding CD3 and / or CD28. In some embodiments, the T cell activator is a polypeptide capable of binding CD3. In some embodiments, the T cell activator is a polypeptide capable of binding CD28. In some embodiments, the T cell activator is a lymphoproliferative factor. In some embodiments, the T cell activator is a cytokine or cytokine receptor or signaling domain thereof that activates the STAT3 pathway, the STAT4 pathway, and / or the Jak / STAT5 pathway. In some embodiments, the T cell activator is a T cell survival motif. In some embodiments, the T cell survival motif is an IL-7 receptor, an IL-15 receptor, or CD28, or a functional portion thereof. In some embodiments, the T cell activator is a microRNA (miRNA) or a short hairpin RNA (shRNA). In some embodiments, the miRNA or shRNA stimulates the STAT5 pathway.In some embodiments, the miRNA or shRNA inhibits the SOCS pathway. In some embodiments, the miRNA or shRNA stimulates the STAT5 pathway and inhibits the SOCS pathway.

[0044] In some embodiments, the lentiviral vector does not contain or encode an inhibitory RNA molecule. In some embodiments, the inhibitory RNA molecule targets mRNA transcribed from a gene expressed by T cells. In some embodiments, the inhibitory RNA molecule targets a gene encoding a component of the T cell receptor (TCR). In some embodiments, the gene is PD-1, CTLA4, TCRα, TCRβ, CD3ζ, SOCS1, SMAD2, miR-155 target, IFNγ, TRAIL2, and / or ABCG1.

[0045] In some embodiments, the lentiviral vector comprises or encodes an inhibitory RNA molecule. In some embodiments, the inhibitory RNA molecule targets mRNA transcribed from a gene expressed by T cells. In some embodiments, the inhibitory RNA molecule targets a gene encoding a component of the T cell receptor (TCR). In some embodiments, the gene is PD-1, CTLA4, TCRα, TCRβ, CD3ζ, SOCS1, SMAD2, miR-155 target, IFNγ, TRAIL2, and / or ABCG1.

[0046] In some of the provided embodiments, the CD4 binding agent is an anti-CD4 antibody or antigen-binding fragment. In some of the provided embodiments, the anti-CD4 antibody or antigen-binding fragment is murine, rabbit, human, or humanized. In some embodiments, the antigen-binding fragment is a single-chain variable fragment (scFv). In some embodiments, the antigen-binding fragment is an anti-CD4 scFv.

[0047] In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 149, 150, and 151, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 152, 153, and 154, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 149, 150, and 151, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 152, 153, and 154, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 207, 208, and 209, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 210, 211, and 154, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 207, 208, and 209, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 210, 211, and 154, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 212, 213, and 209, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 210, 211, and 154, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 212, 213, and 209, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 210, 211, and 154, respectively. In some embodiments, the anti-CD4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 155.In some embodiments, the anti-CD4 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 156. In some embodiments, the anti-CD4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 155; and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 156. In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the anti-CD4 scFv comprises the amino acid sequence set forth in SEQ ID NO: 157.

[0048] In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 158, 159, and 160, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 161, 162, and 163, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 158, 159, and 160, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 161, 162, and 163, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 214, 215, and 216, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 217, 218, and 163, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 214, 215, and 216, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 217, 218, and 163, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 219, 220, and 216, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 217, 218, and 163, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 219, 220, and 216, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 217, 218, and 163, respectively. In some embodiments, the anti-CD4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 164.In some embodiments, the anti-CD4 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 165. In some embodiments, the anti-CD4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 164; and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 165. In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the anti-CD4 scFv comprises the amino acid sequence set forth in SEQ ID NO: 166.

[0049] In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 167, 168, and 169, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 170, 171, and 172, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 167, 168, and 169, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 170, 171, and 172, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 221, 222, and 223, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 224, 225, and 172, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 221, 222, and 223, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 224, 225, and 172, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 226, 227, and 223, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 224, 225, and 172, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 226, 227, and 223, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 224, 225, and 172, respectively. In some embodiments, the anti-CD4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 173.In some embodiments, the anti-CD4 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 174. In some embodiments, the anti-CD4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 173; and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 174. In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the anti-CD4 scFv comprises the amino acid sequence set forth in SEQ ID NO: 175.

[0050] In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 176, 177, and 178, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 179, 180, and 181, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 176, 177, and 178, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 179, 180, and 181, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 228, 229, and 230, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 231, 232, and 181, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 228, 229, and 230, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 231, 232, and 181, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 233, 234, and 230, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 231, 232, and 181, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 233, 234, and 230, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 231, 232, and 181, respectively. In some embodiments, the anti-CD4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 182.In some embodiments, the anti-CD4 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 183. In some embodiments, the anti-CD4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 182; and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 183. In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the anti-CD4 scFv comprises the amino acid sequence set forth in SEQ ID NO: 184.

[0051] In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 185, 186, and 187, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 188, 171, and 189, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 185, 186, and 187, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 188, 171, and 189, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 235, 236, and 237, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 238, 239, and 189, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 235, 236, and 237, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 238, 239, and 189, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 240, 241, and 237, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 238, 239, and 189, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 240, 241, and 237, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 238, 239, and 189, respectively. In some embodiments, the anti-CD4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 190.In some embodiments, the anti-CD4 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 191. In some embodiments, the anti-CD4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 190; and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 191. In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the anti-CD4 scFv comprises the amino acid sequence set forth in SEQ ID NO: 192.

[0052] In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 193, 194, and 195, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 196, 197, and 198, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 193, 194, and 195, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 196, 197, and 198, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 242, 243, and 244, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 245, 246, and 198, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 242, 243, and 244, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 245, 246, and 198, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 247, 248, and 244, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 245, 246, and 198, respectively. In some embodiments, the anti-CD4 scFv comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 247, 248, and 244, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 245, 246, and 198, respectively. In some embodiments, the anti-CD4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 199.In some embodiments, the anti-CD4 scFv comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 200. In some embodiments, the anti-CD4 scFv comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 199; and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 200. In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the anti-CD4 scFv comprises the amino acid sequence set forth in SEQ ID NO: 201.

[0053] In some embodiments, the anti-CD4 antibody or antigen-binding fragment is a single-domain antibody. In some embodiments, the anti-CD4 antibody or antigen-binding fragment is a camelid (e.g., llama, alpaca, camel) anti-CD4 antibody or antigen-binding fragment (e.g., VHH). In some embodiments, the anti-CD4 antibody or antigen-binding fragment is an anti-CD4 VHH. In some embodiments, the anti-CD4 VHH comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 145, 146, and 147, respectively. In some embodiments, the anti-CD4 VHH comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 202, 203, and 204, respectively. In some embodiments, the anti-CD4 VHH comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 205, 206, and 204, respectively. In some embodiments, the anti-CD4 VHH comprises the amino acid sequence set forth in SEQ ID NO:148.

[0054] In some of the provided embodiments, the CD4 binding agent is exposed on the surface of the lentiviral vector, hi some embodiments, the CD4 binding agent is fused to a transmembrane domain incorporated into the viral envelope.

[0055] In some embodiments, the lentiviral vector is pseudotyped with a viral fusion protein. In some embodiments, the viral fusion protein is a VSV-G protein or a functional variant thereof. In some embodiments, the viral fusion protein is a cocalvirus G protein or a functional variant thereof. In some embodiments, the viral fusion protein is an alphavirus fusion protein (e.g., Sindbis virus) or a functional variant thereof. In some embodiments, the viral fusion protein is a Paramyxoviridae fusion protein (e.g., a morbillivirus or a henipavirus) or a functional variant thereof. In some embodiments, the viral fusion protein is a morbillivirus fusion protein (e.g., measles virus (MeV), canine distemper virus, cetacean morbillivirus, peste des petits ruminants virus, phocine distemper virus, rinderpest virus) or a functional variant thereof. In some embodiments, the viral fusion protein is a henipavirus fusion protein (e.g., Nipah virus, Hendra virus, Cedar virus, Kumasi virus, Mojiang virus) or a functional variant thereof.

[0056] In some of any of the provided embodiments, the viral fusion protein comprises one or more modifications to reduce binding to its native receptor.

[0057] In some of the provided embodiments, the viral fusion protein is fused to a CD4-binding agent. In some embodiments, the viral fusion protein is or comprises a canine distemper virus protein. In some embodiments, the viral fusion protein is a canine distemper virus protein or a functional variant thereof. In some embodiments, the viral fusion protein comprises a canine distemper virus F protein or a biologically active portion thereof. In some embodiments, the CD4-binding agent is fused to a canine distemper virus F protein or a biologically active portion thereof. In some embodiments, the viral fusion protein comprises a canine distemper virus F protein or a biologically active portion thereof, and the CD4-binding agent is fused to a canine distemper virus F protein or a biologically active portion thereof. In some embodiments, the CD4-binding protein is fused directly or via a peptide linker.

[0058] In some of any of the provided embodiments, the viral fusion protein is fused to a CD4-binding agent. In some embodiments, the viral fusion protein is or includes a paramyxovirus (e.g., measles virus or Nipah virus) fusion protein (e.g., a paramyxovirus G protein). In some embodiments, the viral fusion protein is a Nipah virus fusion protein or a functional variant thereof. In some embodiments, the viral fusion protein includes a Nipah virus F glycoprotein (NiV-F), or a biologically active portion thereof, and a Nipah virus G glycoprotein (NiV-G), or a biologically active portion thereof. In some embodiments, the CD4-binding agent is fused to NiV-G, or a biologically active portion thereof. In some embodiments, the viral fusion protein includes a Nipah virus F glycoprotein (NiV-F), or a biologically active portion thereof, and a Nipah virus G glycoprotein (NiV-G), or a biologically active portion thereof, and the CD4-binding agent is fused to NiV-G, or a biologically active portion thereof. In some embodiments, the CD4-binding agent is fused to the C-terminus of the Nipah virus G glycoprotein, or a biologically active portion thereof. In some embodiments, the CD4 binding protein is fused directly or via a peptide linker.

[0059] In some embodiments, the NiV-G protein or biologically active portion thereof is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof.

[0060] In some embodiments, the NiV-G protein or biologically active portion thereof is truncated and lacks up to 40 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5). In some embodiments, the NiV-G protein or biologically active portion thereof has a 5 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), and optionally the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:12, or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:12. In some embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the NiV-G protein or biologically active portion thereof has a 5 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5). In some embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 12, or a sequence of amino acids that exhibits at least 80%, 85%, 90%, or 95%, or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 12. In some embodiments, the NiV-G protein or biologically active portion thereof has a 10 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 5), and optionally the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 44, or a sequence of amino acids that exhibits at least 80%, 85%, 90%, or 95%, or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 44. In some embodiments, the NiV-G protein or biologically active portion thereof has a 10 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 5).In some embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 44, or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 44. In some embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, the NiV-G protein or biologically active portion thereof has a 15 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 4, or SEQ ID NO: 5), optionally, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 45, or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 45. In some embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the NiV-G protein or biologically active portion thereof has a 15 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:9, SEQ ID NO:4, or SEQ ID NO:5). In some embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:45, or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:45. In some embodiments, the NiV-G protein or biologically active portion thereof has a 20 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), and optionally the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:13, or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:13.In some embodiments, the NiV-G protein or biologically active portion thereof has a 20 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5). In some embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:13, or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:13. In some embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:13. In some embodiments, the NiV-G protein or biologically active portion thereof has a 25 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), and optionally the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:14, or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:14. In some embodiments, the NiV-G protein or biologically active portion thereof has a 25 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5). In some embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:14, or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:14. In some embodiments, the NiV-G protein, or a biologically active portion thereof, has the amino acid sequence set forth in SEQ ID NO:14.In some embodiments, the NiV-G protein or biologically active portion thereof has a 30 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5), and optionally the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:43, or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:43. In some embodiments, the NiV-G protein or biologically active portion thereof has a 30 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5). In some embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:43, or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:43. In some embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the NiV-G protein or biologically active portion thereof has a 34 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 5), and optionally the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 42, or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 42. In some embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the NiV-G protein or biologically active portion thereof has a 34 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 5).In some embodiments, the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 42, or a sequence of amino acids that exhibits at least 80%, 85%, 90%, or 95%, or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 42. In some embodiments, the NiV-G protein or biologically active portion thereof has a 34 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 5), optionally the NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO: 42, or a sequence of amino acids that exhibits at least 80%, 85%, 90%, or 95%, or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 42. In some embodiments, the NiV-G protein or biologically active portion thereof has a 34 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 5). In some embodiments, the NiV-G protein or a biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:42, or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:42.

[0061] In some embodiments, the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein. In some of the provided embodiments, the NiV-G protein or biologically active portion thereof is a mutant NiV-G protein that exhibits reduced binding to ephrinB2 or ephrinB3. In some of the provided embodiments, the mutant NiV-G protein or biologically active portion thereof comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to the numbering set forth in SEQ ID NO:4. In some embodiments, the mutant NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:17 or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:17. In some embodiments, the mutant NiV-G protein or biologically active portion thereof has the amino acid sequence set forth in SEQ ID NO:17. In some embodiments, the NiV-G protein or biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18 or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 18. In some embodiments, the NiV-G protein or biologically active portion has the amino acid sequence set forth in SEQ ID NO: 18.

[0062] In some of any of the provided embodiments, the NiV-F protein or biologically active portion thereof is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof. In some of the provided embodiments, the NiV-F protein or biologically active portion thereof has a 20 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO:41 or SEQ ID NO:40 without the signal sequence), and optionally the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:20 or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:20. In some of the provided embodiments, the NiV-F protein or biologically active portion thereof has a 20 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO:41). In some embodiments, the NiV-F protein, or biologically active portion thereof, has the sequence set forth in SEQ ID NO: 20 or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 20. In some embodiments, the NiV-F protein, or biologically active portion thereof, has the sequence set forth in SEQ ID NO: 20. In some of the provided embodiments, the NiV-F protein, or biologically active portion thereof, comprises i) a 20 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 41); and ii) a point mutation on an N-linked glycosylation site, optionally, the NiV-F protein, or biologically active portion thereof, has the sequence set forth in SEQ ID NO: 15 or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 15.In some of any of the provided embodiments, the NiV-F protein, or biologically active portion thereof, comprises i) a 20 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO:41 or SEQ ID NO:40 without the signal sequence); and ii) a point mutation on the N-linked glycosylation site. In some embodiments, the NiV-F protein, or biologically active portion thereof, has the sequence set forth in SEQ ID NO:15 or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO:15. In some embodiments, the NiV-F protein, or biologically active portion thereof, has the sequence set forth in SEQ ID NO:15. In some of any of the provided embodiments, the NiV-F protein, or biologically active portion thereof, has a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 4 or SEQ ID NO: 40 without the signal sequence), and optionally the NiV-F protein, or biologically active portion thereof, has a sequence set forth in SEQ ID NO: 16 or 21, or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 16 or 21. In some of the provided embodiments, the NiV-F protein, or biologically active portion thereof, has a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 41). In some embodiments, the NiV-F protein or biologically active portion thereof has a sequence set forth in SEQ ID NO: 16 or 21 or a sequence of amino acids that exhibits at least or about 80%, 85%, 90% or 95% sequence identity to the sequence set forth in SEQ ID NO: 16 or 21. In some embodiments, the NiV-F protein or biologically active portion thereof has a sequence set forth in SEQ ID NO: 16 or a sequence of amino acids that exhibits at least or about 80%, 85%, 90% or 95% sequence identity to the sequence set forth in SEQ ID NO: 16.In some embodiments, the NiV-F protein, or biologically active portion thereof, has the sequence set forth in SEQ ID NO: 16. In some embodiments, the NiV-F protein, or biologically active portion thereof, has the sequence set forth in SEQ ID NO: 21 or a sequence of amino acids that exhibits at least or about 80%, 85%, 90%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 21. In some embodiments, the NiV-F protein, or biologically active portion thereof, has the sequence set forth in SEQ ID NO: 21.

[0063] In some embodiments, the NiV-G protein or biologically active portion thereof comprises the amino acid sequence set forth in SEQ ID NO: 17, and the NiV-F protein or biologically active portion thereof comprises the sequence set forth in SEQ ID NO: 21. In some embodiments, the NiV-G protein or biologically active portion thereof consists of the amino acid sequence set forth in SEQ ID NO: 17, and the NiV-F protein or biologically active portion thereof consists of the sequence set forth in SEQ ID NO: 21.

[0064] In some of any of the provided embodiments, the lentiviral vector comprises a transgene. In some embodiments, the transgene comprises a nucleic acid sequence encoding an RNA sequence capable of RNA interference (e.g., a pre-miRNA, siRNA, or shRNA). In some embodiments, the transgene is selected from the group consisting of a therapeutic gene, a reporter gene, a gene encoding an enzyme, a gene encoding a prodrug enzyme, a gene encoding an apoptosis inducer, a gene encoding a fluorescent protein, a gene encoding a prodrug-activating enzyme, a gene encoding an apoptotic protein, a gene encoding an apoptotic enzyme, a gene encoding a suicide protein, a gene encoding a cytokine, a gene encoding an anti-immunosuppressive protein, a gene encoding an epigenetic regulator, a gene encoding a T cell receptor (TCR), a gene encoding a chimeric antigen receptor (CAR), a gene encoding a protein that modifies the cell surface of transduced cells, a gene encoding a protein that modifies expression of an endogenous TCR, and a gene encoding a switch receptor that converts tumor-promoting signals into anti-tumor signals. In some embodiments, the transgene encodes an engineered receptor that binds to or recognizes a protein or antigen expressed by a cell or lesion (e.g., a tumor) associated with a disease or condition, and optionally the engineered receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In some embodiments, the transgene encodes an engineered receptor that binds to or recognizes a protein or antigen expressed by a cell or lesion (e.g., a tumor) associated with a disease or condition. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

[0065] In some embodiments, the transgene encodes a chimeric antigen receptor (CAR). In some embodiments, the transgene encodes an engineered T cell receptor (TCR).

[0066] In some embodiments, the contacting is performed by ex vivo administration of the lentiviral vector to the subject using a closed fluid circuit. In some embodiments, the administration is performed by ex vivo administration of the lentiviral vector to the subject using a closed fluid circuit. In some embodiments, the ex vivo administration comprises: (a) obtaining whole blood from the subject; (b) collecting a fraction of the blood containing a white blood cell component including T cells (e.g., CD4+ T cells); (c) contacting the white blood cell component including T cells (e.g., CD4+ T cells) with a composition comprising the lentiviral vector; and (d) reinfusing the contacted white blood cell component including T cells (e.g., CD4+ T cells) into the subject, wherein steps (a)-(d) are performed in-line in a closed fluid circuit. In some embodiments, the contacting in step (c) is for 24 hours or less, 18 hours or less, 12 hours or less, or 6 hours or less.

[0067] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication was individually disclosed by reference. To the extent that a definition set forth herein contradicts or otherwise conflicts with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0068] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. [Brief explanation of the drawings]

[0069] [Figure 1] 1 shows an exemplary system for ex vivo dosing. [Figure 2A]1 shows tumor burden at day 21 in CD19+ tumor-bearing mice treated with 2.5E6, 5E6, or 1E7 integration units (IU) of CD4-targeted CD19 CAR fusosomes, as assessed by bioluminescence imaging. [Figure 2B] Shown is the percentage of CD4+ T cells expressing CAR at day 15 in CD19+ tumor-bearing mice treated with 2.5E6, 5E6, or 1E7 integration units (IU) of CD4-targeted CD19 CAR fusosomes, as assessed by flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0070] I. Definition Unless otherwise defined, all terms of the art, notations, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms having a commonly understood meaning have been defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from the meaning commonly understood in the art.

