Targeted lipid particles and compositions and their use

Abstract

This invention provides targeted lipid particles for delivering virus-like particles and lipid particles containing viral vectors to target cells. [Solution] A lipid particle is provided comprising a lipid bilayer surrounding a lumen or cavity, a henipavirus F protein molecule or its biologically active portion, and a targeted envelope protein containing a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion and a binding domain, for example, a single-domain antibody (sdAb) variable domain. Also provided is a targeted envelope protein containing a binding domain, for example, a G protein fused to or linked to an sdAb variable domain, and a polynucleotide encoding such a protein. Furthermore, producer cells and compositions containing such targeted lipid particles, as well as methods for producing and using targeted lipid particles, are provided.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 003,168, entitled "Targeted Lipid Particles and Compositions and Uses Thereof," filed Mar. 31, 2020, and U.S. Provisional Application No. 63 / 154,341, entitled "Targeted Lipid Particles and Compositions and Uses Thereof," filed Feb. 26, 2021, the contents of each of which are hereby incorporated by reference in their entirety for all purposes.

[0002] Incorporation by Reference of Sequence Listing This application is filed with a sequence listing in electronic form. This sequence listing is provided as a file entitled 186152003640SeqList.TXT, created on Mar. 29, 2021, with a size of 2,071,683 bytes. The electronic form information of the sequence listing is hereby incorporated by reference in its entirety.

[0003] Field The present disclosure relates to lipid particles containing a lipid bilayer surrounding a lumen or cavity, a henipavirus F protein molecule or a biologically active portion thereof, and a targeted envelope protein containing a henipavirus envelope - binding glycoprotein G (G protein) or a biologically active portion and binding domain thereof, such as a single - domain antibody (sdAb) variable domain. The present disclosure also provides a targeted envelope protein containing a G protein fused or linked to a binding domain, such as an sdAb variable domain, and a polynucleotide encoding such a protein. Also disclosed are producer cells and compositions containing such targeted lipid particles and methods of making and using the targeted lipid particles.

Background Art

[0004] Background Lipid particles, including virus-like particles and viral vectors, are commonly used for the delivery of exogenous substances to cells. However, the delivery of lipid particles to designated target cells can present challenges. In the case of lentiviral vectors, the host range can be altered by pseudotyped with heterologous envelope proteins. The designated retargeting envelope protein may not be sufficiently stable or even expressed on the surface of the lipid particle. Improved lipid particles, including virus-like particles and viral vectors, are needed to target desired cells. The provided disclosure addresses this need. [Overview of the Initiative]

[0005] overview This specification provides targeted lipid particles comprising (a) a lipid bilayer surrounding a lumen, (b) a henipavirus F protein molecule or a biologically active moiety thereof, and (c) a targeted envelope protein comprising (i) a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active moiety thereof and (ii) a single-domain antibody (sdAb) variable domain, wherein the sdAb variable domain is bound to the C-terminus of the G protein or a biologically active moiety thereof, and the F protein molecule or a biologically active moiety thereof and the targeted envelope protein are embedded in the lipid bilayer. In some embodiments, the single-domain antibody is bound to the G protein via a linker. In some embodiments, the linker is a peptide linker.

[0006] This specification provides targeted lipid particles comprising (a) a lipid bilayer surrounding a lumen, (b) a henipavirus F protein molecule or its biologically active moiety, and (c) a targeted envelope protein comprising a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active moiety, conjugated via a peptide linker to a variable domain of a single-domain antibody (sdAb), wherein the single-domain antibody is conjugated to a cell surface molecule of a target cell, and the F protein molecule or its biologically active moiety and the targeted envelope protein are embedded in the lipid bilayer. In some embodiments, the N-terminus of the F protein molecule or its biologically active moiety is exposed to the outside of the lipid bilayer. In some embodiments, the C-terminus of the G protein is exposed to the outside of the lipid bilayer.

[0007] In some embodiments, the single-domain antibody binds to a cell surface molecule present on the target cell. In some embodiments, the cell surface molecule is a protein, glycan, lipid, or low molecular weight molecule. In some of the optional embodiments, the single-domain antibody binds to an antigen or a portion thereof present on the target cell. In some embodiments, the antigen is a cell surface molecule or a portion thereof containing an epitope recognized by the single-domain antibody. In some of the optional embodiments, the target cell is selected from the group consisting of tumor-infiltrating lymphocytes, T cells, neoplasms or tumor cells, virus-infected cells, stem cells, central nervous system (CNS) cells, hematopoietic stem cells (HSCs), liver cells, or fully differentiated cells. In some embodiments, the target cells are selected from the group consisting of CD3+ T cells, CD4+ T cells, CD8+ T cells, hepatocytes, hematopoietic stem cells, CD34+ hematopoietic stem cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD105+ endothelial cells, B cells, CD20+ B cells, CD19+ B cells, cancer cells, CD133+ cancer cells, EpCAM+ cancer cells, CD19+ cancer cells, Her2 / Neu+ cancer cells, GluA2+ neurons, GluA4+ neurons, NKG2D+ natural killer cells, SLC1A3+ astrocytic cells, SLC7A10+ adipocytes, or CD30+ lung epithelial cells. In some of the embodiments, the target cells are hepatocytes. In some of the embodiments, the cell surface molecules or antigens are selected from the group consisting of ASGR1, ASGR2, and TM4SF5.

[0008] In some of the optional embodiments, the target cells are T cells. In some of the optional embodiments, the cell surface molecule or antigen is CD8 or CD4.

[0009] In some of the various embodiments, the cell surface molecule or antigen is LDL-R.

[0010] This specification provides a targeted lipid particle comprising (a) a lipid bilayer surrounding the lumen, (b) a henipavirus F protein molecule or its biologically active portion, and (c) a targeted envelope protein comprising (i) a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion and (ii) a binding domain, wherein the binding domain is bound to the C-terminus of the G protein or its biologically active portion, the binding domain binds to a cell surface molecule selected from the group consisting of ASGR1, ASGR2, and TM4SF5, and optionally human ASGR1, human ASGR2, and human ASGR2, and the F protein molecule or its biologically active portion and the targeted envelope protein are embedded in the lipid bilayer.

[0011] This specification provides a targeted lipid particle comprising (a) a lipid bilayer surrounding a lumen, (b) a henipavirus F protein molecule or its biologically active portion, and (c) a targeted envelope protein comprising (i) a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion and (ii) a binding domain, wherein the binding domain is bound to the C-terminus of the G protein or its biologically active portion, the binding domain binds to a cell surface molecule selected from the group consisting of CD8 and CD4, optionally human CD8 or human CD4, and the F protein molecule or its biologically active portion and the targeted envelope protein are embedded in the lipid bilayer.

[0012] This specification provides a targeted lipid particle comprising (a) a lipid bilayer surrounding a lumen, (b) a henipavirus F protein molecule or its biologically active portion, and (c) a targeted envelope protein comprising (i) a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion and (ii) a binding domain, wherein the binding domain is bound to the C-terminus of the G protein or its biologically active portion, the binding domain binds to a cell surface molecule which is a low-density lipoprotein receptor (LDL-R), optionally human LDL-R, and the F protein molecule or its biologically active portion and the targeted envelope protein are embedded in the lipid bilayer.

[0013] In some of the optional embodiments, the lipid particles are lentiviral vectors. In some of the optional embodiments, the binding domain is linked to a G protein via a linker. In some of the optional embodiments, the linker is a peptide linker.

[0014] This specification provides a lentiviral vector comprising a binding domain that targets a cell surface molecule selected optionally from the group consisting of ASGR1, ASGR2, and TM4SF5, and is pseudotyped with a retargeting viral fusion protein, wherein the retargeting viral fusion protein comprises (a) a henipavirus F protein molecule or its biologically active portion, and (b) a targeted envelope protein comprising a binding domain bound to henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion.

[0015] This specification provides a lentiviral vector comprising a binding domain that targets cell surface molecules selected from the group consisting of CD8 and CD4, optionally human CD8 and human CD4, and which is pseudotyped with a retargeting viral fusion protein, wherein the retargeting viral fusion protein comprises (a) a henipavirus F protein molecule or its biologically active portion, and (b) a targeted envelope protein comprising a binding domain bound to henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion.

[0016] This specification provides a lentiviral vector comprising a binding domain that targets a low-density lipoprotein receptor (LDL-R), wherein the LDL-R is optionally human LDL-R, and the lentiviral vector is pseudotyped with a retargeting viral fusion protein comprising (a) a henipavirus F protein molecule or its biologically active portion, and (b) a targeted envelope protein comprising a binding domain bound to henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion.

[0017] In some of the available embodiments, the binding domain is bound to the C-terminus of the G protein or its biologically active portion.

[0018] In this specification, (a) Henipavirus F protein molecule or its biologically active portion, (b) A targeted envelope protein comprising (i) a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active moiety and (ii) a binding domain, wherein the binding domain is bound to the C-terminus of the G protein or its biologically active moiety, and the binding domain binds to CD4, (c) A cargo comprising a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR is (i) an extracellular antigen-binding domain that binds to an extracellular antigen (e.g., CD19 or BCMA), and (ii) Intracellular signaling domains comprising a CD3 zeta signaling domain and optionally a 4-1BB or CD28 costimulatory signaling domain Including cargo and A lentiviral vector is provided that includes the following. In some embodiments, the extracellular antigen-binding domain of the CAR is scFv.

[0019] In some of the available embodiments, the lentiviral vector is capable of delivering CAR-encoding nucleic acids to T cells. In some embodiments, the T cells are in vivo in the subject.

[0020] This specification provides a lentiviral vector comprising (a) a henipavirus F protein molecule or a biologically active moiety thereof, and (b)(i) a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active moiety thereof, and (ii) a targeted envelope protein comprising a binding domain, wherein the binding domain is bound to the C-terminus of the G protein or the biologically active moiety thereof, the binding domain binds to ASGR1, and the lentiviral vector is capable of targeting hepatocytes. In some of the various embodiments, the lentiviral vector further comprises an exogenous substance for delivery to hepatocytes.

[0021] In some of the optional embodiments, the lentiviral vector is capable of delivering exogenous material to hepatocytes, which are optionally in vivo in the subject.

[0022] In some of the embodiments, the binding domain is bound to a G protein via a linker. In some of the embodiments, the linker is a peptide linker. In some of the embodiments, the binding domain is a single-domain antibody. In some of the embodiments, the binding domain is a single-stranded variable fragment (scFv).

[0023] In some of the optional embodiments, the peptide linker has a length of up to 65 amino acids. In some of the optional embodiments, the peptide linker has a length of up to 50 amino acids. In some of the optional embodiments, the peptide linker has a length of approximately 2-65 amino acids, 2-60 amino acids, 2-56 amino acids, 2-52 amino acids, 2-48 amino acids, 2-44 amino acids, 2-40 amino acids, 2-36 amino acids, 2-32 amino acids, 2-28 amino acids, 2-24 amino acids, 2-20 amino acids, 2-18 amino acids, 2-14 amino acids, 2-12 amino acids, 2-10 amino acids, 2-8 amino acids, 2-6 amino acids, 6-65 amino acids, 6-60 amino acids, 6-56 amino acids, 6-52 amino acids, 6-48 amino acids Amino acids, 6-44 amino acids, 6-40 amino acids, 6-36 amino acids, 6-32 amino acids, 6-28 amino acids, 6-24 amino acids, 6-20 amino acids, 6-18 amino acids, 6-14 amino acids, 6-12 amino acids, 6-10 amino acids, 6-8 amino acids, 8-65 amino acids, 8-60 amino acids, 8-56 amino acids, 8-52 amino acids, 8-48 amino acids, 8-44 amino acids, 8-40 amino acids, 8-36 amino acids, 8-32 amino acids, 8-28 amino acids, 8-24 amino acids, 8-20 amino acids, 8-18 amino acids, 8-14 amino acids, 8-12 amino acids, 8-10 amino acids, 10-65 amino acids, 10-60 amino acids, 10-56 amino acids, 10-52 amino acids, 10-48 amino acids, 10-44 amino acids, 10-40 amino acids, 10-36 amino acids, 10-32 amino acids, 10-28 amino acids, 10-24 amino acids, 10-20 amino acids, 10-18 amino acids, 10-14 amino acids, 10-12 amino acids, 12-65 amino acids, 12-60 amino acids, 12-56 amino acids, 12-52 amino acids, 12-48 amino acids, 1 2-44 amino acids, 12-40 amino acids, 12-36 amino acids, 12-32 amino acids, 12-28 amino acids, 12-24 amino acids, 12-20 amino acids, 12-18 amino acids, 12-14 amino acids, 14-65 amino acids, 14-60 amino acids, 14-56 amino acids, 14-52 amino acids, 14-48 amino acids, 14-44 amino acids, 14-40 amino acids, 14-36 amino acids, 14-32 amino acids, 14-28 amino acids, 14-24 amino acids, 14-20 amino acids, 14-18 amino acids, 18-65 amino acids,18-60 amino acids, 18-56 amino acids, 18-52 amino acids, 18-48 amino acids, 18-44 amino acids, 18-40 amino acids, 18-36 amino acids, 18-32 amino acids, 18-28 amino acids, 18-24 amino acids, 18-20 amino acids, 20-65 amino acids, 20-60 amino acids, 20-56 amino acids, 20-52 amino acids, 20-48 amino acids, 20-44 amino acids, 20-40 amino acids, 20-36 amino acids, 20-32 amino acids, 20-28 amino acids, 20-2 6 amino acids, 20-24 amino acids, 24-65 amino acids, 24-60 amino acids, 24-56 amino acids, 24-52 amino acids, 24-48 amino acids, 24-44 amino acids, 24-40 amino acids, 24-36 amino acids, 24-32 amino acids, 24-30 amino acids, 24-28 amino acids, 28-65 amino acids, 28-60 amino acids, 28-56 amino acids, 28-52 amino acids, 28-48 amino acids, 28-44 amino acids, 28-40 amino acids, 28-36 amino acids, 28-34 amino acids Acid, 28-32 amino acids, 32-65 amino acids, 32-60 amino acids, 32-56 amino acids, 32-52 amino acids, 32-48 amino acids, 32-44 amino acids, 32-40 amino acids, 32-38 amino acids, 32-36 amino acids, 36-65 amino acids, 36-60 amino acids, 36-56 amino acids, 36-52 amino acids, 36-48 amino acids, 36-44 amino acids, 36-40 amino acids, 40-65 amino acids, 40-60 amino acids, 40-56 amino acids, 40-52 amino acids, 40 Contains ~48 amino acids, 40~44 amino acids, 44~65 amino acids, 44~60 amino acids, 44~56 amino acids, 44~52 amino acids, 44~48 amino acids, 48~65 amino acids, 48~60 amino acids, 48~56 amino acids, 48~52 amino acids, 50~65 amino acids, 50~60 amino acids, 50~56 amino acids, 50~52 amino acids, 54~65 amino acids, 54~60 amino acids, 54~56 amino acids, 58~65 amino acids, 58~60 amino acids, or 60~65 amino acids. In some of the optional embodiments, the peptide linker has lengths of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,The peptide contains polypeptides having 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 amino acids. In some of the embodiments, the peptide linker is a flexible linker containing GS, GGS, GGGGS (SEQ ID NO: 43), GGGGGS (SEQ ID NO: 41), or a combination thereof. In some of the embodiments, the peptide linker contains (GGS)n (n is 1 to 10). In some of the embodiments, the peptide linker contains (GGGGS)n (SEQ ID NO: 42) (n is 1 to 10). In some of the embodiments, the peptide linker contains (GGGGGS)n (SEQ ID NO: 27) (n is 1 to 6).