[0071] Unless otherwise indicated, abbreviations and symbols for chemical and biochemical names follow IUPAC-IUB nomenclature. Unless otherwise indicated, all numerical ranges include the values ​​defining the range as well as all integer values ​​therebetween.

[0072] As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0073] As used herein, the term "about" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which it is used. As used herein, "about," when referring to a measurable value, e.g., an amount, a temporal duration, etc., is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate for practicing the disclosed methods.

[0074] The term "CDR" refers to a complementarity determining region as defined by at least one means of identification to one skilled in the art. The precise amino acid sequence boundaries of a given CDR or FR can be determined using the methods described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol. 2003 Jan;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol,2001 Jun 8;309(3):657-70, ("Aho" numbering scheme); and Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS,1989,86(23):9268-9272, ("AbM" numbering scheme).

[0075] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering for both the Kabat and Chothia schemes is based on the length of the most common antibody region sequences, with insertions provided by insert characters, e.g., "30a," and deletions that appear in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions, based on those used by Oxford Molecular's AbM antibody modeling software.

[0076] In some embodiments, CDRs can be defined according to any of the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the contact definition. VHHs contain three CDRs designated CDR1, CDR2, and CDR3. Table 1 below lists exemplary position boundaries for CDR-H1, CDR-H2, and CDR-H3, as identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between the CDRs; for example, FR-H1 precedes CDR-H1, FR-H2 is located between CDR-H1 and CDR-H2, and FR-H3 is located between CDR-H2 and CDR-H3, etc. Note that the Kabat numbering scheme shown places the insertion at H35A and H35B, so the ends of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention shown, vary between H32 and H34 depending on the length of the loop.

[0077] [Table 1]

[0078] Thus, unless otherwise specified, the "CDR" or "complementarity determining region" of a given antibody or a region thereof, such as a variable region thereof, or each specific CDR (e.g., CDR-H1, CDR-H2, CDR-H3) should be understood to encompass the complementarity determining region defined (or specified) by any of the aforementioned schemes. For example, when a specific CDR (e.g., CDR-H3) is described as containing the amino acid sequence of the corresponding CDR in a given VHH amino acid sequence, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) in the VHH, as defined by any of the aforementioned schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of the provided antibodies are described using various numbering schemes (see, e.g., Table 1), however, it is understood that the provided antibodies may include CDRs described according to any of the other aforementioned numbering schemes or other numbering schemes known to those of skill in the art.

[0079] As used herein, "fusosome" refers to a particle containing a bilayer of amphiphilic lipids surrounding a lumen or cavity, and a fusogen that interacts with the amphiphilic lipid bilayer. In embodiments, fusosomes contain nucleic acid. In some embodiments, fusosomes are membrane-enveloped preparations. In some embodiments, fusosomes are derived from a source cell. In some embodiments, fusosomes are derived from a vector, e.g., a viral vector (e.g., a lentiviral vector).

[0080] As used herein, a "fusosome composition" refers to a composition comprising one or more fusosomes.

[0081] As used herein, "fusogen" refers to an agent or molecule that creates an interaction between two membrane-enclosed lumens. In embodiments, the fusogen facilitates membrane fusion. In other embodiments, the fusogen creates a connection (e.g., a pore) between two lumens (e.g., the lumen of the retroviral vector and the cytoplasm of the target cell). In some embodiments, the fusogen comprises a complex of two or more proteins, e.g., where neither protein has fusogenic activity alone. In some embodiments, the fusogen comprises a targeting domain.

[0082] As used herein, a "retargeted fusogen" refers to a fusogen that includes a targeting site with a sequence that is not part of the naturally occurring form of the fusogen. In embodiments, the fusogen includes a targeting site that is different from the targeting site in the naturally occurring form of the fusogen. In embodiments, the naturally occurring form of the fusogen lacks a targeting domain, and the retargeted fusogen includes a targeting site that is not present in the naturally occurring form of the fusogen. In embodiments, the fusogen is modified to include a targeting site. In embodiments, the fusogen includes one or more sequence modifications outside the targeting site relative to the naturally occurring form of the fusogen, for example, in the transmembrane domain, the fusogenically active domain, or the cytoplasmic domain.

[0083] The term "corresponding to" in reference to a protein position, such as a statement that a nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence, such as those shown in the sequence listing, refers to a nucleotide or amino acid position identified by alignment with the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. For example, corresponding residues of similar sequences (e.g., fragments or species variants) can be determined by alignment to a reference sequence by structural alignment methods. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved identical amino acid residues as a guide.

[0084] The term "effective amount," as used herein, means the amount of a pharmaceutical composition sufficient to significantly and favorably modify the symptoms and / or condition being treated (e.g., provide a favorable clinical response). The effective amount of active ingredient for use in a pharmaceutical composition will vary within the knowledge and expertise of the attending physician, depending on the particular condition being treated, the severity of the condition, the duration of treatment, the nature of any concurrent therapy, the particular active ingredient(s) used, the particular pharmaceutically acceptable excipient(s) and / or carrier(s) employed, and similar factors.

[0085] "Exogenous material," as used herein with respect to viral vectors, refers to material that is not contained in or encoded by the corresponding wild-type virus or fusogen produced from the corresponding wild-type source cell. In some embodiments, the exogenous material is not naturally occurring, e.g., a protein or nucleic acid having a sequence that is altered (e.g., by insertion, addition, or substitution) relative to a naturally occurring protein. In some embodiments, the exogenous material is not naturally occurring in the source cell. In some embodiments, the exogenous material is not naturally occurring in the source cell but is exogenous to the virus. In some embodiments, the exogenous material is not naturally occurring in the recipient cell. In some embodiments, the exogenous material is naturally present in the recipient cell but is not present at the desired level or for the desired time. In some embodiments, the exogenous material comprises RNA or protein.

[0086] As used herein, "promoter" refers to a cis-regulatory DNA sequence that, when operably linked to a gene coding sequence, induces transcription of the gene. A promoter may contain transcription factor binding sites. In some embodiments, a promoter acts in conjunction with one or more enhancers distal to the gene.

[0087] As used herein, "operably linked" or "operably associated" includes reference to the functional linkage of at least two sequences. For example, operably linked includes a linkage between a promoter and a second sequence, where the promoter sequence initiates and mediates transcription of a DNA sequence corresponding to the second sequence. Operatively associated includes a linkage between an inducing or repressing element and a promoter, where the inducing or repressing element acts as a transcriptional activator of the promoter.

[0088] As used herein, "retroviral nucleic acid" refers to a nucleic acid that contains at least the minimum sequence requirements for packaging into a retrovirus or retroviral vector, alone or in combination with a helper cell, helper virus, or helper plasmid. In some embodiments, the retroviral nucleic acid further comprises or encodes an exogenous substance, a positive target cell-specific regulatory element, a non-target cell-specific regulatory element, or a negative TCSSE. In some embodiments, the retroviral nucleic acid comprises one or more of the following: a 5'LTR (e.g., to promote integration), a U3 (e.g., to activate viral genome RNA transcription), an R (e.g., a Tat binding region), a U5, a 3'LTR (e.g., to promote integration), a packaging site (e.g., psi (Ψ)), and an RRE (e.g., to bind Rev and promote nuclear export). Retroviral nucleic acid can comprise RNA (e.g., as part of a virion) or DNA (e.g., when introduced into a source cell or after reverse transcription in a recipient cell). In some embodiments, the retroviral nucleic acid is packaged using a helper cell, helper virus, or helper plasmid containing one or more (eg, all) of gag, pol, and env.

[0089] As used herein, the term "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound and that is relatively non-toxic, i.e., the substance may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0090] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound of the present invention with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickeners, and / or excipients. Pharmaceutical compositions facilitate the administration of a compound to an organism. Multiple techniques for administering compounds exist in the art, including, but not limited to, intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.

[0091] As used herein, the terms "treat," "treating," or "treatment" refer to ameliorating a disease or disorder, e.g., slowing or halting or reducing the progression of a disease or disorder, e.g., the primary cause of the disorder, or at least one of its clinical symptoms.

[0092] As used herein, the terms "effective amount" and "pharmaceutically effective amount" refer to a non-toxic but sufficient amount of an agent or drug to provide a desired biological result. The result may be reduction and / or alleviation of the signs, symptoms, or causes of a disease or disorder, imaging or monitoring of an in vitro or in vivo system (including a living organism), or any other desired modification of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0093] II. Method Provided herein are methods of transducing T cells or a population thereof, comprising contacting non-activated T cells or a population thereof with a lentiviral vector comprising a CD4 binding agent, wherein the lentiviral vector transduces the non-activated T cell(s). In some embodiments, the population of non-activated T cells is transduced with an efficiency of at least 1%.

[0094] Also provided herein is a method for in vivo transduction of T cells, comprising administering to a subject a composition comprising a lentiviral vector comprising a CD4 binding agent, wherein the lentiviral vector transduces T cells in the subject. Also provided herein is a method for treating a subject having a disease or condition, comprising administering to the subject a composition comprising a lentiviral vector comprising a CD4 binding agent. Also provided herein is a method for expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof, comprising administering to the subject a composition comprising a lentiviral vector comprising a CD4 binding agent. In some embodiments, the subject is not administered a T cell activation treatment in conjunction with (e.g., before, after, or simultaneously with) administration of the composition.

[0095] In some embodiments, resting or non-activated T cells are contacted with a viral vector (e.g., a retroviral or lentiviral vector) comprising a CD4 binding agent. Contacting can be performed in vitro (e.g., in T cells derived from a healthy donor or a donor in need of cell therapy) or in vivo by administration of the viral vector to a subject.

[0096] In some embodiments, resting or resting T cells are not treated with one or more T cell stimulatory molecules (e.g., anti-CD3 antibodies), one or more T cell costimulatory molecules, and / or one or more T cell activating cytokines. In some embodiments, resting or resting T cells are not treated with any of one or more T cell stimulatory molecules (e.g., anti-CD3 antibodies), one or more T cell costimulatory molecules, and / or one or more T cell activating cytokines.

[0097] In additional aspects, the applications include methods of administration to a subject, including any of those described in Sections VI and VIII. In some embodiments, the methods include administering to the subject a viral vector comprising an anti-CD4 binding agent, wherein the subject has not or has not been administered a T cell activation treatment. In some embodiments, the T cell activation treatment includes one or more T cell stimulatory molecules (e.g., an anti-CD3 antibody), one or more T cell costimulatory molecules, and / or one or more T cell activation cytokines. In some embodiments, the subject has not or has not been administered one or more T cell stimulatory molecules (e.g., an anti-CD3 antibody), one or more T cell costimulatory molecules, and / or one or more T cell activation cytokines. In some embodiments, the T cell activation treatment is lymphodepletion. In some embodiments, the subject has not or has not been administered a lymphodepletion therapy. In certain embodiments, the subject has not or has not been administered a T cell activation treatment within one month before or after administration of the viral vector. In some embodiments, the subject does not receive or has not received a T cell activation treatment within one month prior to administration of the viral vector, e.g., within 4, 3, 2, or 1 week or at or about 4, 3, 2, or 1 week, e.g., 1, 2, 3, 4, 5, 6, or 7 days, or about 1, 2, 3, 4, 5, 6, or 7 days, before administration of the viral vector. In some embodiments, the subject does not receive or have not received a T cell activation treatment within one month after administration of the viral vector, e.g., within 4, 3, 2, or 1 week or at or about 4, 3, 2, or 1 week, e.g., 1, 2, 3, 4, 5, 6, or 7 days, or about 1, 2, 3, 4, 5, 6, or 7 days, after administration of the viral vector.

[0098] In some aspects, the viral vector does not comprise or encode a T cell activator. In some embodiments, the viral vector does not comprise or encode a membrane-bound T cell activator. In some embodiments, the viral vector does not comprise or encode a surface-displayed T cell activator. In some embodiments, the T cell activator is an anti-CD3 antibody (e.g., an anti-CD3 scFv), a T cell activating cytokine (e.g., IL-2, IL-7, IL-15, or IL-21), or a T cell costimulatory molecule (e.g., an anti-CD28 antibody, CD80, CD86, CD137L, or ICOS-L. In some embodiments, the T cell activator is a polypeptide capable of binding CD3, a polypeptide capable of binding CD28, or both. In some aspects, the viral vector does not comprise one or more T cell stimulatory molecules (e.g., an anti-CD3 antibody), one or more T cell costimulatory molecules, and / or one or more T cell activating cytokines.

[0099] The use of anti-CD3 antibodies is well known for activating T cells. The anti-CD3 antibodies can be of any species, e.g., murine, rabbit, human, humanized, or camelid. Exemplary antibodies include OKT3, CRIS-7, I2C, an anti-CD3 antibody found in DYNABEADS human T-activator CD3 / CD28 (Thermo Fisher), and the anti-CD3 domains of approved and clinically studied molecules such as blinatumomab, catumaxomab, fotetuzumab, teclistamab, ertumaxomab, epcolitamab, talquetamab, odronextamab, civistamab, oblindatamab, tidutamab, duvortuxizumab, solitomab, elbixtamab, pavlutamab, tepoditamab, bibecotamab, pramotamab, glofitamab, eteburitamab, and tarlatamab.

[0100] In some embodiments, the one or more T cell costimulatory molecules include CD28 ligands (e.g., CD80 and CD86); antibodies that bind to CD28, e.g., DYNABEADS human T-activating factor CD3 / CD28 (Thermo and anti-CD28 domains disclosed in US2020 / 0199234, US2020 / 0223925, US2020 / 0181260, US2020 / 0239576, US2020 / 0199233, US2019 / 0389951, US2020 / 0299388, US2020 / 0399369, and US2020 / 0140552; CD137 ligand (CD137L); anti-CD137 antibodies such as urelumab and utomilumab; ICOS ligand (ICOS-L); and anti-ICOS domains of anti-ICOS antibodies such as ferazilimab, vopratelimab, and izularimab.

[0101] In some embodiments, the one or more T cell activating cytokines include IL-2, IL-7, IL-15, IL-21, interferon (e.g., interferon-gamma), and functional variants and modified versions thereof.

[0102] In some aspects, the viral vector does not comprise or encode a T cell activator. In some embodiments, the viral vector does not comprise or encode a membrane-bound T cell activator. In some embodiments, the viral vector does not comprise or encode a surface-displayed T cell activator. In some embodiments, the T cell activator is a lymphoproliferative element. In some embodiments, the lymphoproliferative element is a cytokine or cytokine receptor or signaling domain thereof that activates the STAT3 pathway, the STAT4 pathway, and / or the Jak / STAT5 pathway. In some embodiments, the lymphoproliferative element is a T cell survival motif, e.g., IL-7 receptor, IL-15 receptor, or CD28, or a functional portion thereof. In some embodiments, the lymphoproliferative element is a microRNA (miRNA) or short hairpin RNA (shRNA) that stimulates the STAT5 pathway, inhibits the SOCS pathway, or both.

[0103] In some embodiments, the vector does not contain or encode an inhibitory RNA molecule. In some embodiments, the inhibitory RNA molecule targets mRNA transcribed from a gene expressed by T cells, a gene encoding a component of the T cell receptor (TCR), or both. In some embodiments, the gene is PD-1, CTLA4, TCRα, TCRβ, CD3ζ, SOCS1, SMAD2, miR-155 target, IFNγ, TRAIL2, and / or ABCG1.

[0104] In some embodiments, the vector comprises or encodes an inhibitory RNA molecule. In some embodiments, the inhibitory RNA molecule targets mRNA transcribed from genes expressed by T cells, genes encoding components of the T cell receptor (TCR), or both. In some embodiments, the genes are PD-1, CTLA4, TCRα, TCRβ, CD3ζ, SOCS1, SMAD2, miR-155 target, IFNγ, TRAIL2, and / or ABCG1.

[0105] In some embodiments, the method further includes administering a lymphodepleting therapy to the subject. In some embodiments, the T cell activation treatment includes administering a lymphodepleting therapy to the subject. Lymphodepletion can be induced by various treatments that destroy lymphocytes and T cells in the subject. For example, lymphodepletion can include myeloablative chemotherapy, such as fludarabine, cyclophosphamide, bendamustine, and combinations thereof. Lymphodepletion can also be induced by irradiation (e.g., total body irradiation) of the subject. In some embodiments, the lymphodepleting therapy includes cyclophosphamide and / or fludarabine. In some embodiments, the method further includes administering cyclophosphamide and / or fludarabine.

[0106] III. Viral Vectors Provided herein are viral vectors, for example, for transducing T cells. In some embodiments, viral vectors that bind cell surface receptors for delivery of exogenous substances (e.g., transgenes) via membrane fusion are provided as "fusosomes." Thus, in some cases, fusosomes refer to the viral vectors disclosed herein.

[0107] In some embodiments, the viral vectors disclosed herein are retroviral vectors (e.g., lentiviral vectors). In some embodiments, the retroviral vectors have long terminal repeats (LTRs), such as retroviral vectors derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus-forming virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any avian or mammalian cell source. Retroviruses are typically amphotropic, meaning they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the gag, pol, and / or env sequences of the retrovirus. Numerous exemplary retroviral systems have been described (e.g., U.S. Patent Nos. 5,219,740; 6,207,453; 5,219,740).

[0108] Lentiviral transduction methods are known. Exemplary methods are described, for example, in Wang et al., J. Immunother. 35(9):689-701, 2012; Cooper et al., Blood. 101:1637-1644, 2003; Verhoeyen et al., Methods Mol Biol. 506:97-114, 2009; and Cavalieri et al., Blood. 102(2):497-505, 2003.

[0109] In some embodiments, the retroviral nucleic acid comprises one or more (e.g., all) of a 5' promoter (e.g., for controlling expression of the entire packaged RNA), a 5' LTR (e.g., a U5 containing an R (including a polyadenylation tail signal) and / or a primer activation signal), a primer binding site, a Psi packaging signal, an RRE element for nuclear export, a promoter directly upstream of the transgene to control transgene expression, the transgene (or other exogenous element), a polypurine tract, and a 3' LTR (e.g., containing a mutated U3, R, and U5). In some embodiments, the retroviral nucleic acid further comprises one or more of a cPPT, a WPRE, and / or an insulator element.

[0110] Retroviruses typically replicate by reverse transcription of their genomic RNA into a linear, double-stranded DNA copy, which then covalently integrates into the host genome. Exemplary retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spuma virus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), as well as lentiviruses.

[0111] In some embodiments, the retrovirus is a gamma retrovirus. In some embodiments, the retrovirus is an epsilon retrovirus. In some embodiments, the retrovirus is an alpha retrovirus. In some embodiments, the retrovirus is a beta retrovirus. In some embodiments, the retrovirus is a delta retrovirus. In some embodiments, the retrovirus is a lentivirus. In some embodiments, the retrovirus is a spumaretrovirus. In some embodiments, the retrovirus is an endogenous retrovirus.

[0112] Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visna-Maedi virus (VMV); Caprine Arthritis-Encephalitis Virus (CAEV); Equine Infectious Anemia Virus (EIAV); Feline Immunodeficiency Virus (FIV); Bovine Immunodeficiency Virus (BIV); and Simian Immunodeficiency Virus (SIV). In some embodiments, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is used. In some embodiments, the viral particle is derived from a lentivirus. In some embodiments, the lentiviral vector particle is human immunodeficiency virus-1 (HIV-1).

[0113] In some embodiments, the viral vector, e.g., retroviral or lentiviral vector, comprises one or more of a gag polyprotein, a polymerase (e.g., pol), an integrase (e.g., a functional or non-functional variant), a protease, and a fusogen. In some embodiments, the vector further comprises rev. In some embodiments, one or more of the foregoing proteins are encoded in the retroviral genome, and in some embodiments, one or more of the foregoing proteins are provided in trans, e.g., by a helper cell, helper virus, or helper plasmid. In some embodiments, the retroviral nucleic acid comprises one or more of the following nucleic acid sequences: a 5' LTR (e.g., comprising a U5 and lacking a functional U3 domain), a Psi packaging element (Psi), a central polypurine tract (cPPT) promoter operably linked to a payload gene, a payload gene (optionally including an intron before the open reading frame), a polyA tail sequence, a WPRE, and a 3' LTR (e.g., comprising a U5 and lacking a functional U3). In some embodiments, the non-retroviral nucleic acid further comprises one or more insulator elements. In some embodiments, the recognition site is located between the polyA tail sequence and the WPRE.

[0114] 1. Transfer Vector In some embodiments, viral vectors typically comprise nucleic acid molecules (e.g., transfer plasmids) that contain virus-derived nucleic acid elements that facilitate the transfer or integration of the nucleic acid molecule into a cell's genome or into a viral particle that mediates nucleic acid transfer. In some aspects, vector particles will typically include various viral and sometimes host cell components in addition to the nucleic acid(s). In some embodiments, viral vectors comprise, for example, viruses or viral particles capable of transferring nucleic acids into cells or transferred nucleic acids (e.g., naked mRNA). In some embodiments, viral vectors and transfer plasmids comprise structural and / or functional genetic elements that are primarily derived from viruses. Retroviral vectors can comprise viral vectors or plasmids that contain structural and / or functional genetic elements, or portions thereof, that are primarily derived from retroviruses. Lentiviral vectors can comprise viral vectors or plasmids that contain structural and / or functional genetic elements, or portions thereof, that are primarily derived from lentiviruses, including LTRs.

[0115] In embodiments, a lentiviral vector (e.g., a lentiviral expression vector) can comprise a lentiviral transfer plasmid (e.g., as naked DNA) or an infectious lentiviral particle. With respect to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it should be understood that the sequences of these elements can be present in the lentiviral particle in RNA form or in the DNA plasmid in DNA form.

[0116] In some embodiments, at least a portion of one or more protein coding regions that contribute to or are essential for replication in the vectors described herein may be absent compared to the corresponding wild-type virus. In some embodiments, the viral vector is replication-deficient. In some embodiments, the vector is capable of transducing target non-dividing host cells and / or integrating its genome into the host genome.

[0117] In some embodiments, the structure of wild-type retroviral genomes often includes a 5' long terminal repeat (LTR) and a 3' LTR, between which or within which are located the gag, pol, and env genes, which encode packaging components that facilitate assembly of viral particles, including a packaging signal to enable genome packaging, a primer binding site, an integration site to enable integration into the host cell genome, and packaging components that facilitate assembly of viral particles. More complex retroviruses possess additional features, such as the rev and RRE sequences in HIV, which enable efficient export of integrated proviral RNA transcripts from the nucleus to the cytoplasm of infected target cells. In proviruses, viral genes are flanked on both sides by regions called long terminal repeats (LTRs). In some embodiments, the LTRs are involved in proviral integration and transcription. In some embodiments, the LTRs may function as enhancer-promoter sequences, controlling viral gene expression. In some embodiments, encapsidation of retroviral RNA occurs via a psi sequence located at the 5' end of the viral genome.

[0118] In some embodiments, the LTRs are similar sequences that can be divided into three elements, called U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of the three elements can vary greatly between different retroviruses.

[0119] In some embodiments, for viral genomes, the site of transcription initiation is typically the boundary between U3 and R in one LTR, and the site of poly(A) addition (termination) is the boundary between R and U5 in the other LTR. U3 contains most of the transcriptional control elements of the provirus, including a promoter and multiple enhancer sequences responsive to cellular and, in some cases, viral transcriptional activator proteins. In some embodiments, retroviruses contain any one or more of the following genes that encode proteins involved in regulating gene expression: tat, rev, tax, and rex.

[0120] In some embodiments, the structural genes gag, pol, and env, gag, encode the internal structural proteins of the virus. In some embodiments, the Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid), and NC (nucleocapsid). In some embodiments, the pol gene encodes reverse transcriptase (RT), which contains DNA polymerase, associated RNase H, and integrase (IN), which mediate genome replication. In some embodiments, the env gene encodes the surface (SU) glycoprotein and transmembrane (TM) protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. In some embodiments, the interaction facilitates infection by fusion of the viral membrane with the cellular membrane.

[0121] In some embodiments, the gag, pol, and env regions of the replication-deficient retroviral vector genome may be absent and non-functional. In some embodiments, the R regions at both ends of the RNA are typically repeated sequences. In some embodiments, U5 and U3 represent unique sequences at the 5' and 3' ends of the RNA genome, respectively.

[0122] In some embodiments, retroviruses may also contain additional genes encoding proteins other than gag, pol, and env. Examples of additional genes include (in HIV), one or more of vif, vpr, vpx, vpu, tat, rev, and nef. EIAV has an additional gene, S2 (among others). In some embodiments, proteins encoded by additional genes perform various functions, some of which may overlap with functions provided by cellular proteins. In EIAV, for example, tat acts as a transcriptional activator of the viral LTR (Derse and Newbold 1993 Virology 194:530-6; Maury et al. 1994 Virology 200:632-42). It binds to a stable stem-loop RNA secondary structure called TAR. Rev controls and regulates viral gene expression via the rev-responsive element (RRE) (Martarano et al. 1994 J. Virol. 68:3102-11).

[0123] In some embodiments, in addition to protease, reverse transcriptase, and integrase, non-primate lentiviruses contain a fourth pol gene product that encodes a dUTPase, which in some embodiments plays a role in the ability of these lentiviruses to infect certain non-dividing or slowly dividing cell types.

[0124] In embodiments, a recombinant lentiviral vector (RLV) is a vector that carries sufficient retroviral genetic information to enable packaging of an RNA genome into viral particles capable of infecting target cells in the presence of packaging components. In some embodiments, infection of a target cell can include reverse transcription and integration into the target cell genome. In some embodiments, an RLV typically carries non-viral coding sequences that are delivered to the target cell by the vector. In some embodiments, an RLV is incapable of independent replication to generate infectious retroviral particles in the target cell. In some embodiments, an RLV lacks functional gag-pol and / or env genes and / or other genes involved in replication. In some embodiments, the vector can be configured as a split-intron vector, for example, as described in PCT Patent Application WO 99 / 15683, which is incorporated herein by reference in its entirety.

[0125] In some embodiments, lentiviral vectors comprise a minimal viral genome, e.g., viral vectors that have been engineered to remove non-essential elements and retain essential elements to provide the required functionality to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell, e.g., as described in WO98 / 17815, which is incorporated herein by reference in its entirety.

[0126] In some embodiments, a minimal lentiviral genome can include, for example, (5')R-U5-one or more first nucleotide sequences-U3-R(3'). In some embodiments, a plasmid vector used to generate a lentiviral genome in a source cell can also include a transcriptional regulatory control sequence operably linked to the lentiviral genome to direct transcription of the genome in the source cell. In some embodiments, the regulatory sequence can include a native sequence associated with the transcribed retroviral sequence, e.g., the 5'U3 region, or they can include a heterologous promoter, e.g., another viral promoter, such as a CMV promoter. In some embodiments, the lentiviral genome includes additional sequences to promote efficient virus production. In some embodiments, in the case of HIV, rev and RRE sequences can be included. In some embodiments, codon optimization can be used alternatively or in combination; for example, a gene encoding an exogenous substance can be codon-optimized, as described, for example, in WO 01 / 79518 (incorporated herein by reference in its entirety). In some embodiments, alternative sequences that perform similar or the same function as the rev / RRE system can also be used. In some embodiments, a functional analog of the rev / RRE system is found in the Mason Pfizer monkey virus. In some embodiments, this contains an RRE-type sequence in its genome known as a CTE, which is thought to interact with a factor in infected cells. The cellular factor may be considered a rev analog. In some embodiments, the CTE may be used as an alternative to the rev / RRE system. In some embodiments, the Rex protein of HTLV-I may functionally replace the Rev protein of HIV-I. Rev and Rex have effects similar to those of IRE-BP.

[0127] In some embodiments, the retroviral nucleic acid (e.g., lentiviral nucleic acid, e.g., primate or non-primate lentiviral nucleic acid) comprises: (1) a deleted gag gene, where the deletion in gag removes one or more nucleotides downstream of about nucleotide 350 or 354 of the gag coding sequence; (2) one or more accessory genes not present in the retroviral nucleic acid; (3) lacks the tat gene but includes a leader sequence between the end of the 5' LTR of gag and the ATG; and (4) a combination of (1), (2), and (3). In embodiments, the lentiviral vector comprises all of features (1), (2), and (3). This strategy is described in more detail in WO 99 / 32646, which is incorporated herein by reference in its entirety.

[0128] In some embodiments, the primate lentiviral minimal system does not require any of the additional HIV / SIV genes vif, vpr, vpx, vpu, tat, rev, and nef for vector production or for transduction of dividing and non-dividing cells, hi some embodiments, the EIAV minimal vector system does not require S2 for vector production or for transduction of dividing and non-dividing cells.

[0129] In some embodiments, deletion of additional genes may allow for the generation of vectors without genes associated with disease in lentiviral (e.g., HIV) infection. In some embodiments, tat is associated with disease. In some embodiments, deletion of additional genes allows the vector to package more heterologous DNA. In some embodiments, genes of unknown function, such as S2, may be omitted, thereby reducing the risk of causing unwanted effects. Examples of minimal lentiviral vectors are disclosed in WO99 / 32646 and WO98 / 17815.

[0130] In some embodiments, the retroviral nucleic acid lacks at least tat and S2 (if it is an EIAV vector system), and optionally also vif, vpr, vpx, vpu, and nef. In some embodiments, the retroviral nucleic acid also lacks rev, RRE, or both.

[0131] In some embodiments, the retroviral nucleic acid comprises vpx. The Vpx polypeptide binds to and induces the degradation of the SAMHD1 restriction factor, which degrades free dNTPs in the cytoplasm. In some embodiments, the concentration of free dNTPs in the cytoplasm increases as Vpx degrades SAMHD1 and reverse transcription activity increases, thereby facilitating reverse transcription and integration of the retroviral genome into the target cell genome.

[0132] In some embodiments, different cells differ in their frequency of use of particular codons. In some embodiments, this codon bias corresponds to a bias in the relative abundance of particular tRNAs in a cell type. In some embodiments, expression can be increased by modifying codons in a sequence to match the relative abundance of the corresponding tRNA. In some embodiments, expression can be decreased by deliberately selecting codons whose corresponding tRNAs are known to be rare in a particular cell type. In some embodiments, an additional degree of translational control is possible. Further description of codon optimization can be found, for example, in WO99 / 41397, incorporated herein by reference in its entirety.

[0133] In some embodiments, viruses, including HIV and other lentiviruses, use many rare codons, and by changing these to correspond to commonly used mammalian codons, increased expression of packaging components in mammalian producer cells can be achieved.

[0134] In some embodiments, codon optimization has numerous other advantages. In some embodiments, nucleotide sequences encoding packaging components can have RNA instability sequences (INS) reduced or eliminated therefrom due to modifications in their sequences. At the same time, the amino acid sequences coding for the packaging components are maintained so that the viral components encoded by the sequences remain the same, or at least similar enough, so that the function of the packaging components is not impaired. In some embodiments, codon optimization also overcomes the Rev / RRE requirement for export, rendering the optimized sequences Rev-independent. In some embodiments, codon optimization also reduces homologous recombination between different constructs within a vector system (e.g., between regions of overlap in the gag-pol and env open reading frames). In some embodiments, codon optimization results in increased viral titer and / or improved safety.

[0135] In some embodiments, only the codons for the INS are codon optimized, hi other embodiments, the sequences are codon optimized in their entirety, except for the sequence encompassing the gag-pol frameshift site.

[0136] The gag-pol gene contains two overlapping reading frames encoding gag-pol proteins. Expression of both proteins depends on a frameshift during translation. This frameshift occurs as a result of ribosomal "slippage" during translation. This slippage is thought to be caused, at least in part, by a ribosome-stalling RNA secondary structure. Such a secondary structure exists downstream of the frameshift site in the gag-pol gene. In the case of HIV, the region of overlap extends from 1222 nucleotides downstream of the start of gag (nucleotide 1 is the A of the gag ATG) to the end of gag (nt 1503). Consequently, the 281-bp fragment spanning the frameshift site and the overlapping region of the two reading frames is preferably not codon-optimized. In some embodiments, retaining this fragment allows for more efficient expression of the gag-pol proteins. In the case of EIAV, the start of the overlap is at nt 1262 (nucleotide 1 is the A of the gag ATG). The end of the overlap is at nt 1461. To ensure that the frameshift site and gag-pol overlap are preserved, the wild-type sequence can be retained from nt 1156 to 1465.

[0137] In some embodiments, deviations from optimal codon usage can be made and conservative amino acid changes can be introduced into the gag-pol protein, for example, to provide convenient restriction sites.

[0138] In some embodiments, codon optimization is based on codons with poor codon usage in mammalian systems. The third and sometimes the second and third bases may be changed.

[0139] In some embodiments, due to the degenerate nature of the genetic code, it is understood that numerous gag-pol sequences can be achieved by one skilled in the art. There are also many retroviral variants described that can be used as a stepping stone to generate codon-optimized gag-pol sequences. Lentiviral genomes can be highly variable. For example, there are many quasispecies of HIV-I that are still functional. This is also true for EIAV. These variants can be used to improve specific parts of the transduction process. Examples of HIV-I variants can be found in the HIV database maintained by Los Alamos National Laboratory. Details of EIAV clones can be found in the NCBI database maintained by the National Institutes of Health.

[0140] In some embodiments, the strategy for codon-optimizing gag-pol sequences can be used with any retrovirus, such as EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-1, and HIV-2. This method can also be used to increase expression of genes from HTLV-1, HTLV-2, HFV, HSRV, and human endogenous retroviruses (HERVs), MLV, and other retroviruses.

[0141] In embodiments, retroviral vectors contain a packaging signal comprising 255-360 nucleotides of gag in vectors that still retain the env sequence, or about 40 nucleotides of gag in certain combinations of splice donor mutations, gag, and env deletions. In some embodiments, retroviral vectors contain a gag sequence that contains one or more deletions, e.g., the gag sequence comprises about 360 nucleotides derivable from the N-terminus.

[0142] In some embodiments, the retroviral vector, helper cell, helper virus, or helper plasmid can include retroviral structural and accessory proteins, such as gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef proteins, or other retroviral proteins. In some embodiments, the retroviral proteins are derived from the same retrovirus. In some embodiments, the retroviral proteins are derived from multiple retroviruses, e.g., 2, 3, 4, or more retroviruses.

[0143] In some embodiments, the gag and pol coding sequences are typically organized as a Gag-Pol precursor in native lentiviruses. The gag sequence encodes the 55 kD Gag precursor protein, also called p55. p55 is cleaved by a virally encoded protease (the product of the pol gene) during maturation into four smaller proteins designated MA (matrix [p17]), CA (capsid [p24]), NC (nucleocapsid [p9]), and p6. The pol precursor protein is cleaved from Gag by the virally encoded protease and further digested to separate the protease (p10), RT (p50), RNase H (p15), and integrase (p31) activities.

[0144] In some embodiments, the lentiviral vector is integration-deficient. In some embodiments, the pol vector is integrase-deficient, such as by encoding it with a mutation in the integrase gene. For example, the pol coding sequence can contain an inactivating mutation in integrase, e.g., by mutation of one or more amino acids involved in catalytic activity, i.e., one or more of asparagine 64, aspartic acid 116, and / or glutamic acid 152. In some embodiments, the integrase mutation is a D64V mutation. In some embodiments, the mutation in integrase allows for packaging of viral RNA into lentivirus. In some embodiments, the mutation in integrase allows for packaging of viral proteins into lentivirus. In some embodiments, the mutation in integrase reduces the likelihood of insertional mutagenesis. In some embodiments, the mutation in integrase reduces the likelihood of generating replication-competent recombinants (RCRs) (Wanisch et al. 2009. Mol Ther. 1798):1316-1332). In some embodiments, native Gag-Pol sequences may be utilized in a helper vector (e.g., a helper plasmid or helper virus), or modifications may be made, including chimeric Gag-Pol, in which the Gag and Pol sequences may be derived from different viruses (e.g., different species, subspecies, strains, clades, etc.), and / or in which the sequences have been modified to improve transcription and / or translation and / or to reduce recombination.

[0145] In some embodiments, the retroviral nucleic acid includes a polynucleotide encoding a 150-250 (e.g., 168) nucleotide portion of the gag protein that (i) includes a mutated INS1 inhibitory sequence that reduces restriction of RNA nuclear export compared to wild-type INS1, (ii) contains a two-nucleotide insertion that results in a frameshift and premature termination, and / or (iii) does not include the INS2, INS3, and INS4 inhibitory sequences of gag.

[0146] In some embodiments, the vectors described herein are hybrid vectors that contain both retroviral (e.g., lentiviral) and non-lentiviral viral sequences, hi some embodiments, the hybrid vectors contain retroviral, e.g., lentiviral, sequences for reverse transcription, replication, integration, and / or packaging.

[0147] In some embodiments, most or all of the viral vector backbone sequence is derived from a lentivirus, e.g., HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used or combined, and numerous substitutions and modifications in a given lentiviral sequence can be provided without impairing the ability of the transfer vector to perform the functions described herein. A variety of lentiviral vectors have been described in Naldini et al. (1996a, 1996b, and 1998); Zufferey et al. (1997); Dull et al., 1998, U.S. Patent Nos. 6,013,516; and 5,994,136, many of which can be adapted to generate retroviral nucleic acid.

[0148] In some embodiments, long terminal repeats (LTRs) are typically found at each end of the provirus. LTRs typically contain domains located at the ends of retroviral nucleic acids that are direct repeats in their natural sequence context and contain U3, R, and U5 regions. LTRs usually promote retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs may contain multiple regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences for viral genome replication and integration. Viral LTRs are typically divided into three regions, designated U3, R, and U5. The U3 region typically contains enhancer and promoter elements. The U5 region is typically a sequence between the primer binding site and the R region and may contain a polyadenylation sequence. The R (repeat) region may be flanked by the U3 and U5 regions. LTRs are typically composed of U3, R, and U5 regions and may appear at both the 5' and 3' ends of the viral genome. In some embodiments, the 5'LTR is flanked by sequences for reverse transcription of the genome (tRNA primer binding sites) and for efficient packaging of viral RNA into particles (Psi sites).

[0149] In some embodiments, the packaging signal may comprise a sequence located within the retroviral genome that mediates insertion of viral RNA into the viral capsid or particle, see, e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109. Some retroviral vectors use a minimal packaging signal (psi [Ψ] sequence) for encapsidation of the viral genome.