[0024] In some of the optional embodiments, the G protein or its biologically active portion is the wild-type Nipah virus G (NiV-G) protein or the Hendra virus G protein. In some of the optional embodiments, the G protein or its biologically active portion is the wild-type NiV-G protein or a functionally active variant or biologically active portion thereof. In some of the embodiments, the mutant NiV-G protein or its functionally active variant or biologically active moiety contains an amino acid sequence 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%, 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 with SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44.

[0025] In some of the available embodiments, the NiV-G protein is a cleaved and biologically active portion of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) that lacks up to 40 consecutive amino acid residues at or near its N-terminus.

[0026] In some of the arbitrary embodiments, the NiV-G protein is a biologically active portion cleaved at the N-terminus of wild-type NiV-G, and is a sequence shown in any of SEQ ID NOs: 10-15, 35-40 or 45-50, or a sequence with 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 8 It has an amino acid sequence having sequence identity of 6%, 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%.

[0027] In some of the available embodiments, the NiV-G protein is a biologically active portion of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) that has a 5-amino acid cleavage at or near its N-terminus. In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 10. In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 35.In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 45.

[0028] In some of the optional embodiments, the NiV-G protein is a biologically active portion of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) that has a 10-amino acid cleavage at or near its N-terminus. In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 36. In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 11.In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 46.

[0029] In some of the optional embodiments, the NiV-G protein or the biologically active moiety has a 15-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44). In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 12. In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 37.In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 47.

[0030] In some of the optional embodiments, the NiV-G protein is a biologically active portion of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) that has a 20-amino acid cleavage at or near its N-terminus. In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 13. In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 38.In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 48.

[0031] In some of the optional embodiments, the NiV-G protein is a biologically active portion of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) having a 25-amino acid cleavage at or near its N-terminus. In some of the various embodiments, the NiV-G protein has an amino acid sequence that has 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 with SEQ ID NO: 14. In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 39.In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 49.

[0032] In some of the optional embodiments, the NiV-G protein is a biologically active portion of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) that has a 30-amino acid cleavage at or near its N-terminus. In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 15. In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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 with SEQ ID NO: 40.

[0033] In some of the optional embodiments, the NiV-G protein is a biologically active portion of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) that has a 34-amino acid cleavage at or near its N-terminus. In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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%, 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%, 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 with SEQ ID NO: 22. In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence 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%, 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%, 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 with SEQ ID NO: 53.

[0034] In some of the various embodiments, the G protein, its biologically active portion, is a functionally active variant, which is a mutant NiV-G protein exhibiting reduced binding to ephrin B2 or ephrin B3.

[0035] In some of the optional embodiments, the mutant NiV-G protein contains one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A, with reference to the numbering shown in Sequence ID No. 28.

[0036] In some of the embodiments, the mutant NiV-G protein or the biologically active portion has an amino acid sequence 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%, 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 with SEQ ID NO: 16. In some of the embodiments, the mutant NiV-G protein or the biologically active portion has an amino acid sequence 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%, 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 with SEQ ID NO: 51.

[0037] In some of the optional embodiments, the F protein or its biologically active portion is the wild-type Nipah virus F (NiV-F) protein or the Hendra virus F protein, or a functionally active variant or biologically active portion thereof. In some of the embodiments, the NiV-F protein or its functionally active variant or biologically active moiety includes the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence 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%, 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 with SEQ ID NO: 2.

[0038] In some of the optional embodiments, the NiV-F protein is the biologically active portion of the wild-type NiV-F protein (SEQ ID NO: 2) having a 20-amino acid cleavage at or near its C-terminus.

[0039] In some of the embodiments, the NiV-F protein or the biologically active portion has an amino acid sequence 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%, 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 with SEQ ID NO: 5.

[0040] In some of the optional embodiments, the NiV-F protein is the biologically active portion of the wild-type NiV-F protein (SEQ ID NO: 2) that includes i) a 20-amino acid cleavage at or near the C-terminus, and ii) a point mutation at the N-linked glycosylation site.

[0041] In some of the embodiments, the NiV-F protein or the biologically active portion has an amino acid sequence 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%, 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 with SEQ ID NO: 7.

[0042] In some of the optional embodiments, the NiV-F protein is the biologically active portion of the wild-type NiV-F protein (SEQ ID NO: 2) having a 22-amino acid cleavage at or near its C-terminus.

[0043] In some of the various embodiments, the NiV-F protein or the biologically active portion has an amino acid sequence encoded by a sequence of nucleotides encoding the sequence shown in SEQ ID NO: 8, or a sequence 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%, 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 with SEQ ID NO: 8.

[0044] In some of the embodiments, the NiV-F protein or the biologically active portion has an amino acid sequence 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%, 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 with SEQ ID NO: 23. In some of the various embodiments, the F-protein or its biologically active portion includes an F1 subunit or its fusion portion.

[0045] In some of the optional embodiments, the F protein comprises the sequence shown in SEQ ID NO: 23, and the G protein comprises the sequence shown in SEQ ID NO: 16.

[0046] In some of the various embodiments, the F protein consists of, or is essentially, the sequence shown in SEQ ID NO: 23, and / or the G protein consists of, or is essentially, the sequence shown in SEQ ID NO: 16.

[0047] In some of the various embodiments, the F1 subunit is a proteolytically cleaved portion of the F0 precursor. In some of the various embodiments, the F1 subunit has an amino acid sequence 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%, 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 with SEQ ID NO: 4.

[0048] In some of the optional embodiments, the lipid bilayer is derived from the membrane of a host cell used to generate a retrovirus or retrovirus-like particle. In some of the optional embodiments, the host cell is selected from the group consisting of 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 some of the optional embodiments, the host cell includes 293T cells. In some of the optional embodiments, the lipid bilayer is or contains a viral envelope. In some of the optional embodiments, the retrovirus-like particle is replication-deficient.

[0049] In some of the optional embodiments, the targeted lipid particles include one or more viral components other than F protein molecules and G proteins. In some of the optional embodiments, one or more viral components are from retroviruses. In some of the optional embodiments, the retrovirus is a lentivirus. In some of the optional embodiments, one or more viral components include a viral packaging protein selected from one or more of Gag, Pol, Rev, and Tat. In some of the optional embodiments, one or more viral components include one or more (e.g., all) of the following nucleic acid sequences: 5'LTR (e.g., containing U5 and lacking a functional U3 domain), psi packaging element (psi), central polypurine sequence (cPPT) / central termination sequence (CTS) (e.g., DNA flap), poly-A tail sequence, post-transcriptional regulatory element (e.g., WPRE), Rev reaction element (RRE), and 3'LTR (e.g., containing U5 and lacking a functional U3).

[0050] In some of the optional embodiments, the targeted lipid particles are lentiviral vectors.

[0051] In some of the optional embodiments, the targeted lipid particles or lentiviral vectors are replication-deficient.

[0052] In some of the optional embodiments, the targeted lipid particles or lentiviral vector further comprise exogenous material. In some of the optional embodiments, the targeted lipid particles further comprise exogenous material. In some embodiments, the lentiviral vector further comprises exogenous material.

[0053] In some of the optional embodiments, the exogenous substance is present in the lumen. In some of the optional embodiments, the exogenous substance is a protein or nucleic acid. In some embodiments, the nucleic acid is DNA or RNA.

[0054] In some of the optional embodiments, the exogenous substance is a nucleic acid encoding cargo for delivery to target cells. In some of the optional embodiments, the exogenous substance encodes a therapeutic or diagnostic agent.

[0055] In some of the various embodiments, the exogenous substance encodes a membrane protein. In some embodiments, the protein is an antigen receptor for targeting cells expressed by or associated with a disease or pathological condition. In some embodiments, the membrane protein is a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises (i) an extracellular antigen-binding domain that binds to an extracellular antigen (e.g., CD19 or BCMA), optionally the extracellular antigen-binding domain being an scFv; (ii) a transmembrane domain; and (iii) an intracellular signaling region comprising a CD3 zeta signaling domain and optionally a co-stimulatory signaling domain, e.g., a 4-1BB or CD28 co-stimulatory signaling domain. In some embodiments, the target cell is a T cell. In some embodiments, the cell surface molecule on the target cell is CD4 or CD8. In some embodiments, the binding domain is an scFv that binds to CD4 (e.g., human CD4). In some embodiments, the binding domain is a single-domain antibody that binds to CD4 (e.g., human CD4). In some embodiments, the binding domain is an scFv that binds to CD8 (e.g., human CD8). In some embodiments, the binding domain is a single-domain antibody that binds to CD8 (e.g., human CD8).

[0056] In some of the optional embodiments, the exogenous substance is a nucleic acid containing a gene defect, optionally a payload gene for correcting the gene defect in target cells. In some embodiments, the gene defect is associated with liver cells or hepatocytes. In some embodiments, the target cells are hepatocytes. In some embodiments, the cell surface molecule is a molecule selected from the group consisting of ASGR1, ASGR2, and TM4SF5. In some embodiments, the binding domain is an scFv that binds to ASGR1 (e.g., human ASGR1). In some embodiments, the binding domain is a single-domain antibody that binds to ASGR1 (e.g., human ASGR1). In some embodiments, the binding domain is an scFv that binds to ASGR2 (e.g., human ASGR2). In some embodiments, the binding domain is a single-domain antibody that binds to ASGR2 (e.g., human ASGR2). In some embodiments, the binding domain is an scFv that binds to TM4SF5 (e.g., human TM4SF5). In some embodiments, the binding domain is a single-domain antibody that binds to TM4SF5 (e.g., human TM4SF5).

[0057] In some of the optional embodiments, the single-domain antibody binds to a cell surface molecule present on the target cell. In some of the optional embodiments, the cell surface molecule is a protein, glycan, lipid, or low molecular weight molecule. In some of the optional embodiments, the target cell is selected from the group consisting of tumor-infiltrating lymphocytes, T cells, neoplasms or tumor cells, virus-infected cells, stem cells, central nervous system (CNS) cells, hematopoietic stem cells (HSCs), liver cells, or fully differentiated cells. In some of the optional embodiments, the target cells are selected from the group consisting of CD3+ T cells, CD4+ T cells, CD8+ T cells, hepatocytes, hematopoietic stem cells, CD34+ hematopoietic stem cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD105+ endothelial cells, B cells, CD20+ B cells, CD19+ B cells, cancer cells, CD133+ cancer cells, EpCAM+ cancer cells, CD19+ cancer cells, Her2 / Neu+ cancer cells, GluA2+ neurons, GluA4+ neurons, NKG2D+ natural killer cells, SLC1A3+ astrocytocytes, SLC7A10+ adipocytes, or CD30+ lung epithelial cells.

[0058] In some of the optional embodiments, a single-domain antibody binds to an antigen or a portion thereof present on a target cell. In some of the optional embodiments, the cell surface molecule or antigen is selected from the group consisting of ASGR1, ASGR2, and TM4SF5. In some embodiments, the antigen or a portion thereof is human ASGR1. In some embodiments, the antigen or a portion thereof is human ASGR2. In some embodiments, the antigen or a portion thereof is human TM4SF5.

[0059] This specification provides polynucleotides comprising a nucleic acid sequence encoding (i) a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion thereof, and (ii) a binding domain that binds to a cell surface molecule selected from the group consisting of ASGR1, ASGR2, and TM4SF5. In some embodiments, the cell surface molecule is human ASGR1. In some embodiments, the cell surface molecule is human ASGR2. In some embodiments, the cell surface molecule is human TM4SF5. In some of the various embodiments, the cell surface molecule or antigen is CD8 or CD4.

[0060] This specification provides nucleic acid sequences encoding (i) a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion thereof, and (ii) a binding domain that binds to a cell surface molecule selected from the group consisting of CD4 and CD8. In some embodiments, the cell surface molecule is human CD4. In some embodiments, the cell surface molecule is human CD8. In some embodiments, the cell surface molecule or antigen is a low-density lipoprotein receptor (LDL-R). In some embodiments, the cell surface molecule or antigen is human LDL-R.

[0061] This specification provides polynucleotides comprising a nucleic acid sequence encoding (i) a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion thereof and (ii) a binding domain that binds to a low-density lipoprotein receptor (LDL-R). In some embodiments, the binding domain binds to human LDL-R. In some of the embodiments, the binding domain is a single-domain antibody (sdAb). In some of the embodiments, the binding domain is a single-stranded variable fragment (scFv).

[0062] This specification provides a polynucleotide comprising a nucleic acid sequence encoding (i) a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active moiety thereof and (ii) a single-domain antibody (sdAb) variable domain, wherein the sdAb variable domain is conjugated to the C-terminus of the G protein or the biologically active moiety thereof. In some of the various embodiments, the polynucleotide further comprises (iii) a nucleic acid sequence encoding a henipavirus F protein molecule or a biologically active moiety thereof.

[0063] In some embodiments, the nucleic acid sequence is a first nucleic acid sequence, and the polynucleotide further comprises a second nucleic acid sequence encoding the henipavirus F protein molecule or a biologically active portion thereof. In some embodiments, the polynucleotide comprises an IRES or a sequence encoding a linking peptide between the first and second nucleic acid sequences. In some embodiments, the linking peptide is a self-cleaving peptide or a peptide that causes ribosome skipping, optionally a T2A peptide.

[0064] In some of the optional embodiments, the polynucleotide includes at least one promoter functionally linked to control the expression of the nucleic acid. In some of the optional embodiments, the promoter is functionally linked to control the expression of a first nucleic acid sequence and a second nucleic acid sequence. In some of the optional embodiments, the promoter is a constitutive promoter. In some of the optional embodiments, the promoter is an inductive promoter.