[0150] In various embodiments, the retroviral nucleic acid comprises a modified 5'LTR and / or 3'LTR. Either or both of the LTRs may contain one or more modifications, including, but not limited to, one or more deletions, insertions, or substitutions. Modification of the 3'LTR is often performed to improve the safety of lentiviral or retroviral systems by rendering the virus replication-deficient (e.g., a virus incapable of complete and efficient replication so that infectious virions are not produced (e.g., replication-deficient lentiviral progeny)).

[0151] In some embodiments, the vector is a self-inactivating (SIN) vector, e.g., a replication-deficient vector, e.g., a retroviral or lentiviral vector, in which the right (3') LTR enhancer-promoter region (known as the U3 region) has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the right (3') LTR U3 region can be used as a template for the left (5') LTR U3 region during viral replication, and thus the absence of the U3 enhancer-promoter inhibits viral replication. In embodiments, the 3' LTR is modified so that the U5 region has been removed, altered, or replaced, for example, with an exogenous poly(A) sequence. The 3' LTR, the 5' LTR, or both the 3' and 5' LTRs can be modified LTRs.

[0152] In some embodiments, the U3 region of the 5' LTR is replaced with a heterologous promoter to induce transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include, for example, the viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. In some embodiments, the promoter can induce high levels of transcription in a Tat-independent manner. In certain embodiments, heterologous promoters have the additional advantage of controlling the manner in which the viral genome is transcribed. For example, the heterologous promoter can be inducible, such that transcription of all or part of the viral genome occurs only in the presence of an inducer. Inducers include, but are not limited to, one or more chemical compounds or physiological conditions, such as temperature or pH, under which the host cells are cultured.

[0153] In some embodiments, the viral vector contains a TAR (transactivation response) element, located, for example, in the R region of the lentiviral (e.g., HIV) LTR. This element interacts with the lentiviral transactivator (tat) gene element to enhance viral replication. However, this element is not required, for example, in embodiments in which the U3 region of the 5' LTR is replaced by a heterologous promoter.

[0154] In some embodiments, the R region, e.g., the region within the retroviral LTR beginning at the start of the capping group (i.e., the start of transcription) and ending just before the start of the polyA tract, can be flanked by the U3 and U5 regions. The R region plays a role during reverse transcription in the transfer of nascent DNA from one end of the genome to the other.

[0155] In some embodiments, the retroviral nucleic acid can also contain a FLAP element, e.g., a nucleic acid whose sequence includes the central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173, incorporated herein by reference in their entireties. During HIV-1 reverse transcription, central initiation of plus-strand DNA at the central polypurine tract (cPPT) and central termination at the central termination sequence (CTS) can result in the formation of a triple-stranded DNA structure: the HIV-1 central DNA flap. In some embodiments, the retroviral or lentiviral vector backbone contains one or more FLAP elements upstream or downstream of the gene encoding the exogenous substance. For example, in some embodiments, a transfer plasmid contains a FLAP element, e.g., a FLAP element derived from or isolated from HIV-1.

[0156] In embodiments, the retroviral or lentiviral nucleic acid comprises one or more export elements, e.g., cis-acting posttranscriptional regulatory elements that control the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, e.g., Cullen et al., 1991. J. Virol. 65:1053; and Cullen et al., 1991. Cell 58:423), and the hepatitis B virus posttranscriptional regulatory element (HPRE), which are incorporated herein by reference in their entirety. Typically, the RNA export element is located within the 3'UTR of a gene and can be inserted in one or more copies.

[0157] In some embodiments, expression of heterologous sequences in viral vectors is increased by incorporating into the vector one or more, e.g., all, of a posttranscriptional regulatory element, a polyadenylation site, and a transcription termination signal. Various posttranscriptional regulatory elements, such as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the posttranscriptional regulatory element (HPRE) present in hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864); and analogs (Liu et al., 1995, Genes Dev., 9:1766) (each of which is incorporated herein by reference in its entirety), can increase expression of heterologous nucleic acids as proteins. In some embodiments, the retroviral nucleic acids described herein include a posttranscriptional regulatory element, e.g., a WPRE or HPRE.

[0158] In some embodiments, the retroviral nucleic acids described herein lack or do not include a post-transcriptional regulatory element, eg, a WPRE or HPRE.

[0159] In some embodiments, elements directing termination and polyadenylation of heterologous nucleic acid transcripts can be included, for example, to increase expression of exogenous material. A transcription termination signal can be found downstream of the polyadenylation signal. In some embodiments, the vector contains a polyadenylation sequence 3' to the polynucleotide encoding the exogenous material. A polyA site can contain a DNA sequence that directs both termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. A polyadenylation sequence can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thereby contributing to increased translation efficiency. Illustrative examples of polyA signals that can be used in retroviral nucleic acids include AATAAA, ATTAAA, AGTAAA, bovine growth hormone polyA sequence (BGHpA), rabbit β-globin polyA sequence (rβgpA), or another suitable heterologous or endogenous polyA sequence.

[0160] In some embodiments, the retroviral or lentiviral vector further comprises one or more insulator elements, eg, an insulator element described herein.

[0161] In various embodiments, the vector comprises a promoter operably linked to a polynucleotide encoding an exogenous substance. The vector may have one or more LTRs, any of which may contain one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. The vector may further comprise one of more accessory elements to increase transduction efficiency (e.g., cPPT / FLAP), viral packaging (e.g., psi (Ψ) packaging signal, RRE), and / or other elements to increase exogenous gene expression (e.g., poly(A) sequence), and may comprise a WPRE or HPRE.

[0162] In some embodiments, the lentiviral nucleic acid includes, e.g., from 5' to 3', one or more, e.g., all, of a promoter (e.g., CMV), an R sequence (e.g., including TAR), a U5 sequence (e.g., for integration), a PBS sequence (e.g., for reverse transcription), a DIS sequence (e.g., for genome dimerization), a Psi packaging signal, a partial gag sequence, an RRE sequence (e.g., for nuclear export), a cPPT sequence (e.g., for nuclear import), a promoter for driving expression of an exogenous substance, a gene encoding the exogenous substance, a WPRE sequence (e.g., for efficient transgene expression), a PPT sequence (e.g., for reverse transcription), an R sequence (e.g., for polyadenylation and termination), and a U5 signal (e.g., for integration).

[0163] Some lentiviral vectors possess strong splicing and polyadenylation signals that can be integrated within active genes, leading to the formation of aberrant, possibly truncated, transcripts.

[0164] The mechanism of proto-oncogene activation may involve the generation of chimeric transcripts resulting from the interaction of promoter elements or splice sites contained in the genome of the insertional mutagen with the cellular transcription unit targeted by integration (Gabriel et al. 2009. Nat Med 15:1431-1436; Bokhoven, et al. J Virol 83:283-29). Chimeric fusion transcripts containing vector sequences and cellular mRNAs can be generated either by read-through transcription, initiating from the vector sequences and proceeding to an adjacent cellular gene, or vice versa.

[0165] In some embodiments, the lentiviral nucleic acids described herein comprise a lentiviral backbone in which at least two of the splice sites have been eliminated, e.g., to improve the safety profile of the lentiviral vector. Species and methods for identifying such splice sites are described in WO2012156839A2, all of which are incorporated by reference.

[0166] 2. Packaging Vector Large-scale vector particle production is often useful to achieve a desired concentration of vector particles. Particles can be produced by transfecting transfer vectors into packaging cell lines containing viral structural and / or accessory genes, such as the gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes or other retroviral genes.

[0167] In some embodiments, the packaging vector is an expression vector or viral vector that lacks a packaging signal and contains a polynucleotide encoding one, two, three, four, or more viral structural and / or accessory genes. Typically, the packaging vector is contained in a producer cell and introduced into the cell via transfection, transduction, or infection. Retroviruses, e.g., lentiviral transfer vectors, can be introduced into producer cell lines via transfection, transduction, or infection to generate source cells or cell lines. The packaging vector can be introduced into human cells or cell lines by standard methods, including, for example, calcium phosphate transfection, lipofection, or electroporation. In some embodiments, the packaging vector is introduced into cells with a dominant selectable marker, e.g., neomycin, hygromycin, puromycin, blasticidin, zeocin, thymidine kinase, DHFR, Gln synthetase, or ADA, followed by selection in the presence of the appropriate drug to isolate clones. The selectable marker gene can be physically linked to the encoding gene by the packaging vector, e.g., by an IRES or a self-cleaving viral peptide.

[0168] In some embodiments, producer cell lines include cell lines that do not contain a packaging signal but that stably or transiently express viral structural proteins and replicative enzymes (e.g., gag, pol, and env) that are capable of packaging viral particles. Any suitable cell line can be used, e.g., mammalian cells, e.g., human cells. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In embodiments, the packaging cells are 293 cells, 293T cells, or A549 cells.

[0169] In some embodiments, source cell lines include producer cell lines and cell lines capable of generating recombinant retroviral particles containing transfer vector constructs containing packaging signals. Methods for preparing viral stock solutions are described, for example, by Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633 and N.R. Landau et al. (1992) J. Virol. 66:5110-5113 (incorporated herein by reference). Infectious viral particles can be collected from producer cells, for example, by cell lysis or collection of cell culture supernatant. The collected viral particles can be concentrated or purified.

[0170] In some embodiments, the source cell contains one or more plasmids encoding viral structural proteins and replication enzymes (e.g., gag, pol, and env) capable of packaging viral particles. In some embodiments, the sequences encoding at least two of the gag, pol, and env precursors are on the same plasmid. In some embodiments, the sequences encoding the gag, pol, and env precursors are on different plasmids. In some embodiments, the sequences encoding the gag, pol, and env precursors have the same expression signal, e.g., promoter. In some embodiments, the sequences encoding the gag, pol, and env precursors have different expression signals, e.g., different promoters. In some embodiments, expression of the gag, pol, and env precursors is inducible. In some embodiments, the plasmids encoding the viral structural proteins and replication enzymes are transfected at the same time or at different times. In some embodiments, the plasmids encoding the viral structural proteins and replication enzymes are transfected at the same time or at different times as the packaging vector.

[0171] In some embodiments, the source cell line comprises one or more stably integrated viral structural genes, hi some embodiments, expression of the stably integrated viral structural genes is inducible.

[0172] In some embodiments, expression of viral structural genes is regulated at the transcriptional level. In some embodiments, expression of viral structural genes is regulated at the translational level. In some embodiments, expression of viral structural genes is regulated at the post-translational level.

[0173] In some embodiments, expression of viral structural genes is controlled by a tetracycline (Tet)-dependent system, in which the Tet-controlled transcriptional repressor (Tet-R) binds to DNA sequences contained in the promoter and represses transcription through steric hindrance (Yao et al., 1998; Jones et al., 2005). Addition of doxycycline (dox) releases Tet-R, allowing transcription. Several other suitable transcriptionally controlled promoters, transcription factors, and small molecule inducers are suitable for controlling transcription of viral structural genes.

[0174] In some embodiments, the third generation lentiviral components human immunodeficiency virus type 1 (HIV) Rev, Gag / Pol, and envelope, under the control of a Tet-regulated promoter and linked to an antibiotic resistance cassette, are separately integrated into the source cell genome, in some embodiments, the source cell has only one copy each of the Rev, Gag / Pol, and envelope proteins integrated into its genome.

[0175] In some embodiments, nucleic acid encoding the exogenous agent (eg, a retroviral nucleic acid encoding the exogenous agent) also integrates into the source cell genome.

[0176] In some embodiments, the retroviral nucleic acid described herein is incapable of undergoing reverse transcription. Such nucleic acids, in embodiments, are capable of transiently expressing exogenous material. The retrovirus or VLP may comprise a disabled reverse transcriptase protein or may not comprise a reverse transcriptase protein. In embodiments, the retroviral nucleic acid comprises a disabled primer binding site (PBS) and / or att site. In embodiments, one or more viral accessory genes, including rev, tat, vif, nef, vpr, vpu, vpx, and S2, or functional equivalents thereof, are disabled or absent from the retroviral nucleic acid. In embodiments, one or more accessory genes selected from S2, rev, and tat are disabled or absent from the retroviral nucleic acid.

[0177] In some embodiments, the retroviral vector systems described herein comprise (1) a viral genome carrying cis-acting vector sequences for transcription, reverse transcription, integration, translation, and packaging of viral RNA into viral particles, and (2) a producer cell line expressing the trans-acting retroviral gene sequences (e.g., gag, pol, and env) required for the generation of viral particles. In some embodiments, complete separation of cis- and trans-acting vector sequences prevents the virus from sustaining replication for multiple cycles of infection. The generation of viable virus can be avoided by several strategies, for example, by minimizing overlap between cis- and trans-acting sequences to avoid recombination.

[0178] In some embodiments, viral vector particles lacking or containing sequences lacking viral RNA may be the result of removing or eliminating viral RNA from that sequence. In one embodiment, this may be achieved by using the endogenous packaging signal binding site on gag. In some embodiments, the endogenous packaging signal binding site is on pol. In this embodiment, the delivered RNA contains the cognate packaging signal. In another embodiment, a heterologous binding domain (heterologous to gag) located on the delivered RNA and a cognate binding site located on gag or pol may be used to ensure packaging of the delivered RNA. In some embodiments, the heterologous sequence may be non-viral, or it may be viral, in which case it may be derived from a different virus. In some embodiments, vector particles are used to deliver therapeutic RNA, in which case functional integrase and / or reverse transcriptase are not required. In some embodiments, vector particles may also be used to deliver a therapeutic gene of interest, in which case pol is typically included.

[0179] In some embodiments, gag-pol is modified, and packaging signal is replaced with corresponding packaging signal.In this embodiment, particle can package RNA with new packaging signal.The advantage of this approach is that it can package RNA sequence that lacks viral sequence, such as RNAi.

[0180] In some embodiments, an alternative approach is to rely on overexpression of the packaged RNA. In one embodiment, the packaged RNA is overexpressed in the absence of any RNA containing a packaging signal. This can result in significant levels of packaged therapeutic RNA, sufficient to transduce cells and have a biological effect.

[0181] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a viral gag protein or a retroviral gag and pol protein, wherein the gag protein or pol protein comprises a heterologous RNA-binding domain capable of recognizing a corresponding sequence in an RNA sequence to facilitate packaging of the RNA sequence into a viral vector particle.

[0182] In some embodiments, the heterologous RNA-binding domain comprises an RNA-binding domain derived from a bacteriophage coat protein, a Rev protein, a protein of the U1 small nuclear ribonucleoprotein particle, a Nova protein, a TFl 11A protein, a TIS11 protein, a trp RNA-binding attenuation protein (TRAP), or a pseudouridine synthase.

[0183] In some embodiments, the methods herein include detecting or confirming the absence of a replication-competent retrovirus. The methods may include assessing the RNA level of one or more target genes, such as viral genes (e.g., structural or packaging genes) whose gene products are expressed in a given cell infected with a replication-competent retrovirus, e.g., a gammaretrovirus or lentivirus, but are not present in the viral vector used to transduce the cell with a heterologous nucleic acid, and which are not present and / or expressed, or are not expected to be present and / or expressed, in cells that do not contain a replication-competent retrovirus. If the RNA level of one or more target genes is higher than a reference value, which can be measured directly or indirectly, for example, from a positive control sample containing the target gene, it can be determined that a replication-competent retrovirus is present. For further disclosure, see WO2018023094A1.

[0184] IV. Fusogen In some embodiments, the viral vector is provided as a fusosome. In some embodiments, the viral vector comprises one or more fusogens. In some embodiments, the fusogens facilitate fusion of the viral vector to a membrane. In some embodiments, the membrane is a plasma cell membrane.

[0185] In some embodiments, the viral vector containing the fusogen (also referred to herein as a "fusosome") is incorporated intramembrane into the lipid bilayer of the target cell. In some embodiments, one or more of the fusogens described herein may be included in the viral vector.

[0186] A. Protein fusogens In some embodiments, the fusogen is a protein fusogen, e.g., a mammalian protein or a homolog of a mammalian protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity), a non-mammalian protein, e.g., a viral protein or a homolog of a viral protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity), a naturally occurring protein or a derivative of a naturally occurring protein, a synthetic protein, a fragment thereof, a variant thereof, a protein fusion or fragment comprising one or more fusogens, and any combination thereof.

[0187] In some embodiments, the fusogen causes intermixing between lipids in the viral vector and lipids in the target cell, hi some embodiments, the fusogen causes the formation of one or more pores between the interior of the viral vector and the cytosol of the target cell.

[0188] 1. Mammalian Proteins In some embodiments, the fusogen can include a mammalian protein. Examples of mammalian fusogens include SNARE family proteins, such as vSNAREs and tSNAREs, syncytin proteins, such as syncytin-1 (DOI: 10.1128 / JVI.76.13.6442-6452.2002), and syncytin-2, myomaker (biorxiv.org / content / early / 2017 / 04 / 02 / 123158, doi.org / 10.1101 / 123158, doi:10.1096 / fj.201600945R, doi:10.10 38 / nature12343), myomixer (www.nature.com / nature / journal / v499 / n7458 / full / nature12343.html, doi:10.1038 / nature12343), myomerger (science.sciencemag.org / content / early / 2017 / 04 / 05 / science.aam9361, DOI:10.1126 / science.aam9361), FGFRL1 (fibroblast proliferation factor receptor-like 1), Minion (doi.org / 10.1101 / 122697), isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (e.g., those disclosed in US 6,099,857A), gap junction proteins, such as connexin 43, connexin 40, connexin 45, connexin 32, or connexin 37 (e.g., those disclosed in US 2007 / 0224176), Hap2, any protein capable of inducing syncytium formation between heterologous cells ( Fusogens may include, but are not limited to, any protein with fusogenic properties (see Table 3), homologs thereof, fragments thereof, variants thereof, and protein fusions comprising one or more proteins or fragments thereof. In some embodiments, the fusogen is encoded by a human endogenous retroviral element (hERV) found in the human genome. Additional exemplary fusogens are disclosed in US 6,099,857A and US 2007 / 0224176 (the entire contents of which are incorporated herein by reference).

[0189] 2. Viral proteins In some embodiments, the fusogen can include a non-mammalian protein, such as a viral protein. In some embodiments, the viral fusogen is a class I viral membrane fusion protein, a class II viral membrane protein, a class III viral membrane fusion protein, a viral membrane glycoprotein, or other viral fusion protein, or a homolog thereof, a fragment thereof, a variant thereof, or a protein fusion comprising one or more proteins or fragments thereof.

[0190] In some embodiments, class I viral membrane fusion proteins include, but are not limited to, baculovirus F proteins, such as F proteins of the genus nuclear polyhedrosis virus (NPV), e.g., Spodoptera exigua MNPV (SeMNPV) F protein and Lymantria dispar MNPV (LdMNPV), and paramyxovirus F proteins.

[0191] In some embodiments, class II viral membrane proteins include, but are not limited to, tick-borne encephalitis E (TBEV E), Semliki Forest virus E1 / E2.

[0192] In some embodiments, class III viral membrane fusion proteins include, but are not limited to, rhabdovirus G (e.g., vesicular stomatitis virus fusion protein G (VSV-G), coccalvirus G protein), herpesvirus glycoprotein B (e.g., herpes simplex virus 1 (HSV-1) gB)), Epstein-Barr virus glycoprotein B (EBV gB), Thogotovirus G, baculovirus gp64 (e.g., Autographa California multiple NPV (AcMNPV) gp64), and Borna disease virus (BDV) glycoprotein (BDV G).