[0065] In some of the optional embodiments, the sdAb variable domain is bound to the G protein via an encoded peptide linker. In some embodiments, the binding domain is bound to the G protein via an encoded peptide linker. In some of the optional embodiments, the encoded peptide linker has a length of up to 25 amino acids. In some of the optional embodiments, the encoded peptide linker has a length of up to 65 amino acids. In some of the optional embodiments, the encoded peptide linker has a length of approximately 2-65 amino acids, 2-60 amino acids, 2-56 amino acids, 2-52 amino acids, 2-48 amino acids, 2-44 amino acids, 2-40 amino acids, 2-36 amino acids, 2-32 amino acids, 2-28 amino acids, 2-24 amino acids, 2-20 amino acids, 2-18 amino acids, 2-14 amino acids, 2-12 amino acids, 2-10 amino acids, 2-8 amino acids, 2-6 amino acids, 6-65 amino acids. Amino acids, 6-60 amino acids, 6-56 amino acids, 6-52 amino acids, 6-48 amino acids, 6-44 amino acids, 6-40 amino acids, 6-36 amino acids, 6-32 amino acids, 6-28 amino acids, 6-24 amino acids, 6-20 amino acids, 6-18 amino acids, 6-14 amino acids, 6-12 amino acids, 6-10 amino acids, 6-8 amino acids, 8-65 amino acids, 8-60 amino acids, 8-56 amino acids, 8-52 amino acids, 8-48 amino acids, 8-44 Amino acids, 8-40 amino acids, 8-36 amino acids, 8-32 amino acids, 8-28 amino acids, 8-24 amino acids, 8-20 amino acids, 8-18 amino acids, 8-14 amino acids, 8-12 amino acids, 8-10 amino acids, 10-65 amino acids, 10-60 amino acids, 10-56 amino acids, 10-52 amino acids, 10-48 amino acids, 10-44 amino acids, 10-40 amino acids, 10-36 amino acids, 10-32 amino acids, 10-28 amino acids, 10 ~24 amino acids, 10~20 amino acids, 10~18 amino acids, 10~14 amino acids, 10~12 amino acids, 12~65 amino acids, 12~60 amino acids, 12~56 amino acids, 12~52 amino acids, 12~48 amino acids, 12~44 amino acids, 12~40 amino acids, 12~36 amino acids, 12~32 amino acids, 12~28 amino acids, 12~24 amino acids, 12~20 amino acids, 12~18 amino acids, 12~14 amino acids, 14~65 amino acids,14-60 amino acids, 14-56 amino acids, 14-52 amino acids, 14-48 amino acids, 14-44 amino acids, 14-40 amino acids, 14-36 amino acids, 14-32 amino acids, 14-28 amino acids, 14-24 amino acids, 14-20 amino acids, 14-18 amino acids, 18-65 amino acids, 18-60 amino acids, 18-56 amino acids, 18-52 amino acids, 18-48 amino acids, 18-44 amino acids, 18-40 amino acids, 18-36 amino acids, 18-32 amino acids, 18-28 amino acids, 18-24 amino acids, 18-20 amino acids, 20-65 amino acids Amino acids, 20-60 amino acids, 20-56 amino acids, 20-52 amino acids, 20-48 amino acids, 20-44 amino acids, 20-40 amino acids, 20-36 amino acids, 20-32 amino acids, 20-28 amino acids, 20-26 amino acids, 20-24 amino acids, 24-65 amino acids, 24-60 amino acids, 24-56 amino acids, 24-52 amino acids, 24-48 amino acids, 24-44 amino acids, 24-40 amino acids, 24-36 amino acids, 24-32 amino acids, 24-30 amino acids, 24-28 amino acids, 28-65 amino acids, 28-60 amino acids, 28- 56 amino acids, 28-52 amino acids, 28-48 amino acids, 28-44 amino acids, 28-40 amino acids, 28-36 amino acids, 28-34 amino acids, 28-32 amino acids, 32-65 amino acids, 32-60 amino acids, 32-56 amino acids, 32-52 amino acids, 32-48 amino acids, 32-44 amino acids, 32-40 amino acids, 32-38 amino acids, 32-36 amino acids, 36-65 amino acids, 36-60 amino acids, 36-56 amino acids, 36-52 amino acids, 36-48 amino acids, 36-44 amino acids, 36-40 amino acids, 40-65 amino acids It contains 40-60 amino acids, 40-56 amino acids, 40-52 amino acids, 40-48 amino acids, 40-44 amino acids, 44-65 amino acids, 44-60 amino acids, 44-56 amino acids, 44-52 amino acids, 44-48 amino acids, 48-65 amino acids, 48-60 amino acids, 48-56 amino acids, 48-52 amino acids, 50-65 amino acids, 50-60 amino acids, 50-56 amino acids, 50-52 amino acids, 54-65 amino acids, 54-60 amino acids, 54-56 amino acids, 58-65 amino acids, 58-60 amino acids, or 60-65 amino acids.

[0066] In some of the optional embodiments, the encoded peptide linker comprises polypeptides having a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 amino acids. In some of the optional embodiments, the encoded peptide linker comprises GS, GGS, GGGGS (SEQ ID NO: 43), GGGGGS (SEQ ID NO: 41), and combinations thereof. In some of the optional embodiments, the encoded peptide linker includes (GGS)n (where n is 1 to 10). In some of the optional embodiments, the encoded peptide linker includes (GGGGS)n (SEQ ID NO: 42) (where n is 1 to 10). In some of the optional embodiments, the encoded peptide linker includes (GGGGGS)n (SEQ ID NO: 27) (where n is 1 to 4). In some of the optional embodiments, the sequence encoding the G protein is wild-type Nipah virus G (NiV-G) protein or Hendra virus G protein, or a functionally active variant or biologically active moiety thereof. In some embodiments, the variant is a variant of that which exhibits reduced binding for its native binding partner. In some of the optional embodiments, the nucleic acid sequence encoding the G protein is wild-type Nipah virus G (NiV-G) protein or Hendra virus G protein, or a variant of that which exhibits reduced binding for its native binding partner. In some embodiments, the encoded G protein is the wild-type NiV-G protein or a functionally active variant or biologically active portion thereof. In some of the embodiments, the nucleic acid sequence encoding the G protein is the wild-type NiV-G protein. In some of the embodiments, the nucleic acid sequence encoding the G protein is a mutant NiV-G protein exhibiting reduced binding to ephrin B2 or ephrin B3.

[0067] In some of the embodiments, the NiV-G protein or its functionally active variant or biologically active moiety comprises the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44, or comprises 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 less The amino acid sequence contains at least 87% or approximately 87%, at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99% sequence identity. In some of the various embodiments, the NiV-G protein is a cleaved and biologically active portion lacking up to 40 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44).In some of the arbitrary embodiments, the NiV-G protein is a biologically active portion cleaved at the N-terminus of wild-type NiV-G, which is the sequence shown in any of SEQ ID NOs. 10-15, 35-40 or 45-50, or 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%, and It contains an amino acid sequence having sequence identity of 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%.

[0068] In some of the optional embodiments, the NiV-G protein is a biologically active portion of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) that includes a 5-amino acid cleavage at or near its N-terminus. In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 10. In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 35.In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 45.

[0069] In some of the optional embodiments, the NiV-G protein is a biologically active portion of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) that includes a 10-amino acid cleavage at or near its N-terminus. In some of the embodiments, the mutant NiV-G protein or biologically active moiety includes an amino acid sequence 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 with SEQ ID NO: 11. In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 36.In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 46.

[0070] In some of the optional embodiments, the NiV-G protein is a biologically active portion of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) that includes a 15-amino acid cleavage at or near its N-terminus. In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 12. In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 37.In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 47.

[0071] In some of the optional embodiments, the NiV-G protein is a biologically active portion of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) that includes a 20-amino acid cleavage at or near its N-terminus. In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 13. In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 38.In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 48.

[0072] In some of the optional embodiments, the NiV-G protein is a biologically active portion of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) that includes a 25-amino acid cleavage at or near its N-terminus. In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 14. In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 39.In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 49.

[0073] In some of the optional embodiments, the NiV-G protein is a biologically active portion of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) that includes a 30-amino acid cleavage at or near its N-terminus. In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 15. In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 40.In some of the embodiments, the NiV-G protein or the biologically active portion includes an amino acid sequence 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 with SEQ ID NO: 50.

[0074] In some of the optional embodiments, the NiV-G protein is a biologically active portion of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) that has a 34-amino acid cleavage at or near its N-terminus. In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence having 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 with SEQ ID NO: 22. In some of the embodiments, the NiV-G protein or the biologically active portion has an amino acid sequence having 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 with SEQ ID NO: 53.

[0075] In some of the embodiments, the G protein is a mutant NiV-G protein exhibiting reduced binding to ephrin B2 or ephrin B3. In some of the embodiments, the mutant NiV-G protein contains 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 shown in Sequence ID No. 28. In some of the embodiments, the mutant NiV-G protein contains amino acid substitutions E501A, W504A, Q530A and E533A, with reference to the numbering shown in Sequence ID No. 28.

[0076] In some of the various embodiments, the mutant NiV-G protein includes i) a cleavage at or near the N-terminus, and ii) a point mutation selected from the group consisting of E501A, W504A, Q530A, and E533A. In some of the various embodiments, the mutant NiV-G protein contains an amino acid sequence 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 with SEQ ID NO: 16. In some of the embodiments, the mutant NiV-G protein contains an amino acid sequence 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 with SEQ ID NO: 51.

[0077] In some of the optional embodiments, the F protein or its biologically active portion is the wild-type Nipah virus F (NiV-F) protein or the Hendra virus F protein, or a functionally active variant or biologically active portion thereof. In some of the embodiments, the NiV-F protein or its functionally active variant or biologically active moiety includes the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having 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 with SEQ ID NO: 2.

[0078] In some of the optional embodiments, the NiV-F protein is the biologically active portion of the wild-type NiV-F protein (SEQ ID NO: 2) having a 20-amino acid cleavage at or near its C-terminus. In some of the embodiments, the NiV-F protein or the biologically active moiety has an amino acid sequence having 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 with SEQ ID NO: 5. In some of the optional embodiments, the NiV-F protein is the biologically active portion of the wild-type NiV-F protein (SEQ ID NO: 2) that includes i) a 20-amino acid cleavage at or near the C-terminus, and ii) a point mutation at the N-linked glycosylation site.

[0079] In some of the embodiments, the NiV-F protein or the biologically active portion has an amino acid sequence having 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 with SEQ ID NO: 7.

[0080] In some of the optional embodiments, the NiV-F protein is the biologically active portion of the wild-type NiV-F protein (SEQ ID NO: 2) having a 22-amino acid cleavage at or near its C-terminus. In some of the various embodiments, the NiV-F protein or the biologically active portion has an amino acid sequence encoded by a sequence of nucleotides encoding the sequence shown in SEQ ID NO: 8, or a sequence having 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 with SEQ ID NO: 8.

[0081] In some of the various embodiments, the NiV-F protein has an amino acid sequence having 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 with SEQ ID NO: 23. In some of the embodiments, the F protein comprises the sequence shown in SEQ ID NO: 23, and the G protein comprises the sequence shown in SEQ ID NO: 16.

[0082] This specification provides vectors comprising polynucleotides of any of the embodiments described herein. In some of the embodiments, the vector is a mammalian vector, a viral vector, or an artificial chromosome, and optionally the artificial chromosome is a bacterial artificial chromosome (BAC).

[0083] This specification provides plasmids comprising polynucleotides of any of the embodiments described herein. In some of the embodiments, the plasmid further comprises one or more nucleic acids encoding proteins for lentiviral generation.

[0084] This specification provides cells comprising a polynucleotide of any embodiment described herein, a vector of any embodiment described herein, or a plasmid of any embodiment described herein.

[0085] This specification provides a method for producing targeted lipid particles comprising a henipavirus F protein molecule or its biologically active portion, and a targeted envelope protein comprising a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion and a single-domain antibody (sdAb) variable domain, a) To provide a cell comprising a nucleic acid encoding a henipavirus F protein molecule or a biologically active portion thereof, and a nucleic acid encoding a targeted envelope protein, wherein the targeted envelope protein comprises a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion thereof and a single-domain antibody (sdAb) variable domain. b) Culturing cells under conditions that enable the generation of targeted lipid particles, and c) Separating, concentrating, or purifying targeted lipid particles from cells, thereby producing targeted lipid particles. A method including this is provided.

[0086] This specification describes a method for producing a pseudotyped lentiviral vector, a) To provide producer cells comprising lentiviral nucleic acids (multiple), a nucleic acid encoding a henipavirus F protein molecule or its biologically active portion, and a nucleic acid encoding a targeted envelope protein, wherein the targeted envelope protein comprises henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion and a single-domain antibody. b) Culturing cells under conditions that enable the generation of lentiviral vectors, and c) Isolating, concentrating, or purifying lentiviral vectors from cells, thereby producing pseudotyped lentiviral vectors. A method including this is provided.

[0087] In some of the optional embodiments, the single-domain antibody binds to a cell surface molecule present on the target cell. In some of the optional embodiments, the cell surface molecule is a protein, glycan, lipid, or low molecular weight molecule. In some of the optional embodiments, the target cell is selected from the group consisting of tumor-infiltrating lymphocytes, T cells, neoplasms or tumor cells, virus-infected cells, stem cells, central nervous system (CNS) cells, hematopoietic stem cells (HSCs), liver cells, or fully differentiated cells. In some of the optional embodiments, the target cells are selected from the group consisting of CD3+ T cells, CD4+ T cells, CD8+ T cells, hepatocytes, hematopoietic stem cells, CD34+ hematopoietic stem cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD105+ endothelial cells, B cells, CD20+ B cells, CD19+ B cells, cancer cells, CD133+ cancer cells, EpCAM+ cancer cells, CD19+ cancer cells, Her2 / Neu+ cancer cells, GluA2+ neurons, GluA4+ neurons, NKG2D+ natural killer cells, SLC1A3+ astrocytic cells, SLC7A10+ adipocytes, or CD30+ lung epithelial cells. In some of the optional embodiments, the single-domain antibody binds to an antigen or a portion thereof present on the target cells.

[0088] This specification describes a method for producing targeted lipid particles comprising a henipavirus F protein molecule or its biologically active portion, and a targeted envelope protein comprising a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion and binding domain, a) To provide a cell comprising a nucleic acid encoding a henipavirus F protein molecule or a biologically active portion thereof, and a nucleic acid encoding a targeted envelope protein, wherein the targeted envelope protein comprises a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion thereof and a binding domain, and the binding domain binds to (i) a cell surface molecule selected from the group consisting of ASGR1, ASGR2, and TM4SF5, optionally human ASGR1, human ASGR2, and human ASGR2; (ii) a cell surface molecule selected from the group consisting of CD4 or CD8, optionally human CD4 or human CD8; or (iii) a cell surface molecule that is a low-density lipoprotein receptor (LDL-R), optionally human LDL-R. b) Culturing cells under conditions that enable the generation of targeted lipid particles, and c) Separating, concentrating, or purifying targeted lipid particles from cells, thereby producing targeted lipid particles. A method including this is provided.

[0089] This specification provides a method for producing a pseudotyped lentiviral vector, comprising: a) lentiviral nucleic acid(s); a nucleic acid encoding a henipavirus F protein molecule or a biologically active portion thereof; and a nucleic acid encoding a targeted envelope protein, wherein the targeted envelope protein comprises a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion and a binding domain, and the binding domain is (i) ASGR1, ASGR2, and TM4SF5, optionally human ASGR1, human ASG A method is provided comprising: a) providing a cell surface molecule that binds to (ii) a cell surface molecule selected from the group consisting of R2 and human ASGR2; b) a cell surface molecule that binds to CD4 or CD8, optionally selected from the group consisting of human CD4 or human CD8; or c) a cell surface molecule that binds to a low-density lipoprotein receptor (LDL-R), optionally human LDL-R; b) culturing producer cells under conditions that enable the production of a lentiviral vector; and c) isolating, concentrating, or purifying a lentiviral vector from cells to produce a pseudotyped lentiviral vector.

[0090] In some of the embodiments, the binding domain is a single-domain antibody. In some of the embodiments, the binding domain is a single-stranded variable fragment (scFv). In some of the embodiments, the cell surface molecule is selected from the group consisting of ASGR1, ASGR2, and TM4SF5. In some of the embodiments, the cell surface molecule is CD8 or CD4. In some of the embodiments, the cell surface molecule is LDL-R.

[0091] This specification provides a method for producing targeted lipid particles comprising a henipavirus F protein molecule or a biologically active portion thereof and a targeted envelope protein, a) To provide cells comprising a polynucleotide of any embodiment provided herein, a vector of any embodiment described herein, or a plasmid of any embodiment described herein. b) Culturing cells under conditions that enable the generation of targeted lipid particles, and c) Separating, concentrating, or purifying targeted lipid particles from cells, thereby producing targeted lipid particles. A method including this is provided.

[0092] This specification describes a method for producing a pseudotyped lentiviral vector, a) To provide producer cells comprising lentiviral nucleic acid(s) and a polynucleotide of any of the embodiments listed herein or a vector of any of the embodiments listed herein, b) Culturing cells under conditions that enable the generation of lentiviral vectors, and c) Isolating, concentrating, or purifying lentiviral vectors from cells, thereby producing pseudotyped lentiviral vectors. A method is provided which includes the following. In some of the optional embodiments, prior to step (b), the method further includes providing cells with a henipavirus F protein molecule or a polynucleotide encoding a biologically active portion thereof.

[0093] In some of the arbitrary embodiments, the cells are mammalian cells.

[0094] In some of the optional embodiments, the cells are producer cells containing viral nucleic acids. In some of the optional embodiments, the viral nucleic acids are retroviral nucleic acids or lentiviral nucleic acids, and the targeted lipid particles are viral particles or virus-like particles. In some of the optional embodiments, the viral particles or virus-like particles are retroviral particles or retroviral-like particles. In some embodiments, the viral particles or virus-like particles are lentiviral particles or lentiviral-like particles.

[0095] In some of the optional embodiments, the viral nucleic acid(s) lacks one or more genes involved in viral replication. In some of the optional embodiments, the viral nucleic acid comprises nucleic acid encoding a viral packaging protein selected from one or more of Gag, Pol, Rev, and Tat. In some of the optional embodiments, the viral nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences: 5'LTR (e.g., containing U5 and lacking a functional U3 domain), pseudopackaging element (psi), central polypurine sequence (cPPT) / central termination sequence (CTS) (e.g., DNA flap), poly-A tail sequence, post-transcriptional regulatory element (e.g., WPRE), Rev reaction element (RRE), and 3'LTR (e.g., containing U5 and lacking a functional U3).

[0096] This specification provides producer cells comprising a polynucleotide of any of the embodiments listed herein, a vector of any of the embodiments listed herein, or a plasmid of any of the embodiments described herein.

[0097] In some of the optional embodiments, the producer cells further include nucleic acids that encode the henipavirus F protein or a biologically active portion thereof.

[0098] In some of the optional embodiments, the cell further comprises viral nucleic acid. In some of the optional embodiments, the viral nucleic acid is lentiviral nucleic acid. This specification provides a producer cell comprising (i) viral nucleic acid(s), (ii) a nucleic acid encoding a henipavirus F protein molecule or a biologically active portion thereof, and (iii) a nucleic acid encoding a targeted envelope protein comprising henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion thereof and a single-domain antibody (sdAb) variable domain, wherein the viral nucleic acid(s) is optionally lentiviral nucleic acid. In some of the optional embodiments, the single-domain antibody binds to a cell surface molecule present on the target cell. In some of the optional embodiments, the cell surface molecule is a protein, glycan, lipid, or low molecular weight molecule.

[0099] In some of the optional embodiments, the target cells are selected from the group consisting of tumor-infiltrating lymphocytes, T cells, neoplasms or tumor cells, virus-infected cells, stem cells, central nervous system (CNS) cells, hematopoietic stem cells (HSCs), liver cells, or fully differentiated cells. In some of the optional embodiments, the target cells are selected from the group consisting of CD3+ T cells, CD4+ T cells, CD8+ T cells, hepatocytes, hematopoietic stem cells, CD34+ hematopoietic stem cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD105+ endothelial cells, B cells, CD20+ B cells, CD19+ B cells, cancer cells, CD133+ cancer cells, EpCAM+ cancer cells, CD19+ cancer cells, Her2 / Neu+ cancer cells, GluA2+ neurons, GluA4+ neurons, NKG2D+ natural killer cells, SLC1A3+ astrocytic cells, SLC7A10+ adipocytes, or CD30+ lung epithelial cells. In some of the optional embodiments, the single-domain antibody binds to an antigen or a portion thereof present on the target cells.

[0100] This specification provides producer cells comprising (i) viral nucleic acids(or more), (ii) nucleic acids encoding a henipavirus F protein molecule or a biologically active portion thereof, and (iii) nucleic acids encoding a targeted envelope protein including a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion and a binding domain thereof, wherein the binding domain binds to (i) cell surface molecules selected from the group consisting of ASGR1, ASGR2, and TM4SF5, optionally human ASGR1, human ASGR2, and human ASGR2, (ii) cell surface molecules selected from the group consisting of CD4 or CD8, optionally human CD4 or human CD8, or (iii) cell surface molecules that are low-density lipoprotein receptors (LDL-R), optionally human LDL-R. In some of the various embodiments, the viral nucleic acids(or more) are lentiviral nucleic acids.

[0101] In some of the optional embodiments, the cell surface molecule or antigen is selected from the group consisting of ASGR1, ASGR2, and TM4SF5. In some of the optional embodiments, the cell surface molecule or antigen is CD8 or CD4. In some of the optional embodiments, the cell surface molecule or antigen is LDL-R.

[0102] In some of the optional embodiments, the viral nucleic acid(s) lacks one or more genes involved in viral replication. In some of the optional embodiments, the viral nucleic acid comprises a nucleic acid encoding a viral packaging protein selected from one or more of Gag, Pol, Rev, and Tat.

[0103] In some of the various embodiments, the viral nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences: 5'LTR (e.g., containing U5 and lacking a functional U3 domain), psipackaging elements (psi), central polypurine sequences (cPPT) / central termination sequences (CTS) (e.g., DNA flap), poly(A) tail sequences, post-transcriptional regulatory elements (e.g., WPRE), Rev reaction elements (RRE), and 3'LTR (e.g., containing U5 and lacking a functional U3).

[0104] In some of the embodiments, the henipavirus F protein molecule or its biologically active portion comprises (i) the sequence shown in SEQ ID NO: 2, and (ii) an amino acid sequence 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%, 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% sequence identity with SEQ ID NO: 2. In some of the various embodiments, the henipavirus F protein molecule or its biologically active portion comprises (i) the sequence shown in SEQ ID NO: 5, and (ii) an amino acid sequence having 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% sequence identity with SEQ ID NO: 5.

[0105] In some of the embodiments, the henipavirus F protein molecule or its biologically active portion comprises (i) the sequence shown in SEQ ID NO: 7, and (ii) an amino acid sequence 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%, 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% sequence identity with SEQ ID NO: 7. In some of the embodiments, the henipavirus F protein molecule or its biologically active portion is (i) a sequence encoded by a nucleotide sequence encoding the sequence shown in SEQ ID NO: 8, (ii) 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 It comprises an amino acid sequence encoded by a nucleotide sequence encoding a sequence having approximately 88%, or at least 89%, at least 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%.

[0106] In some of the embodiments, the henipavirus F protein molecule or its biologically active portion is (i) the sequence shown in SEQ ID NO: 23, and (ii) SEQ ID NO: 23 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%. It contains amino acid sequences having sequence identity of approximately 88%, or at least 89%, at least 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%.

[0107] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44, or (ii) SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44 in proportion to 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 less It also contains amino acid sequences having sequence identity of 87% or approximately 87%, at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0108] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 10, (ii) SEQ ID NO: 10 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0109] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 35, (ii) SEQ ID NO: 35 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0110] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 45, (ii) SEQ ID NO: 45 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0111] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 11, (ii) SEQ ID NO: 11 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0112] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 36, (ii) SEQ ID NO: 36 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0113] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 46, (ii) SEQ ID NO: 46 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0114] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 12, (ii) SEQ ID NO: 12 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0115] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 37, (ii) SEQ ID NO: 37 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0116] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 47, (ii) SEQ ID NO: 47 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0117] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 13, (ii) SEQ ID NO: 13 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0118] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 38, (ii) SEQ ID NO: 38 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0119] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 48, (ii) SEQ ID NO: 48 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0120] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 14, (ii) SEQ ID NO: 14 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0121] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 39, (ii) SEQ ID NO: 39 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0122] In some of the embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 49, (ii) SEQ ID NO: 49 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0123] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 15, (ii) SEQ ID NO: 15 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0124] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 40, (ii) SEQ ID NO: 40 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0125] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 50, (ii) SEQ ID NO: 50 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0126] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 16, (ii) SEQ ID NO: 16 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0127] In some of the various embodiments, the henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion is (i) the sequence shown in SEQ ID NO: 51, (ii) SEQ ID NO: 51 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%. It contains amino acid sequences having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, at least 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0128] In some aspects of the provided embodiments, the targeted lipid particles have more targeted envelope protein expression compared to reference lipid particles in which the same envelope protein is incorporated into a similar lipid bilayer but is fused to an alternative targeting site, optionally the alternative targeting site is a single-chain variable fragment (scFv). In some of the embodiments, the expression increases by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more or above that. In some embodiments, the expression increases by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more or above that, preferably 10-fold or about 10-fold or about more than 10-fold or above that. In some of the embodiments, the titer in the target cells after transduction is 1×10 6 transducing units (TU) / mL or more, 2×10 6 TU / mL or more, 3×10 6 TU / mL or more, 4×10 6 TU / mL or more, 5×10 6 TU / mL or more, 6×10 6 TU / mL or more, 7×10<00,00007>TU / mL or more, 8×10 6 TU / mL or more, 9×10 6 TU / mL or more, or 1×10 7 TU / mL or more. Also herein, compositions are provided in which 50% or about more than 50%, 55% or about more than 55%, 60% or about more than 60%, 65% or about more than 65%, 70% or about more than 70%, or 75% or about more than 75% of the population of lipid particles are surface positive for the targeted envelope protein. In some of the embodiments, the targeted envelope protein is present on the surface of the targeted lipid particles at a density of at least about (0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2 or 0.5) of the targeted envelope protein / nm 2 of the targeted envelope protein / nm.

[0129] This specification provides viral vector particles or virus-like particles generated from producer cells of any of the embodiments provided herein.

[0130] This specification provides compositions comprising a plurality of targeted lipid particles of any of the embodiments provided herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some of the embodiments, the targeted lipid particles have an average diameter of less than 1 μm. In some of the embodiments, the composition contains at least about (0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, or 0.5) targeted envelope proteins / nm 2 It further contains targeted envelope proteins present on the surface of targeted lipid particles at an average density.

[0131] This specification provides producer cells containing membrane (e.g., cell membrane) expression of a targeted envelope protein, which is more abundant than in reference producer cells in which the same envelope protein is incorporated into the membrane (e.g., cell membrane) but is fused to an alternative targeting site, wherein the alternative targeting site is optionally a single-stranded variable fragment (scFv). In some embodiments, the expression is increased by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more. In some embodiments, the expression is increased by 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times or more, preferably 10 times or about 10 times or more than 10 times. In some embodiments, the producer cells have expression of a targeted envelope protein on the producer cell membrane (e.g., cell membrane) of at least 20 proteins per square micron (e.g., at least 50, 100, 200, 500, 1000, 2000, 5000, or 10,000 proteins). In some of the arbitrary embodiments, the targeted envelope protein constitutes at least 0.1% (e.g., at least 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) of the total membrane (e.g., cell membrane) proteins of the producer cells (e.g., relative to total protein weight).

[0132] This specification provides a method for transducing cells, comprising transducing cells with any of the viral vectors or compositions described herein. In some of the optional embodiments, the targeted envelope protein of the lentiviral vector or targeted lipid particle targets CD4, and the cells are CD4+ cells. In some of the optional embodiments, the targeted envelope protein of the lentiviral vector targets CD8, and the cells are CD8+ cells. In some of the optional embodiments, the targeted envelope protein of the lentiviral vector targets ASGR1, ASGR2, or TM4SF5, and the cells are hepatocytes.

[0133] This specification provides a method for delivering an exogenous substance to a subject (e.g., a human subject), comprising administering targeted lipid particles or a composition of any of the embodiments provided herein to the subject, wherein the targeted lipid particles or lentiviral vector contain the exogenous substance.

[0134] This specification provides a method for delivering an exogenous substance to a subject (e.g., a human subject), comprising administering one of the compositions described herein to the subject, wherein a plurality of targeted lipid particles or lentiviral vectors contain the exogenous substance.

[0135] This specification provides a method for delivering a chimeric antigen receptor (CAR) to a cell, comprising contacting the cell with a targeted lipid particle of any of the lentiviral vectors described herein or any of the embodiments described herein, wherein the lentiviral vector or targeted lipid particle comprises a nucleic acid encoding the CAR.

[0136] This specification provides a method for delivering a chimeric antigen receptor (CAR) to a cell, comprising contacting the cell with any of the compositions described herein, wherein a plurality of lentiviral vectors or targeted lipid particles contain nucleic acids encoding the CAR.

[0137] This specification provides a method for delivering an exogenous substance to hepatocytes, comprising contacting the cells with any of the lentiviral vectors described herein or any of the embodiments described herein, or with targeted lipid particles or lentiviral vectors.

[0138] This specification provides a method for delivering an exogenous substance to hepatocytes, comprising contacting the cells with one of the compositions described herein, wherein a plurality of lentiviral vectors or targeted lipid particles contain the exogenous substance for delivery to hepatocytes. In some of the optional embodiments, the contact is transduction of the cells with the lentiviral vectors or targeted lipid particles.