[0193] Examples of other viral fusogens, such as membrane glycoproteins and viral fusion proteins, include viral syncytial proteins such as influenza hemagglutinin (HA) or mutants, or fusion proteins thereof; human immunodeficiency virus type 1 envelope protein (HIV-1 ENV), HIV binding LFA-1-derived gp120 to form lymphocyte syncytia, HIV gp41, HIV gp160, or transactivator of transcription (TAT) of HIV; viral glycoprotein VSV-G, a viral glycoprotein from vesicular stomatitis virus of the Rhabdoviridae family; glycoproteins gB and gH-gL of varicella-zoster virus (VZV); murine leukemia virus (MLV)-10A1; gibbon ape leukemia virus glycoprotein (GaLV); G-type glycoproteins of rabies, Mokola, vesicular stomatitis, and togaviruses; murine hepatitis virus JHM surface spike protein; porcine respiratory coronavirus spike and membrane glycoproteins; avian infectious bronchitis spike glycoprotein and its precursor; bovine enteric coronavirus spike protein; morbilliviruses (e.g., measles virus (MeV), canine distemper virus (CNV)). Examples of proteins that may be used include, but are not limited to, the F and H, HN, or G genes of viruses, cetacean morbillivirus, peste des petits ruminants virus, phocine distemper virus, and rinderpest virus, Newcastle disease virus, human parainfluenza virus 3, simian virus 41, Sendai virus, and human respiratory syncytial virus; gH of human herpesvirus 1 and simian varicella virus, along with the chaperone protein gL; human, bovine, and cercopithecoid herpesvirus gB; envelope glycoproteins of Friend murine leukemia virus and Mason-Pfizer monkey virus; mumps virus hemagglutinin neuraminidase and glycoproteins F1 and F2; membrane glycoproteins from Venezuelan equine encephalomyelitis; paramyxovirus F protein; SIV gp160 protein; Ebola virus G protein; or Sendai virus fusion protein, or homologs, fragments, variants thereof, and protein fusions comprising one or more proteins or fragments thereof.

[0194] Non-mammalian fusogens include viral fusogens, their homologs, fragments thereof, and fusion proteins comprising one or more proteins or fragments thereof. Viral fusogens include class I fusogens, class II fusogens, class III fusogens, and class IV fusogens. In embodiments, class I fusogens, such as human immunodeficiency virus (HIV) gp41, have a characteristic post-fusion conformation with a unique trimer of α-helical hairpins with a central coiled-coil structure. Class I viral fusion proteins include proteins with a central post-fusion six-helix bundle. Class I viral fusion proteins include influenza HA, parainfluenza F, HIV Env, Ebola GP, hemagglutinin from orthomyxoviruses, F proteins from paramyxoviruses (e.g., measles, (Katoh et al. BMC Biotechnology 2010, 10:37)), ENV proteins from retroviruses, and fusogens from filoviruses and coronaviruses. In embodiments, class II viral fusogens, e.g., dengue E glycoprotein, have the structural signature of a β-sheet that forms an elongated ectodomain that refolds to yield a trimer of hairpins. In embodiments, class II viral fusogens lack a central coiled-coil. Class II viral fusogens can be found in alphaviruses (e.g., E1 proteins) and flaviviruses (e.g., E glycoproteins). Class II viral fusogens include fusogens from Semliki Forest virus, Symbiosis virus, rubella virus, and dengue virus. In embodiments, class III viral fusogens, e.g., vesicular stomatitis virus G glycoprotein, combine the structural signatures found in classes I and II. In embodiments, class III viral fusogens resemble class II viral fusogens in that they contain an α-helix (e.g., the protein folds back to form a six-helix bundle, similar to class I viral fusogens) and a β-sheet with an amphipathic fusion peptide at its terminus.Class III viral fusogens can be found in rhabdoviruses and herpesviruses. In embodiments, class IV viral fusogens are fusion-associated small transmembrane (FAST) proteins (doi:10.1038 / sj.emboj.7600767, Nesbitt, Rae L., "Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with Multifunctional FAST proteins" (2012). Electronic Thesis and Dissertation Repository. Paper 388), which are encoded by non-enveloped reoviruses. In embodiments, class IV viral fusogens are small enough not to form hairpins (doi:10.1146 / annurev-cellbio-101512-122422, doi:10.1016 / j.devcel.2007.12.008).

[0195] aG protein In some embodiments, the G protein is a paramyxovirus (e.g., morbillivirus or henipavirus) G protein or a biologically active portion thereof. In some embodiments, the henipavirus G protein is Hendra (HeV) virus G protein, Nipah (NiV) virus G-protein (NiV-G), Cedar (CedPV) virus G-protein, Mojiang virus G-protein, bat paramyxovirus G-protein, or a biologically active portion thereof. A non-limiting list of exemplary G proteins is provided in Table 2.

[0196] The NiV-G-binding protein is a type II transmembrane glycoprotein that includes an N-terminal cytoplasmic tail (e.g., corresponding to amino acids 1-49 of SEQ ID NO: 1), a transmembrane domain (e.g., corresponding to amino acids 50-70 of SEQ ID NO: 1), an extracellular domain containing an extracellular stalk (e.g., corresponding to amino acids 71-187 of SEQ ID NO: 1), and a globular head (e.g., corresponding to amino acids 188-602 of SEQ ID NO: 1). The N-terminal cytoplasmic domain is located within the inner lumen of the lipid bilayer, and the C-terminal portion is the extracellular domain exposed outside the lipid bilayer. The C-terminal region of the stalk (e.g., corresponding to amino acids 159-167 of NiV-G) has been shown to be involved in interaction with the F protein and inducing F protein fusion (Liu et al. 2015 J of Virology 89:1838). In wild-type G proteins, the globular head mediates receptor binding to the henipavirus entry receptors ephrin B2 and ephrin B3 but is dispensable for membrane fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13) e00577-19).

[0197] In certain embodiments herein, the specificity of G protein is modified.The binding of G protein to a binding partner can induce fusion mediated by a compatible F protein or its biologically active portion.The G protein sequences disclosed herein are primarily disclosed as expressed sequences containing the N-terminal methionine required for translation initiation.Since such N-terminal methionine is generally cleaved during or after translation, the mature protein sequences of all G protein sequences disclosed herein are also intended to lack N-terminal methionine.

[0198] The G glycoprotein is highly conserved among henipavirus species. For example, the G proteins of NiV and HeV viruses share 79% amino acid identity. Studies have shown that the G protein is highly compatible with the F proteins of different species, as demonstrated by different types of fusion activation (Brandel-Tretheway et al., Journal of Virology, 2019). As described below, retargeted lipid particles can contain heterologous proteins from different species.

[0199] [Table 2] TIFF2024528981000004.tif204165TIFF2024528981000005.tif110165

[0200] In some embodiments, the G protein has a sequence set forth in any of SEQ ID NOs: 1-11, or has a sequence similar to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 by at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about In some embodiments, the G protein has a sequence set forth in SEQ ID NO: 1, or a functionally active variant or biologically active portion thereof having a sequence that is at least 80%, at least 90%, or about 90%, at least 95%, or about 95%, or at least 99%, or about 99% identical to SEQ ID NO: 1. In some embodiments, the G protein has the sequence set forth in SEQ ID NO: 4, or a functionally active variant or biologically active portion thereof having a sequence that is at least or about 80%, at least 90% or about 90%, at least 95% or about 95%, or at least 99% or about 99% identical to SEQ ID NO: 4. In some embodiments, the G protein has the sequence set forth in SEQ ID NO: 5, or a functionally active variant or biologically active portion thereof having a sequence that is at least or about 80%, at least 90% or about 90%, at least 95% or about 95%, or at least 99% or about 99% identical to SEQ ID NO: 5.

[0201] In certain embodiments, the G protein or functionally active variant or biologically active portion thereof is a protein that retains fusion activity in conjunction with a henipavirus F protein, such as NiV-F or HeV-F. The fusion activity includes the activity of the G protein in conjunction with the henipavirus F protein to promote or facilitate fusion of two membrane spaces, for example, the space of a targeted lipid particle in whose lipid bilayer the henipavirus F and G proteins are embedded, and the cytoplasm of a target cell, for example, a cell containing a surface receptor or molecule recognized or bound by the targeting envelope protein. In some embodiments, the F protein and the G protein are from the same henipavirus species (e.g., NiV-G and NiV-F). In some embodiments, the F protein and the G protein are from different henipavirus species (e.g., NiV-G and HeV-F).

[0202] In certain embodiments, the G protein has the sequence of amino acids set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, or a functionally active variant or biologically active portion thereof that retains fusion activity. In some embodiments, a functionally active variant comprises an amino acid sequence having at least or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, and retains fusion activity in conjunction with a Henipavirus F protein (e.g., NiV-F or HeV-F). In some embodiments, the biologically active portion comprises an amino acid sequence having at least or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11, and retains fusion activity in conjunction with a Henipavirus F protein (e.g., NiV-F or HeV-F).

[0203] Reference to retaining fusion activity includes between or about 10% and 150% or about 150% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, e.g., at least 10% or at least about 10% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 15% or at least about 15% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 20% or at least about 20% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 25% or at least about 25% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 30% or at least about 30% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 35% or at least about 35% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., , at least 40% or at least about 40% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 45% or at least about 45% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 50% or at least about 50% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 55% or at least about 55% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 60% or at least about 60% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 65% or at least about 65% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 70% or at least about 70% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 75% or at least about 75% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 80% or at least about 80% of the level or degree of fusion activity of the corresponding wild-type G protein, for example,This includes activity (in combination with a Henipavirus F protein) that is at least 85% or at least about 85% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 90% or at least about 90% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 95% or at least about 95% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 100% or at least about 100% of the level or degree of fusion activity of the corresponding wild-type G protein, or e.g., at least 120% or at least about 120% of the level or degree of fusion activity of the corresponding wild-type G protein.

[0204] In some embodiments, the G protein is a mutant G protein, which is a functionally active variant or biologically active portion thereof containing one or more amino acid mutations, e.g., one or more amino acid insertions, deletions, substitutions, or truncations. In some embodiments, the mutations described herein relate to amino acid insertions, deletions, substitutions, or truncations of amino acids compared to a reference G protein sequence. In some embodiments, the reference G protein sequence is the wild-type sequence of a G protein or a biologically active portion thereof. In some embodiments, the functionally active variant or biologically active portion thereof is a mutant of the wild-type Hendra (HeV) virus G protein, the wild-type Nipah (NiV) virus G protein (NiV-G), the wild-type Cedar (CedPV) virus G protein, the wild-type Mojiang virus G protein, the wild-type bat paramyxovirus G protein, or a biologically active portion thereof. In some embodiments, the wild-type G protein has the sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0205] In some embodiments, the G protein is a mutant G protein that is a biologically active portion that is an N-terminally and / or C-terminally truncated fragment of the wild-type Hendra (HeV) virus G protein, the wild-type Nipah (NiV) virus G protein (NiV-G), the wild-type Cedar (CedPV) virus G protein, the wild-type Mojiang virus G protein, or the wild-type bat paramyxovirus G protein. In certain embodiments, the truncation is an N-terminal truncation of all or a portion of the cytoplasmic domain. In some embodiments, the mutant G protein is a truncated and biologically active portion that lacks up to 49 consecutive amino acid residues at or near the N-terminus of a wild-type G protein, such as the wild-type G protein set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. In some embodiments, the mutant F protein is truncated and lacks up to 49 consecutive amino acids at the N-terminus of the wild-type G protein, for example, up to 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 30, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 consecutive amino acid(s).

[0206] In some embodiments, the G protein is a wild-type Nipah virus G (NiV-G) protein or a Hendra virus G protein, or a functionally active variant or biologically active portion thereof. In some embodiments, the G protein is a NiV-G protein having a sequence set forth in SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5, or is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or more identical to SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5. or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to the amino acid sequence of the present invention. In some embodiments, the G protein is a NiV-G protein having the sequence set forth in SEQ ID NO: 1, or a functional variant or biologically active portion thereof having an amino acid sequence having at least or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 1.In some embodiments, the G protein is a NiV-G protein having the sequence set forth in SEQ ID NO:1. In some embodiments, the G protein is a NiV-G protein having the sequence set forth in SEQ ID NO: 4, or a functional variant or biologically active portion thereof having an amino acid sequence having at least or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 4. In some embodiments, the G protein is a NiV-G protein having the sequence set forth in SEQ ID NO:4. In some embodiments, the G protein is a NiV-G protein having the sequence set forth in SEQ ID NO: 5, or a functional variant or biologically active portion thereof having an amino acid sequence having at least or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 5. In some embodiments, the G protein is a NiV-G protein having the sequence set forth in SEQ ID NO:5.

[0207] In some embodiments, the G protein is a mutant NiV-G protein that is a biologically active portion of wild-type NiV-G. In some embodiments, the biologically active portion is an N-terminal truncated fragment. In some embodiments, the mutant NiV-G protein is truncated and has up to 5 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 6 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 7 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 8 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 9 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 10 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5). up to 11 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 12 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 13 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 14 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 15 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 16 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 17 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1,up to 18 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 19 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 20 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 21 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 22 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 23 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 24 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 25 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 26 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 27 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 28 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 29 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 30 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 31 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 32 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 33 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5),up to 34 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 35 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 36 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 37 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 38 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), up to 39 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), wild-type The NiV-G protein lacks up to 40 consecutive amino acid residues at or near the N-terminus of the NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 41 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 42 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 43 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), up to 44 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), or up to 45 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5).

[0208] In some embodiments, the mutant NiV-G protein is truncated and lacks 5 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the mutant NiV-G protein is truncated and lacks 5 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:10). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:44. In some embodiments, the mutant NiV-G protein is truncated and lacks 15 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:45. In some embodiments, the mutant NiV-G protein is truncated and lacks 20 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:13. In some embodiments, the mutant NiV-G protein is truncated and lacks 25 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:14. In some embodiments, the mutant NiV-G protein is truncated and lacks 30 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:5). In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:43. In some embodiments, the mutant NiV-G protein is truncated and lacks 34 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 5).In some embodiments, the mutant NiV-G protein comprises the amino acid sequence set forth in SEQ ID NO:42.

[0209] In some embodiments, the NiV-G protein is a biologically active portion that does not contain a cytoplasmic domain. In some embodiments, the NiV-G protein that does not have a cytoplasmic domain is encoded by SEQ ID NO: 22.

[0210] In some embodiments, the mutant NiV-G protein comprises a sequence set forth in any of SEQ ID NOs: 12-14, 17, 18 and 22, or 42-45, or is at least 80% or 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or are functional variants thereof having an amino acid sequence with about 87%, at least 88% or about 88%, or at least 89% or about 89%, about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0211] In some embodiments, the mutant NiV-G protein is as set forth in SEQ ID NO: 12 or a functional variant thereof having at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 12 or SEQ ID NO: 17, or SEQ ID NO: 17. and at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or has a 5 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), as indicated by functional variants thereof having about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.In some embodiments, the mutant NiV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, or at least 92% or about 92%, or at least 93% or about 93%, or at least 94% or about 94%, or at least 95% or about 95%, or at least 96% or about 96%, or at least 97% or about 97%, or at least 98% or about 98%, or at least 99% or about 99%, or at least 100% or about 100%, or at least 101% or about 101%, or at least 102% or about 102%, or at least 103% or about 103%, or at least 104% or about 104%, or at least 105% or about 105%, or at least 106% or about 106%, or at least 107% or about 107%, or at least 108% or about 108%, or at least 109% or about 109%, or at least 110% or about 110%, or at least 111% or about 111%, or at least 112% or about 112%, or at least 113% or about 113%, or at least 114% or about 114%, or at least 115% or about 115%, or at least 116% or about and functional variants thereof having at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), as indicated by functional variants thereof having at or near the N-terminus of 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity. In some embodiments, the mutant NiV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, or at least 92% or about 92%, or at least 93% or about 93%, or at least 94% or about 94%, or at least 95% or about 95%, or at least 96% or about 96%, or at least 97% or about 97%, or at least 98% or about 98%, or at least 99% or about 99%, or at least 100% or about 100%, or at least 101% or about 101%, or at least 102% or about 102%, or at least 103% or about 103%, or at least 104% or about 104%, or at least 105% or about 105%, or at least 106% or about 106%, or at least 107% or about 107%, or at least 108% or about 108%, or at least 109% or about 109%, or at least 110% or about 111%, or at least 112% or about 112%, or at least 113% or about 113%, or at least 114% or about 114%, or at least 115% or about 115%, or at least 116% or about 116%, or at least 117% or about and functional variants thereof having at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), as indicated by functional variants thereof having at or near the N-terminus of 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.In some embodiments, the mutant NiV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, or at least 92% or about 92%, or at least 93% or about 93%, or at least 94% or about 94%, or at least 95% or about 95%, or at least 96% or about 96%, or at least 97% or about 97%, or at least 98% or about 98%, or at least 99% or about 99%, or at least 100% or about 100%, or at least 101% or about 101%, or at least 102% or about 102%, or at least 103% or about 103%, or at least 104% or about 104%, or at least 105% or about 105%, or at least 106% or about 106%, or at least 107% or about 107%, or at least 108% or about 108%, or at least 109% or about 109%, or at least 110% or about 111%, or at least 112% or about 112%, or at least 113% or about 113%, or at least 114% or about 114%, or at least 115% or about 115%, or at least 116% or about 116%, or at least 117% or about and functional variants thereof having at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 5), as indicated by functional variants thereof having at or near the N-terminus of 91%, at or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity. In some embodiments, the mutant NiV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, or at least The wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 5) has a 33 amino acid truncation at or near the N-terminus, as indicated by functional variants thereof having 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.In some embodiments, the mutant NiV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, or at least 92% or about 92%, or at least 93% or about 93%, or at least 94% or about 94%, or at least 95% or about 95%, or at least 96% or about 96%, or at least 97% or about 97%, or at least 98% or about 98%, or at least 99% or about 99%, or at least 100% or about 100%, or at least 101% or about 101%, or at least 102% or about 102%, or at least 103% or about 103%, or at least 104% or about 104%, or at least 105% or about 105%, or at least 106% or about 106%, or at least 107% or about 107%, or at least 108% or about 108%, or at least 109% or about 109%, or at least 110% or about 111%, or at least 112% or about 112%, or at least 113% or about 113%, or at least 114% or about 114%, or at least 115% or about 115%, or at least 116% or about 116%, or at least 117% or about The wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 5) has a 34 amino acid truncation at or near the N-terminus, as indicated by functional variants thereof having 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity. In some embodiments, the mutant NiV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, or at least 92% or about 92%, or at least 93% or about 93%, or at least 94% or about 94%, or at least 95% or about 95%, or at least 96% or about 96%, or at least 97% or about 97%, or at least 98% or about 98%, or at least 99% or about 99%, or at least 100% or about 100%, or at least 101% or about 101%, or at least 102% or about 102%, or at least 103% or about 103%, or at least 104% or about 104%, or at least 105% or about 105%, or at least 106% or about 106%, or at least 107% or about 107%, or at least 108% or about 108%, or at least 109% or about 109%, or at least 110% or about 111%, or at least 112% or about 112%, or at least 113% or about 113%, or at least 114% or about 114%, or at least 115% or about 115%, or at least 116% or about 116%, or at least 117% or about The wild-type NiV-G protein (SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 5) has a 48 amino acid truncation at or near the N-terminus thereof, as indicated by functional variants thereof having 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0212] In some embodiments, the mutant NiV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or has a 15 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), as indicated by functional variants thereof having an amino acid sequence with about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0213] In some embodiments, the mutant NiV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or has a 20 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), as indicated by functional variants thereof having an amino acid sequence with about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0214] In some embodiments, the mutant NiV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or has a 25 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), as indicated by functional variants thereof having an amino acid sequence with about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0215] In some embodiments, the mutant NiV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or has a 30 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), as indicated by functional variants thereof having an amino acid sequence with about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0216] In some embodiments, the mutant NiV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91% identical to SEQ ID NO:42 or SEQ ID NO:42. and functional variants thereof having an amino acid sequence with at least 91% or about 92%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to the wild-type NiV-G protein (SEQ ID NO: 4 or SEQ ID NO: 5).