[0139] This specification provides a method for treating a disease or disorder in a subject (e.g., a human subject), which comprises administering targeted lipid particles of any of the embodiments provided herein or a composition of any of the embodiments provided herein to the subject.

[0140] This specification provides a method for fusing mammalian cells to targeted lipid particles, comprising administering the targeted lipid particles of any embodiment provided herein or a composition of any embodiment provided herein to a subject. In some of the optional embodiments, fusing mammalian cells to targeted lipid particles delivers an exogenous substance to a subject (e.g., a human subject). In some of the optional embodiments, fusing mammalian cells to targeted lipid particles treats a disease or disorder in a subject (e.g., a human subject). In some of the optional embodiments, the targeted envelope protein of the lentiviral vector or targeted lipid particle targets CD4, and the cell is a CD4+ cell. In some of the optional embodiments, the targeted envelope protein of the lentiviral vector targets CD8, and the cell is a CD8+ cell. In some of the optional embodiments, the targeted envelope protein of the lentiviral vector targets ASGR1, ASGR2, or TM4SF5, and the cell is a hepatocyte.

[0141] In some of the optional embodiments, the targeted lipid particles have higher targeted envelope protein expression compared to reference lipid particles in which the same envelope protein is incorporated into a similar lipid bilayer but is fused to an alternative targeting site. In some embodiments, the alternative targeting site is a single-stranded variable fragment (scFv). In some of the optional embodiments, the expression increases by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more. In some of the optional embodiments, the expression increases by 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times or more, preferably 10 times or about 10 times or more than 10 times.

[0142] In some of the optional embodiments, the titer in the target cells after transduction is 1 × 10⁻⁶. 6 Transduction units (TU) / mL or more, 2 × 10 6TU / mL or more, 3×10 6 TU / mL or more, 4×10 6 TU / mL or more, 5×10 6 TU / mL or more, 6×10 6 TU / mL or more, 7×10 6 TU / mL or more, 8×10 6 TU / mL or more, 9×10 6 TU / mL or higher, or 1 × 10 7 It is TU / mL or higher.

[0143] In some of the optional embodiments, within the population of lipid particles or lentiviral vectors in the composition, 50% or more than 50%, 55% or more than 55%, 60% or more than 60%, 65% or more than 65%, 70% or more than 70%, or 75% or more than 75% are surface-positive for the targeted envelope protein. In some of the optional embodiments, the targeted envelope protein is at least about (0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, or 0.5) of targeted envelope protein / nm 2 It is present on the surface of the targeted lipid particles at a density of [value].

[0144] This specification describes a composition comprising a plurality of targeted lipid particles of any embodiment described herein or a plurality of lentiviral vectors of any embodiment described herein, wherein the targeted envelope protein is at least about (0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, or 0.5) of targeted envelope protein / nm. 2 A composition is provided that exists on the surface of targeted lipid particles at an average density.

[0145] In some of the arbitrary embodiments, the producer cells have membrane (e.g., cell membrane) expression of the targeted envelope protein, which is more abundant than in the reference producer cells, where the same envelope protein is incorporated into the membrane (e.g., cell membrane), but it is fused to an alternative targeting site, and the alternative targeting site is optionally a single-stranded variable fragment (scFv). In some of the arbitrary embodiments, the expression increases by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more. In some of the optional embodiments, the expression increases by 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times or more, preferably 10 times or about 10 times or more. In some of the optional embodiments, the producer cells have expression of a targeted envelope protein on the membrane (e.g., cell membrane) of the producer cells, which is at least 20 proteins per square micron (e.g., at least 50, 100, 200, 500, 1000, 2000, 5000, or 10,000 proteins). In some of the arbitrary embodiments, the targeted envelope protein constitutes at least 0.1% (e.g., at least 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) of the total membrane (e.g., cell membrane) proteins of the producer cell (e.g., relative to the total protein weight).

[0146] Detailed explanation This specification provides targeted lipid particles comprising a lipid bilayer surrounding a lumen or cavity, and (1) a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active moiety thereof, and (2) a targeted envelope protein containing a binding domain, e.g., a single-domain antibody (sdAb) variable domain, wherein the targeted envelope protein is embedded in the lipid bilayer of the lipid particle. In certain embodiments, the binding domain, e.g., the single-domain antibody, is an antibody that binds to a desired target molecule, e.g., an antibody having the ability to bind specifically. Exemplary binding domains are described in Section II.A.2. In some embodiments, the targeted lipid particles also contain a henipavirus fusion (F) protein molecule or a biologically active moiety thereof embedded in the lipid bilayer. In certain embodiments, the lipid particles may be virus-like particles, viruses, or viral vectors, e.g., lentiviral vectors.

[0147] In some embodiments, one or both of the G protein and / or F protein are derived from the Hendra (HeV) or Nipah (NiV) virus, or are their biologically active portions, variants, or variants. In certain embodiments, both the G protein and / or F protein are derived from the Hendra (HeV) or Nipah (NiV) virus. In some embodiments, the fusion and binding glycoproteins mediate the entry of Nipah virus into cells.

[0148] F proteins, such as NiV-F, are class I fusion proteins that share structural and functional features with many families of fusion proteins (e.g., HIV-1 gp41 or influenza virus hemagglutinin [HA]), including hydrophobic fusion peptides and ectodomains with two heptad repeat regions (White JM et al. 2008. Crit Rev Biochem Mol Biol 43:189-219). F proteins are synthesized as an inactive precursor F0 and activated by proteolytic cleavage into two disulfide-linked subunits F1 and F2 (Moll M. et al. 2004. J. Virol. 78(18):9705-9712).

[0149] The G protein is a type II transmembrane glycoprotein containing an N-terminal cytoplasmic tail, transmembrane domain, extracellular stalk, and bulbous head, and is the binding protein for henipavirus (e.g., Nipah virus or Hendra virus) (Liu, Q. et al. 2015. Journal of Virology, 89(3):1838-1850). The binding protein NiV-G recognizes receptors ephrin B2 and ephrin B3. Receptor binding to NiV-G triggers a series of structural changes that ultimately lead to NiV-F activation, exposing the NiV-F fusion peptide and enabling another series of structural changes that result in viral cell membrane fusion (Stone JA et al. 2016. J Virol. 90(23):10762-10773). Ephrin B2 was previously identified as the primary NiV receptor (Negrete et al., 2005), and ephrin B3 as an alternative receptor (Negrete et al., 2006). In fact, NiV-G exhibits high affinity for ephrin B2 and B3 with picomolar affinity binding constants (Kd) (Negrete et al., 2006) (Kd = 0.06 nM and 0.58 nM, respectively, for ephrin B2 and B3 expressed on the cell surface).

[0150] The efficiency of transduction of targeted lipid particles can be improved by manipulating superfusion mutations in one or both NiV-F and NiV-G. Several such mutations have been previously described (see, e.g., Lee et al, 2011, Trends in Microbiology). This may be useful, for example, to maintain the specificity and picomolar affinity of NiV-G to ephrin B2 and / or B3. Mutations in NiV-G that completely inactivate ephrin B2 and B3 binding but do not affect the association of NiV-G with NiV-F have also been identified. Methods to improve the targeting of lipid particles can be achieved by fusing a binding molecule with a G protein (e.g., Niv-G containing a Niv-G with a mutation to inactivate ephrin B2 and ephrin B3 binding). This may enable modified G protein directivity that allows targeting of other desired cell types other than ephrin B2+ through the addition of binding molecules directed towards different cell surface molecules.

[0151] Retargeted lipid particles incorporating such binding molecules fused to G proteins have been generated, and it has been found herein that some binding molecules are expressed better on the surface of the lipid particles than others when fused to G proteins (e.g., NiV-G). For example, single-domain antibodies (sdAbs), e.g., VHH, have been found to be expressed 10 times better than single-stranded variable fragments (scFv). While we do not wish to be bound by theory, the increased expression may be attributable to the increased stability of the retargeted G protein on the surface of the lipid particle. This greater expression may improve the ability of the lipid particles to target molecules (e.g., cell surface molecules) compared to similar lipid particles containing alternative binding domains for the same target molecule, e.g., scFv.

[0152] Accordingly, this specification provides targeted lipid particles containing a henipavirus G protein (e.g., Hendra or Nipah, e.g., NiV-G) conjugated to an sdAb variable domain that is directed to or capable of binding to cell surface molecules on target cells. The sdAb variable domain may include sdAb of VL or VH only, nanobodies, camelid VHH domains, shark IgNAR or fragments thereof. In some embodiments, the sdAb is VHH.

[0153] In aspects of the provided embodiments, targeted lipid particles may be engineered to express a henipavirus F protein molecule or its biologically active moiety, and a targeted envelope protein comprising (i) a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active moiety and (ii) a single-domain antibody (sdAb) variable domain, wherein the F protein molecule or its biologically active moiety and the targeted envelope protein are embedded in a lipid bilayer. In some embodiments, the sdAb variable domain is conjugated to the C-terminus of the G protein or its biologically active moiety. In some embodiments, the sdAb variable domain is conjugated to the G protein via a linker.

[0154] Furthermore, targeted lipid particles are provided that additionally contain one or more exogenous substances, including after in vivo administration to a target, for example, for the delivery of diagnostic or therapeutic agents to cells. Also provided herein are methods and uses of targeted lipid particles in diagnostic and therapeutic methods. Also provided are methods for manipulating, preparing, and generating polynucleotides, targeted lipid noncellular particles, compositions containing particles, and kits and devices for using, generating, and administering the particles.

[0155] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated by reference in whole for all purposes to the same extent that each individual publication is disclosed by reference. If any definition provided herein contradicts or otherwise conflicts with any definition provided herein in a patent, application, published application, or other publication incorporated herein by reference, the definition provided herein shall prevail over the definition incorporated herein by reference.

[0156] The section headings used herein are for organizational purposes only and should not be construed as limiting the subjects described. [Brief explanation of the drawing]

[0157] [Figure 1A] Figures 1A–1C illustrate the characterization of cells transfected with constructs containing scFv or VHH-binding modalities. Figure 1A shows the surface expression of cells transfected with constructs containing scFv or VHH-binding modalities, shown as median fluorescence intensity (MFI) quantified by the percentage of His+ cells, analyzed by flow cytometry. Empty vectors and expression vectors without binder domains were used as negative controls. [Figure 1B] Figures 1A–1C show the characterization of cells transfected with constructs containing scFv or VHH-binding modality. Figure 1B shows the binding of cells transfected with constructs containing the VHH-binding modality scFV to the soluble hCD4-Fc protein, as analyzed by flow cytometry and shown as median fluorescence intensity (MFI) quantified by %Fc+ cells. Empty vectors and expression vectors without a binder domain were used as negative controls. [Figure 1C]Figures 1A–1C illustrate the characterization of cells transfected with constructs containing scFv or VHH binding modalities. Figure 1C shows the surface expression of targeted binding sequences on 293 cells transfected with constructs containing VHH binding modalities, compared to the scFv binding modality, as analyzed by flow cytometry and expressed as median fluorescence intensity (MFI), quantified by the percentage of His+ cells. Empty vectors and expression vectors without binder domains were used as negative controls. [Figure 2] We demonstrate the transduction efficacy of four exemplary constructs containing scFV or VHH-binding modalities to PanT cells from peripheral blood that was negatively selected, thawed, and activated with anti-CD3 / anti-CD28 to enrich T cells. Cells were analyzed by flow cytometry, and titer was determined by the percentage of CD4-positive cells that were GFP+. [Figure 3A] Figures 3A-B show the transduction efficiency of CD8 retargeting pseudotyped lentivirus in an in vivo model using activated PBMCs intraperitoneally injected into NOD-scid-IL2rγnull mice, as analyzed by flow cytometry. Transduction efficiency of CD8 retargeting pseudotyped lentivirus is shown for CD8+ (Figure 3A) or CD8- (Figure 3B) T cells, and titers were determined by the percentage of CD8-positive or CD8-negative cells that were GFP+. [Figure 3B] See the explanation in Figure 3A. [Figure 4A] Figures 4A and 4B show the ability of CD8 retargeting pseudotyped lentiviruses containing chimeric antigen receptors (CARs) to kill leukemia cells in vitro. Figure 4A shows the ability to detect CD19+CAR expression on CD8+ cells 4 days after transduction. Figure 4B shows the elimination of Nalm6 cells, assessed at 18 hours after incubation, as analyzed by flow cytometry. [Figure 4B] See the explanation in Figure 4A. [Modes for carrying out the invention]

[0158] I. Definition Unless otherwise defined, all terms, notations, and other technical and scientific or specialized terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art in which the claimed subject matter relates. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or for immediate reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a substantial difference from the commonly understood meaning in the art.

[0159] Unless otherwise defined, all technical and scientific terms, acronyms, and abbreviations used herein have the same meaning as those generally understood by those skilled in the art in the field to which this invention relates. 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 and all integer values ​​between them.

[0160] As used herein, the articles "a" and "an" refer to one or more (i.e., at least one) grammatical objects of the article. For example, "an element" means one or more elements.

[0161] As used herein, “about” is to be understood by those skilled in the art and varies to some extent in the context in which it is used. As used herein, when “about” refers to a measurable value, such as a quantity, duration of time, etc., it includes a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and more preferably ±0.1% from the specified value, as such variation is appropriate for carrying out the disclosed method.

[0162] As used herein, “lipid particle” refers to any biological or synthetic particle containing a bilayer of amphiphilic lipids surrounding its lumen or cavity. Typically, lipid particles do not contain a nucleus. Examples of lipid particles include solid particles, e.g., nanoparticles, virus-derived particles, or cell-derived particles. Such lipid particles include, but are not limited to, virus particles (e.g., lentiviral particles), virus-like particles, viral vectors (e.g., lentiviral vectors), exosomes, enucleated cells, various vesicles, e.g., microvesicles, membrane vesicles, extracellular membrane vesicles, cell membrane vesicles, giant cell membrane vesicles, apoptotic bodies, mitoparticles, pyrenocytes, or lysosomes. In some embodiments, the lipid particle may be a fusosome. In some embodiments, the lipid particle is not a platelet.

[0163] As used herein, with respect to proteins such as G proteins or F proteins, “biologically active moiety” refers to a portion of a protein that exhibits or retains the activity or properties of the full-length protein. For example, the biologically active moiety of an F protein, together with a G protein, retains fusion activity when each is embedded in a lipid bilayer. The biologically active moiety of a G protein, together with an F protein, retains fusion activity when each is embedded in a lipid bilayer. The retained activity may range from 10% to 150% or more of the activity of the full-length or wild-type F protein or G protein. Examples of biologically active regions of F and G proteins include cleavage of the cytoplasmic domain, e.g., cleavage of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35 or more consecutive amino acids; see, for example, Khetawat and Broder 2010 Virology Journal 7:312, Witting et al. 2013 Gene Therapy 20:997-1005, and patent application no. WO / 2013 / 148327.

[0164] As used herein, “fusosome” refers to a particle containing an amphiphilic lipid bilayer surrounding a lumen or cavity and a fusogen that interacts with the amphiphilic lipid bilayer. In embodiments, fusosomes contain nucleic acids. In some embodiments, fusosomes are membrane-enveloped preparations. In some embodiments, fusosomes are derived from source cells.

[0165] As used herein, “fusosome composition” refers to a composition comprising one or more fusosomes.

[0166] As used herein, "fusogen" refers to a substance or molecule that creates an interaction between two membrane-enveloping lumens. In some 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 a retroviral vector and the cytoplasm of a target cell). In some embodiments, the fusogen comprises a complex of two or more proteins, e.g., none of the proteins possess fusion activity individually. In some embodiments, the fusogen comprises a targeting domain.

[0167] As used herein, “retargeted fusogen” refers to a fusogen that includes a targeting site having a sequence that is not part of the naturally occurring form of the fusogen. In embodiments, the fusogen includes a different targeting site compared to the targeting site in the naturally occurring form of the fusogen. In embodiments, the naturally occurring form of the fusogen lacks a retargeting main, 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, for example, one or more sequence modifications outside the targeting site in a transmembrane domain, a fusionally active domain, or a cytoplasmic domain compared to the naturally occurring form of the fusogen.

[0168] As used herein, “targeted envelope protein” refers to a polypeptide containing a single-domain antibody (sdAb) variable domain that targets a molecule to a desired cell type, e.g., a VL or VH-only sdAb, a nanobody, a camelid VHH domain, a shark IgNAR, or a henipavirus G protein conjugated to a fragment thereof. In some such embodiments, conjugation may be direct or indirect, via a linker such as a peptide linker.

[0169] As used herein, “targeted lipid particles” refers to lipid particles containing targeted envelope proteins embedded in a lipid bilayer.

[0170] As used herein, “retroviral nucleic acid” refers to a nucleic acid containing at least the minimum sequence requirements for packaging a retrovirus or retroviral vector, either 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 TCSRE. In some embodiments, the retroviral nucleic acid comprises one or more (e.g., all) of the following: 5'LTR (e.g., for promoting integration), U3 (e.g., for activating viral genomic RNA transcription), R (e.g., a Tat-binding region), U5, 3'LTR (e.g., for promoting integration), a packaging site (e.g., psi(Ψ)), and RRE (e.g., for binding to Rev and promoting nuclear export). The retroviral nucleic acid may comprise RNA (e.g., if it is part of a virion) or DNA (e.g., if it has been introduced into a source cell or after reverse transcription in a recipient cell). In some embodiments, retroviral nucleic acids are packaged using helper cells, helper viruses, or helper plasmids containing one or more (e.g., all) of gag, pol, and env.

[0171] As used herein, “target cells” refers to the type of cells to which targeted lipid particles are desired to deliver exogenous substances. In embodiments, target cells are cells of a specific tissue type or class, e.g., immune effector cells, e.g., T cells. In some embodiments, target cells are diseased cells, e.g., cancer cells. In some embodiments, fusogens, e.g., retargeted fusogens, result in preferential delivery of exogenous substances to target cells compared to non-target cells.

[0172] As used herein, “non-target cells” refers to a type of cell to which targeted lipid particles do not wish to deliver exogenous substances. In some embodiments, non-target cells are cells of a particular tissue type or class. In some embodiments, non-target cells are non-affected cells, e.g., non-cancer cells. In some embodiments, fusogens, e.g., retargeted fusogens, result in lower delivery of exogenous substances to non-target cells compared to target cells.

[0173] As used herein, “single-domain antibody” or “sdAb” refers to an antibody having a single monomeric domain antigen-binding / recognition domain. Such antibodies include nanobodies, camelid antibodies (e.g., VHH), or shark antibodies (e.g., IgNAR). In some embodiments, the variable domain of the sdAb comprises three CDRs and four framework regions (designated as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4). In some embodiments, the sdAb variable domain may be cleaved at the N-terminus or C-terminus to contain only partial FR1 and / or FR4, or to lack one or both of those framework regions, as long as the sdAb variable domain substantially maintains antigen-binding and specificity.

[0174] The term "CDR" refers to a complementarity determination region defined by at least one identification method for those skilled in the art. The precise amino acid sequence boundaries of a given CDR or FR are as follows: 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. The numbering scheme can be easily determined using one of several well-known schemes, including those described in 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).

[0175] The boundaries of a given CDR or FR can 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. For both the Kabat and Chothia schemes, numbering is based on the most common antibody region sequence length, with insertions provided by insertive letters, e.g., "30a", and deletions that appear in some antibodies. These two schemes place given insertions and deletions ("indels") at different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways 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.

[0176] In some embodiments, CDRs may be defined according to one of the following: the Chothia numbering scheme, the Kabat numbering scheme, a combination of Kabat and Chothia, the AbM definition, and / or the Contact definition. The sdAb variable domain includes three CDRs designated as CDR1, CDR2, and CDR3. Table 1 below lists exemplary positional boundaries of 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 CDRs; for example, FR-H1 is located before CDR-H1, FR-H2 is located between CDR-H1 and CDR-H2, FR-H3 is located between CDR-H2 and CDR-H3, and so on. Note that the Kabat numbering scheme shown places insertions at H35A and H35B, so the ends of a Chothia CDR-H1 loop will vary between H32 and H34 depending on the loop length when numbered using the Kabat numbering rules shown.

[0177] (Table 1) Boundaries of CDRs following various numbering schemes TIFF2026067851000002.tif77165

[0178] Therefore, unless otherwise specified, a given antibody or its variable region, or any "CDR" or "complementarity-determining region" in that region, or any individual specific CDR (e.g., CDR-H1, CDR-H2, CDR-H3), should be understood to encompass a certain (or specific) complementarity-determining region as defined by any of the schemes described above. For example, if it is described that a particular CDR (e.g., CDR-H3) contains the amino acid sequence of the corresponding CDR in a given sdAb amino acid sequence, it is understood that such a CDR has the sequence of the corresponding CDR (e.g., CDR-H3) in the sdAb as defined by any of the schemes described above. It is understood that any antibody, e.g., an sdAb, contains a CDR, which can be identified according to any of the other numbering schemes described above or any other numbering scheme known to those skilled in the art.

[0179] As used herein, the term “specifically binds” to a target molecule such as an antigen means that the binding molecule, e.g., a single-domain antibody, reacts or associates with a particular target molecule more frequently, more rapidly, for a longer duration, and / or with a higher affinity than alternative molecules. A binding molecule, e.g., an sdAb variable domain, “specifically binds” to a target molecule if it binds with higher affinity, avidity, more readily, and / or for a longer duration than it would if it bound to other molecules. It is understood that a binding molecule such as an sdAb that specifically binds to a first target may or may not specifically bind to a second target. Thus, “specific binding” does not necessarily require (but may include) exclusive binding.

[0180] As used herein, with respect to peptide, polypeptide, or antibody sequences, “amino acid sequence identity percentage (%)” and “homology” are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, after the sequences have been aligned and gaps introduced as necessary to achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining amino acid sequence identity percentage can be achieved in various ways within the scope of the art, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared.

[0181] Amino acid substitutions may include, but are not limited to, the replacement of one amino acid with another in a polypeptide. Exemplary substitutions are shown in Table 2. Amino acid substitutions are introduced into the antibody of interest, and the product can be screened for desired activity, e.g., retention / improved binding.

[0182] (Table 2) TIFF2026067851000003.tif131165

[0183] Amino acids can be classified according to their common side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, (3) Acidic: Asp, Glu, (4) Basicity: His, Lys, Arg, (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0184] Non-conservative substitution involves swapping one member of one of these classes with one of another.

[0185] The term "corresponds" to a protein location, for example, the statement that a nucleotide or amino acid location "corresponds" to a nucleotide or amino acid location in a disclosed sequence, such as those shown in a sequence listing, refers to a nucleotide or amino acid location 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 in similar sequences (e.g., fragments or species variants) can be determined by alignment with a reference sequence by a structural alignment method. By aligning sequences, a person skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.

[0186] The term “isolated,” as used herein, refers to a molecule that has been separated from at least a portion of its naturally occurring or typically produced components. For example, a polypeptide is referred to as “isolated” if it has been separated from at least a portion of the components of the cell in which it was produced. If a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell in which it was produced is considered “isolation” of the polypeptide. Similarly, a polynucleotide is referred to as “isolated” if it is not part of a larger polynucleotide that is naturally occurring or typically produced (e.g., in the case of a DNA polynucleotide, such as genomic DNA or mitochondrial DNA), or, for example, in the case of an RNA polynucleotide, if it has been separated from at least a portion of the components of the cell in which it was produced. Thus, a DNA polynucleotide contained in a vector inside a host cell may be referred to as “isolated.”

[0187] Where used herein, the term "effective dose" means an amount of a pharmaceutical composition sufficient to significantly and favorably modify (e.g., provide a favorable clinical response) the symptom and / or condition being treated. The effective dose of the active ingredient for use in a pharmaceutical composition varies with the knowledge and expertise of the physician, depending on the specific condition being treated, the severity of the condition, the duration of treatment, the characteristics of the combination therapy, the specific active ingredient(s) used, the specific pharmaceutically acceptable excipient(s) and / or carrier(s) used, and similar factors.

[0188] When used herein in relation to targeted lipid particles, “exogenous substance” refers to a substance that is not contained in, and does not encode, the corresponding wild-type virus or fusogen produced from the corresponding wild-type source cell. In some embodiments, the exogenous substance is a protein or nucleic acid that does not exist naturally and has a sequence modified (e.g., by insertion, addition, or substitution) relative to a naturally occurring protein. In some embodiments, the exogenous substance does not exist naturally in the source cell. In some embodiments, the exogenous substance is naturally present in the source cell but is exogenous with respect to the virus. In some embodiments, the exogenous substance does not exist naturally in the recipient cell. In some embodiments, the exogenous substance is naturally present in the recipient cell but is not present at a desired level or for a desired time. In some embodiments, the exogenous substance includes RNA or a protein.

[0189] As used herein, “promoter” refers to a cis-acting DNA sequence that, when functionally ligated to a gene coding sequence, induces the transcription of a gene. A promoter may include a transcription factor binding site. In some embodiments, a promoter acts in cooperation with one or more enhancers distal to the gene.

[0190] As used herein, composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0191] As used herein, the term “pharmaceutically acceptable” means a substance such as a carrier or diluent that does not inhibit the biological activity or properties of a compound and is relatively non-toxic; that is, the substance can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any of the components of the composition in which it is contained.

[0192] 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, dispersants, suspenders, thickeners, and / or excipients. Pharmaceutical compositions facilitate the administration of compounds to living organisms. Multiple techniques exist in the art for administering compounds, including but not limited to intravenous, oral, aerosol, parenteral, intraocular, pulmonary, and topical administration.

[0193] When used herein, “disease” or “disorder” refers to a condition that requires and / or is desired to be treated.

[0194] As used herein, “to treat,” “to treat,” or “treatment” means to improve a disease or disorder, for example, to slow, stop or reduce the progression of the disease or disorder or to reduce at least one of its clinical symptoms. For the purposes of this disclosure, improving a disease or disorder may include, but is not limited to, obtaining any beneficial or desired clinical outcome, including: reducing one or more symptoms; reducing the severity of the disease; preventing or delaying the spread of the disease (e.g., metastasis, e.g., to the lungs or lymph nodes); preventing or delaying the recurrence of the disease; slowing or delaying the progression of the disease; improving the disease state; inhibiting the disease or the progression of the disease; inhibiting or delaying the disease or its progression; stopping its progression; and remission (whether partial or whole).

[0195] The terms “individual” and “subject” are used interchangeably herein to refer to animals, e.g., mammals. The term “patient” includes humans and veterinary subjects. In some embodiments, methods are provided for treating mammals, including but not limited to humans, rodents, monkeys, cats, dogs, horses, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sports animals, and mammalian pets. Subjects may be male or female and may be of any preferred age, including infants, young, adolescent, adult, and elderly subjects. In some examples, “individual” or “subject” refers to an individual or subject requiring treatment for a disease or disorder. In some embodiments, the subject receiving treatment may be a patient, specifying the fact that the subject has an injury related to the treatment or is identified as being at sufficient risk of developing the disorder. In certain embodiments, the subject is a human, e.g., a human patient.

[0196] II. Targeted lipid particles (e.g., lentiviral vectors) This specification provides targeted lipid particles comprising a henipavirus F protein molecule or a biologically active moiety thereof, and a targeted envelope protein comprising (i) a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active moiety thereof and (ii) a binding domain, wherein the binding domain is bound to the C-terminus of the G protein or the biologically active moiety, and each of (i) and (ii) is exposed on the outer surface of the targeted lipid particle. In some embodiments, the binding domain is a single-domain antibody. In some embodiments, the binding domain is a single-stranded variable fragment. In certain embodiments, the provided lipid particles exhibit fusion activity mediated by the targeted envelope protein, which facilitates binding to target cells and facilitates the integration or fusion of the two lumens of the lipid particle and the target cell membrane, as well as the G protein or its biologically active moiety thereof, and the F glycoprotein.

[0197] This specification provides targeted lipid particles comprising a henipavirus F protein molecule or a biologically active moiety thereof, and a targeted envelope protein comprising (i) a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active moiety thereof and (ii) a single-domain antibody (sdAb) variable domain, wherein the single-domain antibody is bound to the C-terminus of the G protein or the biologically active moiety, and each of (i) and (ii) is exposed on the outer surface of the targeted lipid particle. In certain embodiments, the provided lipid particles exhibit fusion activity mediated by the targeted envelope protein, which facilitates binding to target cells and facilitates the integration or fusion of the two lumens of the lipid particle and the target cell membrane, as well as the G protein or its biologically active moiety thereof, and the F glycoprotein.

[0198] In some of the various embodiments, the targeted lipid particles are viral particles or virus-like particles. In some embodiments, such targeted lipid particles contain viral nucleic acids, e.g., retroviral nucleic acids, e.g., lentiviral nucleic acids. In certain embodiments, any provided targeted lipid particles, e.g., viral particles or virus-like particles, are replication-deficient. In some embodiments, the targeted lipid particles are lentiviral vectors, and the lentiviral vectors are pseudotyped with henipavirus F protein and targeted envelope protein.

[0199] For example, this specification provides a pseudotyped lentiviral vector comprising a henipavirus F protein molecule or a biologically active moiety thereof, and a targeted envelope protein comprising (i) a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active moiety thereof and (ii) a binding domain, wherein the binding domain is bound to the C-terminus of the G protein or the biologically active moiety, and each of (i) and (ii) is exposed on the outer surface of a targeted lipid particle. In some embodiments, the binding domain is a single-domain antibody. In some embodiments, the binding domain is a single-stranded variable fragment.