[0217] In some embodiments, the mutant NiV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or has a 48 amino acid truncation at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:5), as indicated by functional variants thereof having an amino acid sequence with about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0218] In some embodiments, the G protein is a mutant HeV-G protein that is a biologically active portion of wild-type HeV-G. In some embodiments, the biologically active portion is an N-terminal truncated fragment.

[0219] In some embodiments, the G protein is a wild-type HeV-G protein having a sequence set forth in SEQ ID NO: 23 or 24, or a G protein having a sequence similar to SEQ ID NO: 23 or 24 by at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, 88% or about 88%, or at least 89%. and functional variants or biologically active portions thereof having an amino acid sequence with 9% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0220] In some embodiments, the G protein is a mutant HeV-G protein that is a biologically active portion of wild-type HeV-G (SEQ ID NO: 23 or SEQ ID NO: 24). In some embodiments, the biologically active portion is an N-terminally truncated fragment. In some embodiments, the mutant HeV-G protein is truncated and has up to 5 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 6 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 7 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), or up to 8 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 9 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 10 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 11 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 12 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 13 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 14 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 15 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 16 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 17 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 18 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 19 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24),up to 20 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 21 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 22 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 23 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 24 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 25 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 26 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 27 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 28 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 29 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 30 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 31 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 32 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 33 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 34 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 35 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 36 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 37 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24),up to 38 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 39 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 40 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 41 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), wild-type It lacks up to 42 consecutive amino acid residues at or near the N-terminus of the HeV-G protein (SEQ ID NO: 23 or 24), up to 43 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), up to 44 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), or up to 45 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 23 or 24).

[0221] In some embodiments, the HeV-G protein is a biologically active portion that does not contain a cytoplasmic domain. In some embodiments, the mutant HeV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or It lacks the N-terminal cytoplasmic domain of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), as indicated by functional variants thereof having about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity. In some embodiments, the mutant HeV-G protein is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or It lacks the N-terminal cytoplasmic domain of the wild-type HeV-G protein (SEQ ID NO: 23 or 24), as indicated by functional variants thereof having about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity.

[0222] In some embodiments, the G protein, or functionally active variant or biologically active portion thereof, binds to EphrinB2 or EphrinB3. In some aspects, the G protein has the sequence of amino acids set forth in any one of SEQ ID NO:24, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10, or is a functionally active variant or biologically active portion thereof capable of binding to EphrinB2 or EphrinB3. In some embodiments, the functionally active variant or biologically active portion has a similar affinity to any of SEQ ID NO:24, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10 at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, or at least 89%. or at least 99% or about 99% sequence identity, or a functionally active variant or biologically active portion thereof, which retains binding to ephrin B2 or B3.

[0223] In some embodiments, a functionally active variant or biologically active portion has an amino acid sequence having at least about 80%, at least about 85%, at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof, and retains binding to ephrin B2 or B3. Reference to retaining binding to ephrin B2 or B3 includes at least 5% or at least about 5% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof. or a functionally active variant or biologically active portion thereof; 20% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:10; 25% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:10;30% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:8 or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof, 35% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof, 40% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof, 45% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof, SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, 50% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:8 or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof, 55% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof, 60% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof, 65% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof, SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5,70% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:8 or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof, e.g., at least 75% or at least about 75% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof, e.g., at least 80% or at least about 80% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof, e.g., SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: Examples of binding include binding that is at least 85% or at least about 85% of the level or degree of binding of a corresponding wild-type G protein, such as set forth in SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof, e.g., at least 90% or at least about 90% of the level or degree of binding of a corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof, e.g., at least 95% or at least about 95% of the level or degree of binding of a corresponding wild-type G protein, such as set forth in SEQ ID NO:27, SEQ ID NO:23, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10, or a functionally active variant or biologically active portion thereof. In some embodiments, the G protein is NiV-G or a functionally active variant or biologically active portion thereof and binds to ephrinB2 or ephrinB3. In some aspects, the NiV-G has the sequence of amino acids set forth in SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:27, or is a functionally active variant thereof or a biologically active portion thereof that is capable of binding to ephrin B2 or ephrin B3.Functionally active variants or biologically active portions have an amino acid sequence with at least about 80%, at least about 85%, at least or about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:27, and retain binding to ephrin B2 or B3. Exemplary biologically active portions include all or a portion of the cytoplasmic domain, e.g., N-terminal truncation variants lacking one or more, e.g., 1-49 consecutive N-terminal amino acid residues. Reference to retaining binding to ephrin B2 or B3 includes at least 5% or at least about 5% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 10% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 15% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 20% of the level or extent of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 25% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 30% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 35% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 40% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 45% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, 50% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:27, SEQ ID NO:4,55% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 5 or SEQ ID NO: 27, 60% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 27, 65% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 27, 70% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 27, for example, at least 75% or at least about 75% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 27, for example, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 27. The binding includes binding that is at least 80% or at least about 80% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 5 or SEQ ID NO: 27, e.g., at least 85% or at least about 85% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 27, e.g., at least 90% or at least about 90% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 27, or at least 95% or at least about 95% of the level or degree of binding of the corresponding wild-type NiV-G as set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 27.

[0224] In some embodiments, the G protein or biologically active portion thereof is a mutant G protein that exhibits reduced binding to the native binding partner of the wild-type G protein. In some embodiments, the mutant G protein or biologically active portion thereof is a mutant of wild-type Niv-G and exhibits reduced binding to one or both of the native binding partners, ephrin B2 or ephrin B3. In some embodiments, the mutant G protein or biologically active portion thereof, e.g., mutant NiV-G protein, exhibits reduced binding to the native binding partner. In some embodiments, the reduced binding to EphrinB2 or EphrinB3 is at or about 5%, 10% or about 10%, 15% or about 15%, 20% or about 20%, 25% or about 25%, 30% or about 30%, 40% or about 40%, 50% or about 50%, 60% or about 60%, 70% or about 70%, 80% or about 80%, 90% or about 90%, or 100% or more than about 100%.

[0225] In some embodiments, the mutations described herein can improve transduction efficiency. In some embodiments, the mutations described herein enable specific targeting of desired cell types other than EphrinB2 or EphrinB3. In some embodiments, the mutations described herein at least partially disable binding to at least one native receptor, e.g., reduce binding to at least one of EphrinB2 or EphrinB3. In some embodiments, the mutations described herein interfere with native receptor recognition.

[0226] In some embodiments, the G protein is HeV-G, or a functionally active variant or biologically active portion thereof, which binds to ephrin B2 or ephrin B3. In some aspects, HeV-G has the sequence of amino acids set forth in SEQ ID NO: 23 or 24, or a functionally active variant or biologically active portion thereof capable of binding to ephrin B2 or ephrin B3. In some embodiments, the functionally active variant or biologically active portion has an amino acid sequence having at least about 80%, at least about 85%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 23 or 24 and retains binding to ephrin B2 or B3. Exemplary biologically active portions include all or part of the cytoplasmic domain, for example, N-terminal truncation variants lacking one or more, eg, 1-49 consecutive N-terminal amino acid residues. References to retaining binding to ephrin B2 or B3 include at least 5% or at least about 5% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, 10% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, 15% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, 20% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, 25% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, 30% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, 35% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, 40% of the level or extent of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24,45% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, 50% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, 55% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, 60% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, 65% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, 70% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, e.g., For example, at least 75% or at least about 75% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, for example, at least 80% or at least about 80% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, for example, at least 85% or at least about 85% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, for example, at least 90% or at least about 90% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24, or at least 95% or at least about 95% of the level or degree of binding of the corresponding wild-type HeV-G as set forth in SEQ ID NO: 23 or 24.

[0227] In some embodiments, the G protein or biologically active portion thereof is a mutant G protein that exhibits reduced binding to the native binding partner of the wild-type G protein. In some embodiments, the mutant G protein or biologically active portion thereof is a mutant of wild-type Niv-G and exhibits reduced binding to one or both of the native binding partners, ephrin B2 or ephrin B3. In some embodiments, the mutant G protein or biologically active portion thereof, e.g., mutant NiV-G protein, exhibits reduced binding to the native binding partner. In some embodiments, the reduced binding to EphrinB2 or EphrinB3 is at or about 5%, 10% or about 10%, 15% or about 15%, 20% or about 20%, 25% or about 25%, 30% or about 30%, 40% or about 40%, 50% or about 50%, 60% or about 60%, 70% or about 70%, 80% or about 80%, 90% or about 90%, or 100% or more than about 100%.

[0228] In some embodiments, the G protein contains one or more amino acid substitutions at residues involved in interactions with one or both of EphrinB2 and EphrinB3, hi some embodiments, the amino acid substitutions correspond to the mutations E501A, W504A, Q530A, and E533A with reference to the numbering set forth in SEQ ID NO:4.

[0229] In some embodiments, the G protein is a mutant G protein. In some embodiments, the G protein is a mutant G protein containing one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to the numbering set forth in SEQ ID NO: 4. In some embodiments, the G protein is a mutant G protein containing one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to SEQ ID NO: 4, and a biologically active portion thereof containing an N-terminal truncation. In some embodiments, the mutant NiV-G protein or biologically active portion thereof is truncated and includes up to 5 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 6 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 7 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 8 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 9 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), up to 10 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), or 11 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4). 12 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 13 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 14 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), up to 15 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 16 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 17 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 18 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 19 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4),Up to 20 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 21 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 22 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 23 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 24 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), up to 25 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 26 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 27 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 28 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 29 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), up to 30 consecutive amino acid residues, up to 31 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 32 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 33 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 34 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), 35 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), The wild-type NiV-G protein (SEQ ID NO: 4) lacks up to 36 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), up to 37 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), up to 38 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), up to 39 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4), or up to 40 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 4).

[0230] In some embodiments, the mutant NiV-G protein has an amino acid sequence set forth in SEQ ID NO: 17 or 18 or an amino acid sequence having at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 17 or 18. In certain embodiments, the G protein has the amino acid sequence set forth in SEQ ID NO: 17 or 18. In some embodiments, the mutant NiV-G protein has the amino acid sequence set forth in SEQ ID NO: 17 or an amino acid sequence having at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 17. In certain embodiments, the G protein has the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the mutant NiV-G protein has the amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence having at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 18. In certain embodiments, the G protein has the amino acid sequence set forth in SEQ ID NO: 18.

[0231] In some embodiments, the G protein is a mutant G protein containing one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to the numbering set forth in SEQ ID NO: 4. In some embodiments, the G protein is a mutant G protein containing one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to SEQ ID NO: 4, and a biologically active portion thereof containing an N-terminal truncation.

[0232] bF protein In some embodiments, the vector-surface targeting moiety comprises a protein having a hydrophobic fusogenic peptide domain. In some embodiments, the vector-surface targeting moiety comprises a Henipavirus F protein molecule, or a biologically active portion thereof. In some embodiments, the Henipavirus F protein is Hendra (Hev) virus F protein, Nipah (NiV) virus F protein, Cedar (CedPV) virus F protein, Mojiang virus F protein, or a bat paramyxovirus F protein, or a biologically active portion thereof.

[0233] Table 3 provides non-limiting examples of F proteins. In some embodiments, the N-terminal hydrophobic fusion peptide domain of the F protein molecule, or a biologically active portion thereof, is exposed to the outside of the lipid bilayer.

[0234] The F protein of henipaviruses is encoded as an F precursor that includes a signal peptide (e.g., corresponding to amino acid residues 1-26 of SEQ ID NO:28). After cleavage of the signal peptide, the mature F (e.g., SEQ ID NO:29) is transported to the cell surface, then endocytosed and cleaved by cathepsin L to yield the mature fusion subunits F1 and F2. In some embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO:38. In some embodiments, F0 comprises the amino acid sequence set forth in SEQ ID NO:41. In some embodiments, the F1 subunit comprises the amino acid sequence set forth in SEQ ID NO:46. In some embodiments, the F2 subunit comprises the amino acid sequence set forth in SEQ ID NO:39. The F1 and F2 subunits are associated by disulfide bonds and recycled back to the cell surface. The F1 subunit contains a fusion peptide domain located at its N-terminus that can insert into the cell membrane to induce fusion. In some embodiments, fusion is blocked by the association of the F protein with the G protein until the G protein engages the target molecule, resulting in its dissociation from F and exposure of the fusion peptide to mediate membrane fusion.

[0235] The sequence and activity of the F protein are highly conserved among different Henipavirus species. For example, the F proteins of NiV and HeV viruses share 89% amino acid sequence identity. Furthermore, in some cases, Henipavirus F proteins exhibit compatibility with G proteins from other species to induce fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13):e00577-19). In some embodiments or provided retargeted lipid particles, the F protein is heterologous to the G protein, i.e., the F and G proteins or biologically active portions are from different Henipavirus species. For example, the F protein is from Hendra virus and the G protein is from Nipah virus. In other aspects, the F protein can be a chimeric F protein containing regions of F proteins from different Henipavirus species. In some embodiments, exchanging a region of amino acid residues of the F protein from one Henipavirus species with another can result in fusion to the G protein of the species containing the amino acid insertion. (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13):e00577-19). In some cases, the chimeric F protein contains an extracellular domain from one Henipavirus species and a transmembrane and / or cytoplasmic domain from a different Henipavirus species. For example, the F protein contains the extracellular domain of Hendra virus and the transmembrane / cytoplasmic domain of Nipah virus. The F protein sequences disclosed herein are primarily disclosed as expressed sequences that include an N-terminal signal sequence. Because such N-terminal signal sequences are generally cleaved co- or post-translationally, the mature protein sequences for all F protein sequences disclosed herein are also contemplated as lacking an N-terminal signal sequence.

[0236] [Table 3] TIFF2024528981000007.tif168165TIFF2024528981000008.tif100165

[0237] In some embodiments, the F protein is encoded by a nucleotide sequence encoding the sequence set forth in any one of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, or a functionally active variant or biologically active portion thereof having a sequence that is at least 80% or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% identical to any one of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the F protein is encoded by a nucleotide sequence that encodes a sequence set forth in any one of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.

[0238] In certain embodiments, the F protein, or a functionally active variant or biologically active portion thereof, retains fusion activity in conjunction with a henipavirus G protein, e.g., a G protein described in Section IV.A.2 (e.g., NiV-G or HeV-G). Fusion activity includes the activity of the F protein in conjunction with the G protein to promote or facilitate fusion of two membrane spaces, e.g., the space of a targeted lipid particle in whose lipid bilayer the henipavirus F and G proteins are embedded, and the cytoplasm of a target cell, e.g., a cell containing a surface receptor or molecule recognized or bound by the targeting envelope protein. In some embodiments, the F protein and the G protein are from the same henipavirus species (e.g., NiV-G and NiV-F). In some embodiments, the F protein and the G protein are from different henipavirus species (e.g., NiV-G and HeV-F). In certain embodiments, the F protein, or a functionally active variant or biologically active portion thereof, retains a cleavage site that is cleaved by cathepsin L (e.g., corresponding to the cleavage site between amino acids 109 and 110 of SEQ ID NO: 30).

[0239] In certain embodiments, the F protein has the sequence of amino acids set forth in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, or a functionally active variant thereof or a biologically active portion thereof that retains fusion activity. In some embodiments, a functionally active variant comprises an amino acid sequence having at least or about 80%, at least 85% or about 85%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, and retains fusion activity in conjunction with a Henipavirus G protein (e.g., NiV-G or HeV-G). In some embodiments, the biologically active portion comprises an amino acid sequence having at least or about 80%, at least 85%, or about 85%, at least 90%, or about 90%, at least 91%, or about 91%, at least 92%, or about 92%, at least 93%, or about 93%, at least 94%, or about 94%, at least 95%, or about 95%, 96%, or about 96%, at least 97%, or about 97%, at least 98%, or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.

[0240] Reference to retaining fusion activity includes 10% or about 10% to 150% or about 150% or more of the level or degree of binding of the corresponding wild-type F protein as set forth in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37, e.g., at least 10% or at least about 10% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 15% or at least about 15% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 20% or at least about 20% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 25% or at least about 25% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 30% or at least about 30% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 35% or at least about 35% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., For example, at least 40% or at least about 40% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 45% or at least about 45% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 50% or at least about 50% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 55% or at least about 55% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 60% or at least about 60% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 65% or at least about 65% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 70% or at least about 70% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 75% or at least about 75% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 80% or at least about 80% of the level or degree of fusion activity of the corresponding wild-type F protein, for example,The activity includes an activity (in combination with the Nipah virus G protein) that is at least 85% or at least about 85% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 90% or at least about 90% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 95% or at least about 95% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 100% or at least about 100% of the level or degree of fusion activity of the corresponding wild-type F protein, or e.g., at least 120% or at least about 120% of the level or degree of fusion activity of the corresponding wild-type F protein.

[0241] In some embodiments, the F protein is a mutant F protein, which is a functionally active fragment or biologically active portion thereof, containing one or more amino acid mutations, e.g., one or more amino acid insertions, deletions, substitutions, or truncations. In some embodiments, the mutations described herein relate to amino acid insertions, deletions, substitutions, or truncations of amino acids compared to a reference F protein sequence. In some embodiments, the reference F protein sequence is a wild-type sequence of an F protein or a biologically active portion thereof. In some embodiments, the mutant F protein or a biologically active portion thereof is a mutant of a wild-type Hendra (Hev) virus F protein, Nipah (NiV) virus F protein, Cedar (CedPV) virus F protein, Mojiang virus F protein, or bat paramyxovirus F protein. In some embodiments, the wild-type F protein is encoded by a sequence of nucleotides encoding any one of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.

[0242] In some embodiments, the mutant F protein is a biologically active portion of a wild-type F protein that is an N-terminally and / or C-terminally truncated fragment. In some embodiments, the mutant F protein or biologically active portion thereof comprises one or more amino acid substitutions. In some embodiments, the mutations described herein may improve transduction efficiency. In some embodiments, the mutations described herein may increase fusion capability. Exemplary mutations include any of those described, e.g., Khetawat and Broder 2010 Virology Journal 7:312; Witting et al. 2013 Gene Therapy 20:997-1005; published international patent application WO / 2013 / 148327.

[0243] In some embodiments, the mutant F protein is a biologically active portion that is truncated and lacks up to 20 consecutive amino acid residues at or near the C-terminus of a wild-type F protein, such as a wild-type F protein encoded by a sequence of nucleotides encoding the F protein set forth in any one of SEQ ID NOS: 28-37. In some embodiments, the mutant F protein is truncated and lacks up to 20 consecutive amino acids, e.g., up to 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 consecutive amino acid, at the C-terminus of the wild-type F protein. In some embodiments, the mutant F protein comprises the sequence set forth in SEQ ID NO: 15. In some embodiments, the mutant F protein comprises the sequence set forth in SEQ ID NO: 20. In some embodiments, the mutant F protein is truncated and lacks up to 19 consecutive amino acids at the C-terminus of the wild-type F protein, for example, up to 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 consecutive amino acid.