[0200] In some embodiments, the targeted lipid particles (e.g., targeted lentiviral vectors) provided herein have increased or greater targeted envelope protein expression compared to reference lipid particles (e.g., reference lentiviral vectors) that incorporate a similar envelope protein but are fused to an alternative targeting site other than the sdAb variable domain, such as a single-stranded variable fragment (scFv). In some embodiments, such targeted lipid particles are generated by pseudotyping of the lipid particles (e.g., lentiviral particles) after cotransfection of cells with transfer, envelope, and gag-pol plasmids.

[0201] In some embodiments, expression increases by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 40%, 500% or more compared to a reference lipid particle (e.g., a reference lentiviral vector), e.g., a reference lipid particle containing a similar envelope protein but fused to an scFv. In some examples, expression increases by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more compared to a reference lipid particle (e.g., a reference lentiviral vector), e.g., a reference lipid particle containing a similar envelope protein but fused to an scFv. In some embodiments, expression can be assayed in vitro using flow cytometry, e.g., FAC. In some embodiments, expression may be expressed as the number or density of targeted envelope proteins on the surface of targeted lipid particles (e.g., targeted lentiviral vectors). In some embodiments, expression may be expressed as the mean fluorescence intensity (MFI) of surface expression of targeted envelope proteins on the surface of targeted lipid particles (e.g., targeted lentiviral vectors). In some embodiments, expression may be expressed as the percentage of lipid particles (e.g., lentiviral vectors) in a population that is surface-positive for targeted envelope proteins.

[0202] In some embodiments, in a population of targeted lipid particles (e.g., targeted lentiviral vectors), 50% or more than 50% of the lipid particles are surface-positive for the targeted envelope protein. For example, in a population of provided targeted lipid particles (e.g., targeted lentiviral vectors), 55% or more than 55%, 60% or more than 60%, 65% or more than 65%, 70% or more than 70%, and 75% or more than 75% of the cells in the population are surface-positive for the targeted envelope protein.

[0203] In some embodiments, the titer of targeted lipid particles after introduction into target cells, such as by transduction (e.g., transduction cells), increases compared to the titer of reference lipid particles (e.g., reference lentiviral vectors) to the same target cells, which incorporate similar envelope proteins but are fused to alternative targeting sites other than the sdAb variable domain, such as single-stranded variable fragments (scFvs). Typically, the alternative targeting sites recognize or bind to the same target molecules as the sdAb variable domain of the targeted envelope protein of the targeted lipid particles. In some embodiments, the titer increases by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 200%, 300%, 400%, 500% or more compared to the titer of reference lipid particles (e.g., reference lentiviral vectors), such as reference lipid particles containing similar envelope proteins but fused to scFvs. In some examples, the titer increases by 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more compared to the titer of a reference lipid particle (e.g., a reference lentiviral vector), e.g., a reference lipid particle containing a similar envelope protein but fused to an scFv. In some embodiments, the titer of the targeted lipid particle in target cells (e.g., transduced cells) is 1 × 10⁻¹⁶ 6 Transduction units (TU) / mL or approximately 1 x 10⁻¹⁰ 6 The transduction unit (TU) / mL is greater than 2 × 10⁻¹⁰. For example, the titer of targeted lipid particles in target cells (e.g., transduction cells) is 2 × 10⁻¹⁰. 6 TU / mL or approximately 2 × 10 6 TU / mL or higher, 3 x 10 6 TU / mL or approximately 3 × 10 6 TU / mL or higher, 4 x 10 6 TU / mL or approximately 4 × 10 6 TU / mL or higher, 5 x 10 6 TU / mL or approximately 5 × 10 6 TU / mL or higher, 6 x 10 6 TU / mL or approximately 6 × 10 6 TU / mL or higher, 7 x 10 6TU / mL or approximately 7 × 10 6 TU / mL or higher, 8 x 10 6 TU / mL or approximately 8 x 10 6 TU / mL or higher, 9 x 10 6 TU / mL or approximately 9 x 10 6 Greater than TU / mL, or 1 × 10⁻⁶ 7 TU / mL or approximately 1 × 10⁻⁶ 7 It is greater than TU / mL.

[0204] A. Targeted envelope proteins (e.g., henipavirus-binding domain) In some embodiments, the targeted lipid particles (e.g., lentiviral vectors) include targeted envelope proteins exposed on the surface of the targeted lipid particles (e.g., lentiviral vectors).

[0205] In some embodiments, the targeted envelope protein contains a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active moiety and a binding domain that binds to a cell surface molecule on the target cell. In some embodiments, the binding domain is a single-domain antibody (sdAb). In some embodiments, the binding domain is a single-strand variable fragment (scFv). The binding domain may be linked directly or indirectly to the G protein. In certain embodiments, the binding domain is linked to the C-terminus (C-terminal amino acid) of the G protein or its biologically active moiety. Linking may be via a peptide linker, such as a flexible peptide linker.

[0206] 1. G protein In some embodiments, the targeted envelope protein comprises a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active moiety and a single-domain antibody (sdAb) variable domain or its biologically active moiety. In some embodiments, the sdAb binds to a cell surface molecule on the target cell. The sdAb variable domain may be linked directly or indirectly to the G protein. In certain embodiments, the sdAb variable domain is linked to the C-terminus (C-terminal amino acid) of the G protein or its biologically active moiety. Linking may be via a peptide linker, such as a flexible peptide linker.

[0207] In some embodiments, the binding domain (e.g., sdAb) binds to a cell surface antigen of a cell. In some embodiments, the cell surface antigen is characteristic of one type of cell. In some embodiments, the cell surface antigen is characteristic of multiple types of cells.

[0208] In some embodiments, the binding domain (e.g., sdAb) variable domain binds to a cell surface molecule or antigen. In some embodiments, the cell surface molecule is ASGR1, ASGR2, TM4SF5, CD8, CD4, or low-density lipoprotein receptor (LDL-R). In some embodiments, the cell surface molecule is ASGR1. In some embodiments, the cell surface molecule is ASGR2. In some embodiments, the cell surface molecule is TM4SF5. In some embodiments, the cell surface molecule is CD8. In some embodiments, the cell surface molecule is CD4. In some embodiments, the cell surface molecule is LDL-R.

[0209] In some embodiments, the G protein is the henipavirus G protein or its biologically active portion. In some embodiments, the henipavirus G protein is the hendra (HeV) virus G protein, the nipah (NiV) virus G protein (NiV-G), the cedar (CedPV) virus G protein, the Mojiang virus G protein, the bat paramyxovirus G protein or its biologically active portion. Table 3 provides non-limiting examples of G proteins.

[0210] The binding G protein is a type II transmembrane glycoprotein containing an N-terminal cytoplasmic tail (e.g., corresponding to amino acids 1-49 of SEQ ID NO: 9), a transmembrane domain (e.g., corresponding to amino acids 50-70 of SEQ ID NO: 9), an extracellular domain containing an extracellular stalk (e.g., corresponding to amino acids 71-187 of SEQ ID NO: 9), and a spherical head (e.g., corresponding to amino acids 188-602 of SEQ ID NO: 9). The N-terminal cytoplasmic domain is located within the inner lumen of the lipid bilayer, while the C-terminal portion is an extracellular domain exposed outside the lipid bilayer. The stalk region in the C-terminal region (e.g., corresponding to amino acids 159-167 of NiV-G) has been shown to be involved in interaction with the F protein and induction of F protein fusion (Liu et al. 2015 J of Virology 89:1838). In wild-type G proteins, the globular head mediates receptor binding to henipavirus entry receptors ephrin B2 and ephrin B3 but is unnecessary for membrane fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13)e00577-19). In certain embodiments herein, the directivity of a G protein is modified by ligation of the G protein or a biologically active fragment (e.g., a cytoplasmic cleavage) to the sdAb variable domain. Binding of the G protein to a binding partner can induce fusion mediated by a compatible F protein or its biologically active moiety. 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 for all G protein sequences disclosed herein are also intended to lack the N-terminal methionine.

[0211] G glycoproteins are highly conserved among henipavirus species. For example, the G proteins of NiV and HeV viruses share 79% amino acid identity. Studies have shown high compatibility between G proteins and F proteins of different species, as demonstrated by heteromorphic fusion activation (Brandel-Tretheway et al. Journal of Virology. 2019). As further described below, retargeted lipid particles may contain heteromorphic G and F proteins from different species.

[0212] (Table 3) Henipavirus protein G sequence clusters Column 1: The Genbank ID contains the Genbank ID for the entire viral genome sequence, which is the centroid sequence of the cluster. Column 2: The nucleotides in CDS provide the nucleotides corresponding to the CDS of genes throughout the genome. Column 3: Provides the complete gene name, including the Genbank ID, virus species, strain, and protein name. Column 4: A sequence, which provides the amino acid sequence of a gene. Column 5: Number of arrays / cluster, which provides the number of centroid arrays and the number of arrays to cluster. Column 6: Provides the array index for the sequence described. TIFF2026067851000004.tif156165TIFF2026067851000005.tif205165TIFF2026067851000006.tif196165TIFF2026067851000007.tif162165

[0213] In some embodiments, the G protein has a sequence shown in any of SEQ ID NOs: 9, 18, 28, 29, 30, 31, 44, 52, or 54-56, or a sequence with 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 8 The functionally active variant or biologically active moiety has a sequence that is identical to 7%, 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%, 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%. In certain embodiments, the G protein or functionally active variant or biologically active moiety is a protein that maintains fusion activity in combination with a henipavirus F protein, e.g., the F protein shown in section IB (e.g., NiV-F or HeV-F). Fusion activity includes the activity of the G protein in combination with the henipavirus F protein to promote or facilitate the fusion of two membrane lumens, for example, the lumen of a targeted lipid particle having henipavirus F and G proteins embedded in its lipid bilayer, and the cytoplasm of a target cell, for example, a cell containing a surface receptor or molecule recognized or bound by a targeted envelope protein. In some embodiments, the F protein and G protein are from the same henipavirus species (e.g., NiV-G and NiV-F). In some embodiments, the F protein and G protein are from different henipavirus species (e.g., NiV-G and HeV-F).

[0214] In certain embodiments, the G protein has the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 28, SEQ ID NO: 18, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 44, SEQ ID NO: 52, or SEQ ID NO: 54-56, or is a functionally active variant thereof or a biologically active portion thereof that retains fusion activity. In some embodiments, the functionally active variant comprises an amino acid sequence having 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%, 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 with the henipavirus F protein (e.g., NiV-F or HeV-F), and retains fusion activity in combination with the henipavirus F protein (e.g., NiV-F or HeV-F). In some embodiments, the biologically active portion has an amino acid sequence having 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%, 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 with SEQ ID NO: 9, SEQ ID NO: 28, SEQ ID NO: 18, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 44, SEQ ID NO: 52, or SEQ ID NO: 54-56, and retains fusion activity in combination with henipavirus F protein (e.g., NiV-F or HeV-F).

[0215] References to maintaining fusion activity include levels or degrees of binding of the corresponding wild-type G protein between 10% or about 10% and 150% or about 150%, or more, as shown in SEQ ID NOs. 9, SEQ ID NOs. 28, SEQ ID NOs. 18, SEQ ID NOs. 30, SEQ ID NOs. 31, SEQ ID NOs. 44, SEQ ID NOs. 52 or SEQ ID NOs. 54-56, for example, at least or at least about 10% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or at least about 15% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or at least about 20% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or at least about 25% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or at least about 30% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or at least about 35% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or at least about 40% of the level or degree of fusion activity of the corresponding wild-type G protein. For example, at least or about 45% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or about 50% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or about 55% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or about 60% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or about 65% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or about 70% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or about 75% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or about 80% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or about 85% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least or about 90% of the level or degree of fusion activity of the corresponding wild-type G protein,For example, the activity (combined with Nipah virus F protein) includes at least or about 95% of the level or degree of fusion activity of the corresponding wild-type G protein, at least or about 100%, or at least about 120% of the level or degree of fusion activity of the corresponding wild-type G protein.

[0216] In some embodiments, the G protein is a mutant G protein which is a functionally active variant or biologically active moiety containing one or more amino acid mutations, e.g., one or more amino acid insertions, deletions, substitutions, or cleavages. In some embodiments, the mutations described herein relate to amino acid insertions, deletions, substitutions, or cleavages of amino acids compared to a reference G protein sequence. In some embodiments, the reference G protein sequence is the wild-type sequence of the G protein or its biologically active moiety. In some embodiments, the functionally active variant or biologically active moiety is a variant of the wild-type Hendra (HeV) virus G protein, wild-type Nipah (NiV) virus G protein (NiV-G), wild-type Cedar (CedPV) virus G protein, wild-type Mojiang virus G protein, wild-type bat paramyxovirus G protein or its biologically active moiety. In some embodiments, the wild-type G protein has the sequence shown in any one of SEQ ID NOs: 9, 18, 28, 29, 30, 31, SEQ ID NOs: 44, SEQ ID NOs: 52, or SEQ ID NOs: 54-56.

[0217] In some embodiments, the G protein is a mutant G protein, which is a biologically active portion that is a fragment cleaved at the N-terminus and / or C-terminus of a wild-type Hendra (HeV) virus G protein, wild-type Nipah (NiV) virus G protein (NiV-G), wild-type Cedar (CedPV) virus G protein, wild-type Mojiang virus G protein, or wild-type bat paramyxovirus G protein. In certain embodiments, the cleavage is an N-terminal cleavage of all or part of the cytoplasmic domain. In some embodiments, the mutant G protein is a biologically active portion that is cleaved and 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 shown in SEQ ID NOs. 9, 18, 28, 29, 30, 31, SEQ ID NOs. 44, SEQ ID NOs. 52, or SEQ ID NOs. 54-56. In some embodiments, the mutant F protein is cleaved 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.

[0218] In some embodiments, the G protein is the wild-type Nipah virus G (NiV-G) protein or the Hendra virus G protein, or a functionally active variant or biologically active portion thereof. In some embodiments, the G protein is the NiV-G protein having the sequence shown in SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44, or 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 8 It is a functional variant or biologically active moiety having an amino acid sequence having sequence identity of 8% 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%, and at least 99% or about 99%.