[0244] In some embodiments, the F protein, or functionally active variant or biologically active portion thereof, comprises an F1 subunit or a fusion portion thereof. In some embodiments, the F1 subunit is a proteolytically cleaved portion of an F0 precursor. In some embodiments, the F0 precursor is inactive. In some embodiments, cleavage of the F0 precursor forms a disulfide-linked F1+F2 heterodimer. In some embodiments, cleavage exposes the fusion peptide and generates a mature F protein. In some embodiments, cleavage occurs at or around a single basic residue. In some embodiments, cleavage occurs at arginine 109 of the NiV-F protein. In some embodiments, cleavage occurs at lysine 109 of the Hendra virus F protein.

[0245] In some embodiments, the F protein is a wild-type Nipah virus F (NiV-F) protein, or a functionally active variant or biologically active portion thereof. In some embodiments, the F precursor is encoded by a sequence of nucleotides that encodes the sequence set forth in SEQ ID NO:20. The encoding nucleic acid may encode a signal peptide sequence having the sequence MVVILDKRCY CNLLILILMI SECSVG (SEQ ID NO:38). In some examples, the F protein is cleaved into an F1 subunit comprising the sequence set forth in SEQ ID NO:46 and an F2 subunit comprising the sequence set forth in SEQ ID NO:39.

[0246] In some embodiments, the F protein is a NiV-F protein encoded by a sequence of nucleotides that encodes the sequence set forth in SEQ ID NO: 30, or a protein that is at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, or or a functionally active variant or biologically active portion thereof having an amino acid sequence with at least or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity. In some embodiments, the F protein is a NiV-F protein encoded by a sequence of nucleotides encoding the sequence set forth in SEQ ID NO:30. In some embodiments, the NiV-F-protein has the sequence shown in 30, or is a functionally active variant or biologically active portion thereof having an amino acid sequence having at least or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to 30. In some embodiments, the NiV-F-protein has the sequence shown in 30.In certain embodiments, the F protein, or a functionally active variant or biologically active portion thereof, retains a cleavage site that is cleaved by cathepsin L.

[0247] In some embodiments, the F protein or functionally active variant or biologically active portion thereof includes an F1 subunit having a sequence set forth in SEQ ID NO:46, or an amino acid sequence having at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:46.

[0248] In some embodiments, the F protein or functionally active variant or biologically active portion thereof includes an F2 subunit having a sequence set forth in SEQ ID NO:39, or an amino acid sequence having at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:39.

[0249] In some embodiments, the F protein or functionally active variant or biologically active portion thereof includes a Fl subunit having a sequence set forth in SEQ ID NO:46, or an amino acid sequence having at least or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:46.

[0250] In some embodiments, the F protein or functionally active variant or biologically active portion thereof includes an F2 subunit having a sequence set forth in SEQ ID NO:39, or an amino acid sequence having at least or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or about 84%, at least 85% or about 85%, 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, or at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:39.

[0251] In some embodiments, the F protein is a mutant NiV-F protein that is a truncated and biologically active portion of a wild-type NiV-F protein (e.g., as set forth in SEQ ID NO:40) lacking up to 20 consecutive amino acid residues at or near the C-terminus thereof. In some embodiments, the mutant NiV-F protein comprises the amino acid sequence set forth in SEQ ID NO:20. In some embodiments, the mutant NiV-F protein has a sequence having at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:20. In some embodiments, the mutant F protein contains an F1 protein having the sequence set forth in SEQ ID NO:46. In some embodiments, the mutant F protein has a sequence having at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:46.

[0252] In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof having a 20 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 40); and a point mutation in the N-linked glycosylation site. In some embodiments, the mutant NiV-F protein comprises the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the mutant NiV-F protein has a sequence having at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 15.

[0253] In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof having a 25 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 40). In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof having a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 40). In some embodiments, the NiV-F protein is encoded by a nucleotide sequence that encodes the sequence set forth in SEQ ID NO: 20. In some embodiments, the NiV-F protein is encoded by a nucleotide sequence that encodes a sequence having at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 20.

[0254] In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof having a 22 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO:40). In some embodiments, the NiV-F protein comprises the amino acid sequence set forth in SEQ ID NO:21, or an amino acid sequence having at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:21. In some embodiments, the NiV-F protein is encoded by a nucleotide sequence that encodes the sequence set forth in SEQ ID NO:21. In some embodiments, the NiV-F protein is encoded by a nucleotide sequence that encodes a sequence having at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO:21.

[0255] B.CD4 binding substance In some embodiments, the viral vectors described herein are retargeted by a binding agent (e.g., a CD4 binding agent). For example, in some cases, the viral vector includes a fusogen to facilitate fusion of the viral vector to a membrane, and the fusogen is modified to include a CD4 binding agent to retarget the viral vector. In some cases, the fusogen includes the Nipah virus F glycoprotein (NiV-F), or a biologically active portion thereof, and the Nipah virus G glycoprotein (NiV-G), or a biologically active portion thereof. In some embodiments, the CD4 binding agent is fused to the NiV-G protein. Thus, in some cases, the viral vector is retargeted by including a retargeting fusogen including NiV-G fused to a CD4 binding agent.

[0256] The viral vectors disclosed herein include one or more CD4 binding substances. For example, the CD4 binding substance can be fused to or incorporated into a fusogen protein or a viral envelope protein. In another embodiment, the CD4 binding substance can be incorporated into the viral envelope via fusion with a transmembrane domain.

[0257] Exemplary CD4 binding agents include antibodies and fragments thereof (e.g., scFv, VHH) that bind to CD4. Such antibodies can be from any species, for example, murine, rabbit, human, humanized, or camelid antibodies. Exemplary antibodies include ibalizumab, zanolimumab, tregalizumab, priliximab, cedelizumab, clenoliximab, keliximab, and antibodies described in WO2002102853, WO2004083247, WO2004067554, WO2007109052, WO2008134046, WO2010074266, WO2012113348, WO2013188870, WO2017104735, WO2018035001, WO2018170096, WO2 anti-CD4 antibodies disclosed in US Provisional Application No. 63 / 326,269, US Provisional Application No. 63 / 341,681; and antibodies B486A1, RPA-T4, CE9.1 (Novus GK1.5, RM4-5, RPA-T4, OKT4, 4SM95, S3.5, N1UG0 (ThermoFisher); GTX50984, ST0488, 10B5, EP204 (GeneTex); GK1.3, 5A8, 10C12, W3 / 25, 8A5, 13B8.2, 6G5 (Absolute Antibody); VIT4, M-T466, M-T321, REA623, (Miltenyi); MEM115, MT310 (Enzo Life Sciences); H129.19, 5B4, 6A17, 18-46, A-1, C-1, OX68 (Santa Cruz); EP204, D2E6M (Cell Signaling Technology). Other exemplary binding agents include designed ankyrin repeat proteins (DARPins) (e.g., the anti-CD4 DARPin disclosed in WO2017182585) and binding agents based on the fibronectin type III (Fn3) scaffold.Each of US 9,005,963, US Provisional Application No. 63 / 326,269, and US Provisional Application No. 63 / 341,681 is incorporated herein by reference in its entirety.

[0258] In some embodiments, a protein fusogen or viral envelope protein can be retargeted by mutating amino acid residues in the fusion protein or targeting protein (e.g., hemagglutinin (H) protein or G protein). In certain embodiments, the fusogen (e.g., G protein) is mutated to reduce binding of the fusogen to its native binding partner. In some embodiments, the fusogen is or comprises a mutant G protein, or a biologically active portion thereof, that is a mutant of wild-type Niv-G and exhibits reduced binding to one or both of its native binding partners, ephrin B2 or ephrin B3, including any of those described above. Thus, in some aspects, a fusogen can be retargeted to exhibit altered tropism. In some embodiments, binding confers retargeted binding relative to binding of the wild-type surface glycoprotein, conferring new or different binding activity. In certain embodiments, binding confers retargeted binding relative to binding of the wild-type G protein, conferring new or different binding activity. In some embodiments, the fusogen is randomly mutated. In some embodiments, the fusogen is rationally mutated. In some embodiments, the fusogen is subjected to directed evolution. In some embodiments, the fusogen is truncated, and only a subset of the peptides is used in the viral vector. In some embodiments, amino acid residues in the measles hemagglutinin protein can be mutated to alter the binding properties of the protein and redirect fusion (doi:10.1038 / nbt942, Molecular Therapy vol.16 no.8, 1427-1436 Aug.2008, doi:10.1038 / nbt1060, DOI:10.1128 / JVI.76.7.3558-3563.2002, DOI:10.1128 / JVI.75.17.8016-8020.2001, doi:10.1073pnas.0604993103).

[0259] In some embodiments, a protein fusogen can be retargeted by covalently conjugating a CD4 binding agent to a fusion protein or targeting protein (e.g., a hemagglutinin protein). In some embodiments, the fusogen and CD4 binding agent are covalently conjugated by expression of a chimeric protein comprising the fusogen linked to the CD4 binding agent. In some embodiments, single-chain variable fragments (scFvs) can be conjugated to fusogens to redirect fusion activity to cells that present scFv-binding targets (doi:10.1038 / nbt1060, DOI 10.1182 / blood-2012-11-468579, doi:10.1038 / nmeth.1514, doi:10.1006 / mthe.2002.0550, HUMAN GENE THERAPY 11:817-826, doi:10.1038 / nbt942, doi:10.1371 / journal.pone.0026381, DOI 10.1186 / s12896-015-0142-z). In some embodiments, engineered ankyrin repeat proteins (DARPins) can be conjugated to fusogens to redirect fusion activity to cells presenting DARPin-binding targets (doi:10.1038 / mt.2013.16, doi:10.1038 / mt.2010.298, doi:10.4049 / jimmunol.1500956), and combinations of different DARPins (doi:10.1038 / mto.2016.3). In some embodiments, receptor ligands and antigens can be conjugated to fusogens to redirect fusion activity to cells presenting target receptors (DOI:10.1089 / hgtb.2012.054, DOI:10.1128 / JVI.76.7.3558-3563.2002).In some embodiments, targeting proteins can also include antibodies or antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single-domain antibodies such as sdAbs (either VL or VH), nanobodies, or camelid VHH domains), antigen-binding fibronectin type III (Fn3) scaffolds, e.g., fibronectin polypeptide minibodies, ligands, cytokines, chemokines, or T cell receptors (TCRs). In some embodiments, VHH domains can be conjugated to fusogens to redirect fusion activity to cells presenting VHH-binding targets. In some embodiments, protein fusogens can be retargeted by non-covalently conjugating a CD4-binding agent to the fusion protein or targeting protein (e.g., hemagglutinin protein). In some embodiments, fusion proteins can be engineered to bind the Fc region of an antibody that targets an antigen on a target cell, redirecting fusion activity to cells that present the antibody's target (DOI:10.1128 / JVI.75.17.8016-8020.2001, doi:10.1038 / nm1192). In some embodiments, modified and unmodified fusogens can be displayed on the same retroviral vector or VLP (doi:10.1016 / j.biomaterials.2014.01.051).

[0260] In some embodiments, the CD4 binding agent is a humanized antibody molecule, an intact IgA, IgG, IgE, or IgM antibody; a bi- or multispecific antibody (e.g., Zybodies®, etc.); an antibody fragment, e.g., a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fd' fragment, an Fd fragment, and an isolated CDR or set thereof; a single chain Fvs; a polypeptide-Fc fusion; a single domain antibody (e.g., a shark single domain antibody, e.g., an IgNAR or a fragment thereof); a camelid antibody; a masked antibody (e.g., Probodies®); small modular immunopharmaceuticals ("SMIPs™"); a single chain or tagged antibody. These include transduction diabodies (TandAb®); VHHs; Anticalins®; Nanobodies®; minibodies; BiTEs®; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®.

[0261] In some embodiments, the CD4 binding agent is a peptide.

[0262] In some embodiments, the CD4 binding agent is an antibody, eg, a single chain variable fragment (scFv).

[0263] In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 149, 150, and 151, respectively. In some embodiments, a CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 152, 153, and 154, respectively. In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 149, 150, and 151, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 152, 153, and 154, respectively. In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 207, 208, and 209, respectively. In some embodiments, the CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 210, 211, and 154, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 207, 208, and 209, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 210, 211, and 154, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 212, 213, and 209, respectively. In some embodiments, the CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 210, 211, and 154, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 212, 213, and 209, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 210, 211, and 154, respectively. In some embodiments, the CD4 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 155.In some embodiments, the CD4 binding agent comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 156. In some embodiments, the CD4 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 155; and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 156. In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the CD4 binding agent comprises the amino acid sequence set forth in SEQ ID NO: 157.

[0264] In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 158, 159, and 160, respectively. In some embodiments, a CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 161, 162, and 163, respectively. In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 158, 159, and 160, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 161, 162, and 163, respectively. In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 214, 215, and 216, respectively. In some embodiments, the CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 217, 218, and 163, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 214, 215, and 216, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 217, 218, and 163, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 219, 220, and 216, respectively. In some embodiments, the CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 217, 218, and 163, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 219, 220, and 216, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 217, 218, and 163, respectively. In some embodiments, the CD4 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 164.In some embodiments, the CD4 binding agent comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 165. In some embodiments, the CD4 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 164; and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 165. In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the CD4 binding agent comprises the amino acid sequence set forth in SEQ ID NO: 166.

[0265] In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 167, 168, and 169, respectively. In some embodiments, a CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 170, 171, and 172, respectively. In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 167, 168, and 169, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 170, 171, and 172, respectively. In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 221, 222, and 223, respectively. In some embodiments, the CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 224, 225, and 172, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 221, 222, and 223, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 224, 225, and 172, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 226, 227, and 223, respectively. In some embodiments, the CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 224, 225, and 172, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 226, 227, and 223, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 224, 225, and 172, respectively. In some embodiments, the CD4 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 173.In some embodiments, the CD4 binding agent comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 174. In some embodiments, the CD4 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 173; and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 174. In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the CD4 binding agent comprises the amino acid sequence set forth in SEQ ID NO: 175.

[0266] In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 176, 177, and 178, respectively. In some embodiments, a CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 179, 180, and 181, respectively. In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 176, 177, and 178, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 179, 180, and 181, respectively. In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 228, 229, and 230, respectively. In some embodiments, the CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 231, 232, and 181, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 228, 229, 230, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 231, 232, and 181, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 233, 234, 230, respectively. In some embodiments, the CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 231, 232, and 181, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 233, 234, and 230, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 231, 232, and 181, respectively. In some embodiments, the CD4 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 182.In some embodiments, the CD4 binding agent comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 183. In some embodiments, the CD4 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 182; and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 183. In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the CD4 binding agent comprises the amino acid sequence set forth in SEQ ID NO: 184.

[0267] In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 185, 186, and 187, respectively. In some embodiments, a CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 188, 171, and 189, respectively. In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 185, 186, and 187, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 188, 171, and 189, respectively. In some embodiments, a CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 235, 236, and 237, respectively. In some embodiments, the CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 238, 239, and 189, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 235, 236, and 237, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 238, 239, and 189, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 240, 241, and 237, respectively. In some embodiments, the CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 238, 239, and 189, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 240, 241, and 237, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 238, 239, and 189, respectively. In some embodiments, the CD4 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 190.In some embodiments, the CD4 binding agent comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 191. In some embodiments, the CD4 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 190; and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 191. In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the CD4 binding agent comprises the amino acid sequence set forth in SEQ ID NO: 192.

[0268] In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 193, 194, and 195, respectively. In some embodiments, the CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 196, 197, and 198, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 193, 194, and 195, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 196, 197, and 198, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 242, 243, and 244, respectively. In some embodiments, the CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 245, 246, and 198, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 242, 243, and 244, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 245, 246, and 198, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 247, 248, and 244, respectively. In some embodiments, the CD4 binding agent comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 245, 246, and 198, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 247, 248, and 244, respectively; and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 245, 246, and 198, respectively. In some embodiments, the CD4 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 199.In some embodiments, the CD4 binding agent comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 200. In some embodiments, the CD4 binding agent comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 199; and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 200. In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 143. In some embodiments, the CD4 binding agent comprises the amino acid sequence set forth in SEQ ID NO: 201.

[0269] In some embodiments, the CD4 binding agent is an antibody, e.g., a single domain antibody. In some embodiments, the antibody can be human or humanized. In some embodiments, the CD4 binding agent is a VHH. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 145, 146, and 147, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 202, 203, and 204, respectively. In some embodiments, the CD4 binding agent comprises CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 205, 206, and 204, respectively. In some embodiments, the CD4 binding agent comprises the amino acid sequence set forth in SEQ ID NO: 148.

[0270] In some embodiments, the antibody or portion thereof is naturally occurring. In some embodiments, the antibody or portion thereof is synthetic.

[0271] In some embodiments, antibodies can be generated from phage display libraries to have specificity for desired target ligands. In some embodiments, phage display libraries are generated from VHH repertoires of camelids immunized with various antigens, as described in Arbabi et al., FEBS Letters, 414, 521-526 (1997); Lauwereys et al., EMBO J., 17, 3512-3520 (1998); Decaniere et al., Structure, 7, 361-370 (1999). In some embodiments, phage display libraries are generated containing antibody fragments from non-immunized camelids. In some embodiments, libraries of human single-domain antibodies are synthetically generated by introducing diversity into one or more scaffolds.

[0272] In some embodiments, the C-terminus of the CD4 binding agent is linked to the C-terminus of a G protein (e.g., a fusogen) or a biologically active portion thereof. In some embodiments, the N-terminus of the CD4 binding agent is exposed to the outer surface of the lipid bilayer.

[0273] In some embodiments, the CD4 binding agent is a non-viral sequence displayed only on the surface of the viral vector. In some embodiments, the CD4 binding agent is the only membrane-bound non-viral sequence of the viral vector. In some embodiments, the viral vector does not contain any molecules that engage or stimulate T cells other than the CD4 binding agent.

[0274] In some embodiments, viral vectors may present CD4-binding agents that are not conjugated to protein fusogens to redirect fusion activity to cells bound by the targeting moiety or to affect homing.

[0275] In some embodiments, protein fusogens derived from viruses or organisms that do not infect humans have no natural fusion targets in patients and therefore have high specificity.

[0276] V. Engineered Receptor Payloads In some embodiments, the viral vectors disclosed herein encode an engineered receptor. In some embodiments, cells for use in or administered in connection with the provided methods contain, or are engineered to contain, an engineered receptor, e.g., an engineered antigen receptor, e.g., a chimeric antigen receptor (CAR). Also provided are populations of such cells, compositions containing such cells and / or enriched for such cells (e.g., enriched or selected for a given type of cell, e.g., T cells or CD4+ cells). Compositions include pharmaceutical compositions and formulations for administration, e.g., adoptive cell therapy. Also provided are therapeutic methods for administering cells and compositions to a subject, e.g., a patient, according to the provided methods and / or using the provided articles of manufacture or compositions.

[0277] In some embodiments, gene transfer is achieved without first stimulating the cells, e.g., by combining a stimulus that induces a response such as proliferation, survival, and / or activation, as measured by expression of cytokines or activation markers, followed by introducing a nucleic acid into the stimulated cells, e.g., by transduction, and optionally incubating or growing in culture to sufficient numbers for clinical application.

[0278] Viral vectors can express recombinant receptors, such as antigen receptors, including chimeric antigen receptors (CARs), and other antigen-binding receptors, such as transgenic T cell receptors (TCRs), and other chimeric receptors.

[0279] A. Chimeric Antigen Receptor (CAR) In some embodiments of the provided methods and uses, the chimeric receptor, e.g., chimeric antigen receptor, contains one or more domains that combine an antigen or ligand binding domain (e.g., an antibody or antibody fragment) that provides specificity for a desired antigen (e.g., a tumor antigen) with an intracellular signaling domain. In some embodiments, the intracellular signaling domain is a stimulatory or activation intracellular domain portion, e.g., a T cell stimulatory or activation domain (depending on a primary activation signal or primary signal). In some embodiments, the intracellular signaling domain contains, or additionally contains, a costimulatory signaling domain to facilitate effector function. In some embodiments, the chimeric receptor, when genetically engineered into an immune cell, can regulate T cell activity, and in some cases, regulate T cell differentiation or homeostasis, thereby resulting in genetically engineered cells with improved longevity, survival, and / or persistence in vivo, e.g., for use in adoptive cell therapy.

[0280] Exemplary antigen receptors, including CARs, and methods of engineering and introducing such receptors into cells are described, for example, in WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US20130149 337, U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP2537416, and / or Sadelain et al., Cancer Discov. 2013 April;3(4):388-398; Davila et al. (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr. Opin. Immunol., 2012 October;24(5):633-39; Wu et al., Cancer, 2012 March 18(2):160-75. In some embodiments, antigen receptors include CARs described in U.S. Patent No. 7,446,190 and those described in WO / 2014055668. Examples of CARs include those disclosed in any of the publications mentioned above, e.g., WO2014031687, US8,339,645, US7,446,179, US2013 / 0149337, US7,446,190, US8,389,282, Kochenderfer et al., (2013) Nature Reviews Clinical Oncology, 10, 267-276; Wang et al. (2012) J. Immunother. 35(9):689-701; and Brentjens et al., Sci Transl Med. 2013 5(177).See also WO2014031687, US8,339,645, US7,446,179, US2013 / 0149337, US7,446,190, and US8,389,282. Recombinant receptors, e.g., CARs, typically include an extracellular antigen-binding domain, e.g., a portion of an antibody molecule, typically the variable heavy (VH) and / or variable light (VL) chain regions of an antibody, e.g., an scFv antibody fragment. In some embodiments, the antigen-binding domain of the CAR molecule comprises an antibody, antibody fragment, scFv, Fv, Fab, (Fab')2, single-domain antibody (SdAb), VH or VL domain, or camelid VHH domain.

[0281] In some embodiments, the CAR antigen binding domain is or comprises an antibody or an antigen-binding portion thereof. In some embodiments, the CAR antigen binding domain is or comprises an scFv or Fab. In some embodiments, the CAR antigen binding domain is or comprises a CD19 antibody; a CD22 antibody; a T cell alpha chain antibody; a T cell beta chain antibody; a T cell gamma chain antibody; a T cell delta chain antibody; a CCR7 antibody; a CD3 antibody; a CD4 antibody; a CD5 antibody; a CD7 antibody; a CD8 antibody; a CD11b antibody; a CD11c antibody; a CD16 antibody; a CD20 antibody; a CD21 antibody; a CD25 antibody; a CD28 antibody; a CD34 antibody; a CD35 antibody; a CD40 antibody; a CD45RA antibody; a CD45RO antibody; a CD5 2 antibody; CD56 antibody; CD62L antibody; CD68 antibody; CD80 antibody; CD95 antibody; CD117 antibody; CD127 antibody; CD133 antibody; CD137 (4-1BB) antibody; CD163 antibody; F4 / 80 antibody; IL-4Ra antibody; Sca-1 antibody; CTLA-4 antibody; GITR antibody, GARP antibody; LAP antibody; Granzyme B antibody; LFA-1 antibody; MR1 antibody; uPAR antibody; or scFv or Fab fragments of transferrin receptor antibody.

[0282] In some embodiments, the CAR comprises a signaling domain that is a costimulatory domain. In some embodiments, the CAR comprises a second costimulatory domain. In some embodiments, the CAR comprises at least two costimulatory domains. In some embodiments, the CAR comprises at least three costimulatory domains. In some embodiments, the CAR comprises a costimulatory domain selected from one or more of CD27, CD28, 4-1BB, CD134 / OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83. In some embodiments, when the CAR comprises two or more costimulatory domains, the two costimulatory domains are different. In some embodiments, when the CAR comprises two or more costimulatory domains, the two costimulatory domains are the same.

[0283] In addition to the CARs described herein, various chimeric antigen receptors and the nucleotide sequences encoding them are known in the art and would be suitable for fusomal delivery and reprogramming of target cells in vivo and in vitro as described herein. See, e.g., WO2013040557; WO2012079000; WO2016030414; Smith T, et al., Nature Nanotechnology. 2017. DOI: 10.1038 / NNANO.2017.57 (the disclosures of which are incorporated herein by reference).

[0284] In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on diseased or pathological cells, e.g., tumor or pathogenic cells, compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells.

[0285] In some embodiments, the antigen targeted by the receptor includes an antigen associated with a B cell malignancy, e.g., any of a number of known B cell markers. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD47, CD21, CD5, CD33, Ig kappa, Ig lambda, CD79a, CD79b, or CD30.

[0286] In some embodiments, the CAR binds to CD19. In some embodiments, the CAR binds to CD22. In some embodiments, the CAR binds to CD19 and CD22. In some embodiments, the CAR is selected from the group consisting of a first-generation CAR, a second-generation CAR, a third-generation CAR, and a fourth-generation CAR. In some embodiments, the CAR includes a single binding domain that binds to a single target antigen. In some embodiments, the CAR includes a single binding domain that binds to multiple target antigens, e.g., two, three, or more target antigens. In some embodiments, the CAR includes two binding domains, such that each binding domain binds to a different target antigen. In some embodiments, the CAR includes two binding domains, such that each binding domain binds to the same target antigen. Detailed descriptions of exemplary CARs, including CD19-specific, CD22-specific, and CD19 / CD22 bispecific CARs, can be found in WO2012 / 079000, WO2016 / 149578, and WO2020 / 014482 (the disclosures, including sequence listings and figures, are incorporated herein by reference in their entirety).

[0287] In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or antibody fragment. In some aspects, the chimeric antigen receptor comprises an extracellular portion comprising an antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv.

[0288] In some embodiments, the antigen targeted by the antigen-binding domain is CD19. In some aspects, the antigen-binding domain of the recombinant receptor, e.g., CAR, and the antigen-binding domain bind, e.g., specifically binds to, or specifically recognizes CD19, such as human CD19. In some embodiments, the scFv contains a VH and a VL derived from an antibody or antibody fragment specific for CD19. In some embodiments, the antibody or antibody fragment that binds to CD19 is a murine-derived antibody, e.g., FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US2016 / 0152723.

[0289] In some embodiments, the antigen is CD19. In some embodiments, the scFv contains a VH and a VL derived from an antibody or antibody fragment specific for CD19. In some embodiments, the antibody or antibody fragment that binds to CD19 is a murine antibody, e.g., FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US2016 / 0152723.

[0290] In some embodiments, the scFv is derived from FMC63, which generally refers to a murine monoclonal IgG1 antibody raised against Naim-1 and Naim-16 cells expressing CD19 of human origin (Fing, NR, et al. (1987). Leucocyte typing III. 302).

[0291] In some embodiments, the antibody portion of the recombinant receptor (e.g., CAR) further comprises a spacer between the transmembrane domain and the extracellular antigen-binding domain. In some embodiments, the spacer comprises at least a portion of an immunoglobulin constant region, e.g., a hinge region, e.g., an IgG4 hinge region, and / or a CH1 / CL and / or Fc region. In some embodiments, the constant region or portion is that of human IgG, e.g., IgG4 or IgG1. In some aspects, the portion of the constant region functions as a spacer region between the antigen-recognition component (e.g., scFv) and the transmembrane domain. The spacer can be of a length that provides increased cellular reactivity after antigen binding compared to the absence of the spacer. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, WO2014031687, U.S. Patent No. 8,822,647, or published application no. US2014 / 0271635. In some embodiments, the constant region or portion is that of a human IgG, eg, IgG4 or IgG1.

[0292] In some embodiments, an antigen receptor comprises an intracellular domain linked directly or indirectly to an extracellular domain. In some embodiments, a chimeric antigen receptor comprises a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an ITAM. For example, in some aspects, the antigen recognition domain (e.g., the extracellular domain) is typically linked to one or more intracellular signaling components, e.g., signaling components that mimic activation via an antigen receptor complex, e.g., a TCR complex in the case of a CAR, and / or signaling via another cell surface receptor. In some embodiments, a chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g., an scFv) and the intracellular signaling domain. Thus, in some embodiments, an antigen binding component (e.g., an antibody) is linked to one or more transmembrane and intracellular signaling domains.

[0293] In one embodiment, a transmembrane domain that naturally associates with one or more domains in a receptor, such as a CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein, so as to minimize interaction with other members of the receptor complex.

[0294] In some embodiments, the CAR transmembrane domain comprises at least the transmembrane region of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a functional variant thereof. In some embodiments, the transmembrane domain comprises at least the transmembrane region(s) of CD8α, CD8β, 4-1BB / CD137, CD28, CD34, CD4, FcεRIγ, CD16, OX40 / CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRζ, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L / CD154, VEGFR2, FAS, and FGFR2B, or a functional variant thereof. The transmembrane domain, in some embodiments, is derived from either natural or synthetic sources. If the source is natural, the domain, in some aspects, is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e., comprising at least the transmembrane region(s) of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain, in some embodiments, is synthetic. In some aspects, the synthetic transmembrane domain comprises primarily hydrophobic residues, e.g., leucine and valine. In some aspects, triplets of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. In some embodiments, the linkage is by a linker, spacer, and / or transmembrane domain(s). In some aspects, the transmembrane domain contains the transmembrane portion of CD28.

[0295] In some embodiments, the extracellular domain and the transmembrane domain can be directly or indirectly linked. In some embodim...

Claims

1. 1. A method of transducing T cells, the method comprising contacting a population of resting CD4+ T cells with a lentiviral vector comprising a CD4 binding agent, wherein the lentiviral vector transduces the resting T cells, and the population of resting T cells is transduced with an efficiency of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40%.

2. 2. The method of claim 1, wherein the non-activated T cells are surface negative for one or more T cell activation markers selected from the group consisting of CD25, CD44, and CD69.

3. 10. The method of claim 1, wherein the lentiviral vector comprises a transgene encoding an engineered receptor that binds to or recognizes a protein or antigen expressed by or on a cell associated with a disease or pathology, and optionally the cell is a tumor cell.

4. 4. The method of claim 3, wherein the engineered receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

5. The method of claim 4, wherein the engineered receptor is a CAR, and the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising intracellular components of a CD3 zeta signaling domain and a costimulatory signaling domain. (a) the costimulatory signaling domain is (i) a CD28 costimulatory domain, optionally comprising the amino acid sequence set forth in SEQ ID NO:60; or (ii) a 4-1BB signaling domain, optionally comprising the amino acid sequence set forth in SEQ ID NO:59; (b) the CD3 zeta signaling domain comprises the sequence set forth in SEQ ID NO: 61 or SEQ ID NO: 62; and / or (c) the transmembrane domain comprises a sequence set forth in any one of SEQ ID NOs: 56, 57, and 58; The method of claim 5.

7. 6. The method of claim 5, wherein the CAR comprises a hinge domain, and optionally the hinge domain comprises a sequence set forth in any one of SEQ ID NOs: 50, 51, 52, 53, 54, 55, and 142.

8. The method of claim 5, wherein the antigen-binding domain binds to CD19.

9. The antigen-binding domain comprises: (a) CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 70, 71, and 72, respectively, and CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 65, 66, and 67, respectively; (b) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 69, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 64; and / or (c) the amino acid sequence shown in SEQ ID NO: 63 or 73 The method of claim 5 , comprising:

10. 6. The method of claim 5, wherein the CAR comprises the amino acid sequence set forth in SEQ ID NO: 75, 77, 79, or 81 and / or the amino acid sequence encoded by the polynucleotide sequence set forth in SEQ ID NO: 74, 76, 78, or 80.

11. 2. The method of claim 1, wherein at least 75% of the T cells in the population of resting T cells are surface negative for one or more T cell activation marker(s) selected from the group consisting of CD25, CD44, and CD69, and optionally, at least 80%, at least 85%, at least 90%, or at least 95% of the T cells in the population are surface negative for the one or more T cell activation marker(s).

12. The population of resting T cells comprises: (a) an anti-CD3 antibody, optionally OKT3; (b) an anti-CD28 antibody, optionally CD28.2; (c) an anti-CD3 antibody, optionally OKT3, and an anti-CD28 antibody, optionally beads coupled to CD28.2, optionally superparamagnetic beads; (d) a T cell activating cytokine, optionally recombinant IL-2, IL-7, IL-15, IL-21, or a combination thereof; and / or (e) a soluble T cell costimulatory molecule, optionally an anti-CD28 antibody or soluble CD80, soluble CD86, soluble CD137L, or soluble ICOS-L. The method of claim 1, wherein the patient has not been treated with 13. The method of claim 1, further comprising one or more of the following: (a) expanding the population of the transduced T cells; and (b) incubating the transduced cells with one or more T cell activating cytokine(s), optionally wherein the one or more T cell activating cytokine(s) are selected from recombinant IL-2, IL-7, IL-15, and IL-21.

14. 1. Use of a composition comprising a lentiviral vector comprising a CD4 binding agent in the manufacture of a medicament for a method of in vivo transduction of T cells, the method comprising administering the composition to a subject, wherein the lentiviral vector transduces T cells in the subject, and wherein the subject is not administered a T cell activation treatment concomitantly with administration of the composition, and optionally, the subject is not administered a T cell activation treatment before, after, or simultaneously with administration of the composition.

15. A composition comprising a lentiviral vector comprising a CD4 binding agent for use in treating a subject having a disease or condition, optionally cancer.

16. Use of a composition comprising a lentiviral vector containing a CD4 binding substance in the manufacture of a medicament for a method of expanding in vivo T cells capable of recognizing and killing tumor cells in a subject in need thereof, wherein the subject is not administered a T cell activation treatment in conjunction with administration of the composition, and optionally the subject is not administered a T cell activation treatment before, after, or simultaneously with administration of the composition.

17. The method of any one of claims 1 to 13, the use of claim 14 or 16, or the composition of claim 15, wherein the CD4 binding agent is an anti-CD4 antibody or antigen-binding fragment.

18. 18. The method, use, or composition of claim 17, wherein the anti-CD4 antibody or antigen-binding fragment is murine, rabbit, human, or humanized, and / or is a single-chain variable fragment (scFv) or single-domain antibody.

19. 18. The method, use, or composition of claim 17, wherein the anti-CD4 antibody or antigen-binding fragment is a camelid, optionally a llama, alpaca, or camel anti-CD4 antibody or antigen-binding fragment, optionally a VHH.

20. The method of any one of claims 1 to 13, the use of claims 14 or 16, or the composition of claim 15, wherein the CD4 binding agent is an anti-CD4 VHH; is expressed on the surface of the lentiviral vector; and / or is fused to a transmembrane domain incorporated into the viral envelope.

21. 16. The method of any one of claims 1 to 13, the use of claim 14 or 16, or the composition of claim 15, wherein the lentiviral vector is pseudotyped with a viral fusion protein.

22. 22. The method, use, or composition of claim 21, wherein the viral fusion protein comprises Nipah virus F glycoprotein (NiV-F) or a biologically active portion thereof and Nipah virus G glycoprotein (NiV-G) or a biologically active portion thereof, and the CD4 binding agent is fused to the NiV-G or the biologically active portion thereof.

23. 23. The method, use, or composition of claim 22, wherein the CD4 binding agent is fused to the C-terminus of the Nipah virus G glycoprotein or the biologically active portion thereof.

24. 24. The method, use, or composition of claim 22 or 23, wherein the CD4 binding protein is fused to the viral fusion protein directly or via a peptide linker.

25. 24. The method, use, or composition of claim 22 or 23, wherein the NiV-G protein or the biologically active portion thereof is a mutant NiV-G protein that exhibits reduced binding to ephrinB2 or ephrinB3.

26. 26. The method, use, or composition of claim 25, wherein the mutant NiV-G protein or biologically active portion comprises one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A with reference to the numbering set forth in SEQ ID NO:

4.

27. 24. The method, use, or composition of claim 22 or 23, wherein the NiV-F protein or the biologically active portion thereof is a wild-type NiV-F protein, or a functionally active variant or biologically active portion thereof.

28. 24. The method, use, or composition of claim 22 or 23, wherein the NiV-F protein or biologically active portion thereof has a 20 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein, and optionally the NiV-F protein or biologically active portion thereof has the sequence set forth in SEQ ID NO:20 or a sequence of amino acids exhibiting at least or about 80%, 85%, 90% or 95% sequence identity to the sequence set forth in SEQ ID NO:

20.

29. The NiV-F protein or the biologically active portion thereof comprises: i) a 20 amino acid truncation at or near the C-terminus of the wild-type NiV-F protein; and ii) Point mutations on N-linked glycosylation sites and optionally the NiV-F protein or the biologically active portion thereof has the sequence set forth in SEQ ID NO: 15 or a sequence of amino acids exhibiting at least or about 80%, 85%, 90% or 95% sequence identity to the sequence set forth in SEQ ID NO:

15.

30. The subject (a) has not been administered a T cell activation treatment simultaneously with the lentiviral vector; (b) has not been administered a T cell activation treatment within one month prior to said contact with the lentiviral vector or prior to said administration of the composition comprising the lentiviral vector; (c) has not been administered a T cell activation treatment within one, two, three, or four weeks, or at or about one, two, three, or four weeks, optionally at or about one, two, three, four, five, six, or seven days prior to said contact with the lentiviral vector or prior to said administration of the composition comprising the lentiviral vector; (d) has not been administered a T cell activation treatment within one, two, three, or four weeks, or at or about one, two, three, or four weeks, or optionally at or about one, two, three, four, five, six, or seven days prior to said contact with the lentiviral vector or prior to said administration of the composition comprising the lentiviral vector; 19. The method of any one of claims 1 to 13, the use of claim 14 or 16, or the composition of claim 15, wherein (e) a T cell activation treatment is not administered within one month after said contact with the viral vector or after said administration of said composition comprising said lentiviral vector; or (f) a T cell activation treatment is not administered within one, two, three or four weeks, or at or about one, two, three or four weeks, optionally at or about one, two, three, four, five, six or seven days after said contact with the lentiviral vector or after said administration of said composition comprising said lentiviral vector.

31. A population of transduced T cells produced by the method of any one of claims 1 to 13.

32. 32. A composition comprising the population of transduced T cells of claim 31, optionally a pharmaceutical composition.

33. 33. Use of the composition of claim 32 in the manufacture of a medicament for a method of treating a subject having a disease or condition, wherein the subject is not administered a T cell activation treatment in conjunction with administration of the composition, and optionally, the subject is not administered a T cell activation treatment before, after, or simultaneously with administration of the composition.

34. 33. Use of the composition of claim 32 in the manufacture of a medicament for a method of expanding in vivo T cells capable of recognizing and killing tumor cells in a subject in need thereof, wherein the subject is not administered a T cell activation treatment concomitantly with administration of the composition, and optionally, the subject is not administered a T cell activation treatment before, after, or simultaneously with administration of the composition.

35. A composition comprising a lentiviral vector containing a CD4 binding agent for use in expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof.

36. 33. The composition of claim 32 for use in expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof.

Images