[0219] In some embodiments, the G protein is a mutant NiV-G protein, which is the biologically active portion of wild-type NiV-G. In some embodiments, the biologically active portion is a fragment cleaved at the N-terminus. In some embodiments, the mutant NiV-G protein is cleaved and contains up to 5 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 6 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 7 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 8 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 9 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), and up to 10 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44). 0.0 amino acid residues, up to 11 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO 9, SEQ ID NO 28, or SEQ ID NO 44), up to 12 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO 9, SEQ ID NO 28, or SEQ ID NO 44), up to 13 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO 9, SEQ ID NO 28, or SEQ ID NO 44), up to 14 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO 9, SEQ ID NO 28, or SEQ ID NO 44), up to 15 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO 9, SEQ ID NO 28, or SEQ ID NO 44), up to 16 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO 9, SEQ ID NO 28, or SEQ ID NO 44), wild-type NiV-G protein (SEQ ID NO 9,Up to 17 consecutive amino acid residues at or near the N-terminus of SEQ ID NO: 28 or SEQ ID NO: 44, up to 18 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44), up to 19 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44), up to 20 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44) The base, up to 21 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 22 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 23 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) or up to 24 consecutive amino acid residues near or at the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 25 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 26 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 27 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 44) Up to 28 consecutive amino acid residues at or near the N-terminus of (SEQ ID NO. 28 or SEQ ID NO. 44), up to 29 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO. 9, SEQ ID NO. 28 or SEQ ID NO. 44), up to 30 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO. 9, SEQ ID NO. 28 or SEQ ID NO. 44), up to 31 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO. 9, SEQ ID NO. 28 or SEQ ID NO. 44),Up to 32 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 33 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 34 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) Up to 35 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 36 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 37 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 38 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 39 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 40 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 41 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to It lacks 42 consecutive amino acid residues, up to 43 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), up to 44 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), or up to 45 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44).

[0220] 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 without a cytoplasmic domain is encoded by SEQ ID NO: 32.

[0221] In some embodiments, the mutant NiV-G protein contains the sequence shown in any of SEQ ID NOs: 10-15, 35-40, 45-50, 22, 53 or SEQ ID NO: 32, or 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 It is a functional variant having an amino acid sequence with sequence identity of 87% or approximately 87%, at least 88% or approximately 88%, or at least 89% or approximately 89%, approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0222] In some embodiments, the mutant NiV-G protein is cleaved at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), for example, as shown in SEQ ID NO: 10 or SEQ ID NO: 10, with 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%. , a functional variant having sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%, or, for example, as shown in Sequence ID No. 35. or sequence number 35 and at least 80% or approximately 80%, at least 81% or approximately 81%, at least 82% or approximately 82%, at least 83% or approximately 83%, 84% or approximately 84%, at least 85% or approximately 85%, at least 86% or approximately 86%, or at least 87% or approximately 87%, at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93% , a functional variant having sequence identity of at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%, or, for example, the one shown in SEQ ID NO: 45 or SEQ ID NO: 45 with at least 80% or approximately 80%, at least 81% or approximately 81%, at least 82% or approximately 82%, at least 83% or approximately 83%, 84% or approximately 84%, at least 85% or approximately 85%,It has a functional variant having sequence identity of at least 86% or approximately 86%, or at least 87% or approximately 87%, at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%. In some embodiments, the mutant NiV-G protein is cleaved 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%, 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%, and at least 94%. or a functional variant having sequence identity of approximately 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%, or, for example, as shown in sequence number 36 or sequence number 36 with 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%,A functional variant having sequence identity of at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%, or, for example, as shown in SEQ ID NO: 46 or at least 80% or approximately 80%, at least 81% or approximately 81%, at least 82% or approximately 82%, at least 83% or approximately 83%, 84% or approximately 84%, at least 85% or approximately 85%, It has a functional variant with sequence identity of at least 86% or approximately 86%, or at least 87% or approximately 87%, at least 88% or approximately 88%, at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0223] In some embodiments, the mutant NiV-G protein is cleaved at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), for example, as shown in SEQ ID NO: 12 or SEQ ID NO: 12, 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%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%, A functional variant having an amino acid sequence with sequence identity of at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%, as shown in, for example, SEQ ID NO: 37 Those that are included or number 37 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 about 92%, at least 93% or about 9 Its functional variant having sequence identity of 3%, 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%, or, for example, as shown in SEQ ID NO: 47 or 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%,It has a functional variant with sequence identity of at least 86% or approximately 86%, or at least 87% or approximately 87%, at least 88% or approximately 88%, at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%. In some embodiments, the mutant NiV-G protein is cleaved at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), for example, as shown in SEQ ID NO: 13 or SEQ ID NO: 13, 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%. A functional variant having sequence identity of %, at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%, or, for example, as shown in SEQ ID NO: 38. or sequence number 38 and at least 80% or approximately 80%, at least 81% or approximately 81%, at least 82% or approximately 82%, at least 83% or approximately 83%, 84% or approximately 84%, at least 85% or approximately 85%, at least 86% or approximately 86%, or at least 87% or approximately 87%, at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93% , a functional variant having sequence identity of at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%, or, for example, as shown in sequence number 48 or sequence number 48 with at least 80% or approximately 80%, at least 81% or approximately 81%, at least 82% or approximately 82%, at least 83% or approximately 83%, 84% or approximately 84%, at least 85% or approximately 85%,It has a functional variant with sequence identity of at least 86% or approximately 86%, or at least 87% or approximately 87%, at least 88% or approximately 88%, at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%. In some embodiments, the mutant NiV-G protein is cleaved at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), for example, as shown in SEQ ID NO: 14 or SEQ ID NO: 14, 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%. A functional variant having sequence identity of %, at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%, or, for example, as shown in sequence number 39. or sequence number 39 and at least 80% or approximately 80%, at least 81% or approximately 81%, at least 82% or approximately 82%, at least 83% or approximately 83%, 84% or approximately 84%, at least 85% or approximately 85%, at least 86% or approximately 86%, or at least 87% or approximately 87%, at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93% , a functional variant having sequence identity of at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%, or, for example, as shown in sequence number 49 or sequence number 49 with at least 80% or approximately 80%, at least 81% or approximately 81%, at least 82% or approximately 82%, at least 83% or approximately 83%, 84% or approximately 84%, at least 85% or approximately 85%,It has a functional variant with sequence identity of at least 86% or approximately 86%, or at least 87% or approximately 87%, at least 88% or approximately 88%, at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%. In some embodiments, the mutant NiV-G protein is cleaved at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44) 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%, 84% or about 84%, at least 85% or about 85%, at least 86% or about 86%, or at least 87% or about 87%. A functional variant having sequence identity of %, at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%, or, for example, as shown in sequence number 40. or sequence number 40 and at least 80% or approximately 80%, at least 81% or approximately 81%, at least 82% or approximately 82%, at least 83% or approximately 83%, 84% or approximately 84%, at least 85% or approximately 85%, at least 86% or approximately 86%, or at least 87% or approximately 87%, at least 88% or approximately 88%, or at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93% , a functional variant having sequence identity of at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%, or, for example, as shown in SEQ ID NO: 50 or at least 80% or approximately 80%, at least 81% or approximately 81%, at least 82% or approximately 82%, at least 83% or approximately 83%, 84% or approximately 84%, at least 85% or approximately 85%,It has a functional variant with sequence identity of at least 86% or approximately 86%, or at least 87% or approximately 87%, at least 88% or approximately 88%, at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%. In some embodiments, the mutant NiV-G protein is cleaved at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), for example, as shown in SEQ ID NO: 22 or SEQ ID NO: 22, 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 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 a functional variant thereof having at least 99% or approximately 99% sequence identity, or, for example, the one shown in SEQ ID NO: 53 or SEQ ID NO: 53 with at least 80% or approximately 80%, at least 81% or approximately 81%, at least 82% or approximately 82%, at least 83% or approximately 83%, 84% or approximately 84%, at least 85% or approximately 85%, at least 86% or approximately 86%, or at least 87% or approximately 87%, at least 88% or It has a functional variant having sequence identity of approximately 88%, or at least 89%, at least 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%. In some embodiments, the mutant HiV-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% , lacking the N-terminal cytoplasmic domain of the wild-type HiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44), as indicated by its functional variant having at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99% sequence identity.

[0224] In some embodiments, the mutant G protein is a mutant HeV-G protein having the sequence shown in SEQ ID NO: 18 or 52, or 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%, at least It is a functional variant or biologically active moiety having an amino acid sequence having sequence identity of 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%.

[0225] In some embodiments, the G protein is a mutant HeV-G protein, which is the biologically active portion of wild-type HeV-G. In some embodiments, the biologically active portion is a fragment cleaved at the N-terminus. In some embodiments, the mutant HeV-G protein is cleaved 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: 18 or 52), up to 6 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 7 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 8 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 9 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 10 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 11 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein ( Up to 12 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 13 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 14 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 15 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 16 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 17 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 18 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 19 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52),Up to 20 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 21 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 22 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 23 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52) Up to 24 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 25 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 26 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 27 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), and the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52) Up to 28 consecutive amino acid residues at or near the end, up to 29 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 30 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 31 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), and at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52). Up to 32 consecutive amino acid residues, up to 33 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 34 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 35 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 36 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52),Up to 37 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 38 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 39 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 40 consecutive amino acid residues at or near the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52), at the N-terminus of wild-type HeV-G protein (SEQ ID NO: 18 or 52) It lacks up to 41 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 42 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 43 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), up to 44 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52), or up to 45 consecutive amino acid residues at or near the N-terminus of the wild-type HeV-G protein (SEQ ID NO: 18 or 52). 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 has a functional variant that has 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 with SEQ ID NO: 33 or SEQ ID NO: 33, as shown in its functional variant.It lacks the N-terminal cytoplasmic domain of the wild-type HeV-G protein (SEQ ID NO: 18 or 52).

[0226] In some embodiments, the G protein or a functionally active variant or biologically active moiety thereof binds to ephrin B2 or ephrin B3. In some embodiments, the G protein is a functionally active variant or biologically active moiety thereof having the amino acid sequence shown in any one of SEQ ID NOs: 9, SEQ ID NOs: 18 or 28, SEQ ID NOs: 29, SEQ ID NOs: 44, SEQ ID NOs: 30 or 31, or capable of binding to ephrin B2 or ephrin B3. In some embodiments, the functionally active variant or biologically active moiety 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 with SEQ ID NO: 9, SEQ ID NO: 18 or SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 31, or a functionally active variant or biologically active moiety thereof, and retains binding to ephrin B2 or B3. References to retain binding to ephrin B2 or B3 include at least or about 5% of the level or degree of binding of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 9, SEQ ID NO: 18 or SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 31; 10% of the level or degree of binding of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 9, SEQ ID NO: 18 or SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 31; 15% of the level or degree of binding of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 9,20% of the binding level or degree of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 18 or SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 31; 25% of the binding level or degree of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 9, SEQ ID NO: 18 or SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 31; 30% of the binding level or degree of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 9, SEQ ID NO: 18 or SEQ ID NO: 28, SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 31; 35% of the binding level or degree of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 9, SEQ ID NO: 18 or SEQ ID NO: 28, 40% of the binding level or degree of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 31; 45% of the binding level or degree of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 9, SEQ ID NO: 18 or SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 31; 50% of the binding level or degree of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 9, SEQ ID NO: 18 or SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 31; 55% of the binding level or degree of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 9, SEQ ID NO: 18 or SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 44,60% of the level or degree of binding of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 30 or SEQ ID NO: 31; 65% of the level or degree of binding of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 9, SEQ ID NO: 18 or SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 31; 70% of the level or degree of binding of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 9, SEQ ID NO: 18 or SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 31; for example, at least or at least about 75% of the level or degree of binding of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 9, SEQ ID NO: 1 At least or about 80% of the level or degree of binding of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 8 or SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 31, for example, at least or about 85% of the level or degree of binding of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 9, SEQ ID NO: 18 or SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 31, for example, at least or about 90% of the level or degree of binding of the corresponding wild-type G protein, or its functionally active variant or biologically active moiety, as shown in SEQ ID NO: 9, SEQ ID NO: 18 or SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 44, SEQ ID NO: 30 or SEQ ID NO: 31,or the binding includes at least or about 95% of the level or degree of binding of the functionally active variant or biologically active moiety thereof. In some embodiments, the G protein is NiV-G or a functionally active variant or biologically active moiety thereof that binds to ephrin B2 or ephrin B3. In some embodiments, NiV-G is a functionally active variant or biologically active moiety thereof having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44, or that can bind to ephrin B2 or ephrin B3. In some embodiments, a functionally active variant or biologically active moiety 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 with SEQ ID NOs. Exemplary biologically active moieties include, for example, N-terminal cleavage variants lacking all or part of the cytoplasmic domain, e.g., one or more, e.g., 1 to 49 consecutive N-terminal amino acid residues, as shown in any one of SEQ ID NOs. 10-15, 35-40, 45-50, and 32. References to retaining binding to ephrin B2 or B3 include at least or about 5% of the level or degree of binding of the corresponding wild-type NiV-G as shown in SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44, 10% of the level or degree of binding of the corresponding wild-type NiV-G as shown in SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44, 15% of the level or degree of binding of the corresponding wild-type NiV-G as shown in SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44, 20% of the level or degree of binding of the corresponding wild-type NiV-G as shown in SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44,25% of the level or degree of binding of the corresponding wild-type NiV-G as shown in SEQ ID NO: 28 or SEQ ID NO: 44, 30% of the level or degree of binding of the corresponding wild-type NiV-G as shown in SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44, 35% of the level or degree of binding of the corresponding wild-type NiV-G as shown in SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44, 40% of the level or degree of binding of the corresponding wild-type NiV-G as shown in SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44, 45% of the level or degree of binding of the corresponding wild-type NiV-G as shown in SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44, 50% of the level or degree of binding of the corresponding wild-type NiV-G as shown in SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44, 55% of the level or degree of binding of the corresponding wild-type NiV-G as shown in SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44, 60% of the level or degree of binding of the corresponding wild-type NiV-G as shown in SEQ ID NO: 9, SEQ ID NO: 28 or SEQ ID NO: 44 The binding includes levels or degrees of 65% of the corresponding wild-type NiV-G binding, 70% of the corresponding wild-type NiV-G binding level or degree as shown in SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44, for example, at least or at least about 75% of the corresponding wild-type NiV-G binding level or degree as shown in SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44, for example, at least or at least about 80% of the corresponding wild-type NiV-G binding level or degree as shown in SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44, for example, at least or at least about 85% of the corresponding wild-type NiV-G binding level or degree as shown in SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44, for example, at least or at least about 90% of the corresponding wild-type NiV-G binding level or degree as shown in SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44, or for example, at least or at least about 95% of the corresponding wild-type NiV-G binding level or degree as shown in SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44.

[0227] In some embodiments, the G protein is HeV-G or a functionally active variant or bioactive moiety thereof that binds to ephrin B2 or ephrin B3. In some embodiments, HeV-G is a functionally active variant or bioactive moiety thereof having the amino acid sequence shown in SEQ ID NO: 18 or 52, or capable of binding to ephrin B2 or ephrin B3. In some embodiments, a functionally active variant or biologically active moiety 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 with SEQ ID NO: 18 or 52, and retains binding to ephrin B2 or B3. Exemplary biologically active moieties include, for example, N-terminal cleavage variants lacking all or part of the cytoplasmic domain, e.g., one or more, e.g., 1 to 49 consecutive N-terminal amino acid residues, as shown in any one of SEQ ID NOs: 33. References to retain binding to ephrin B2 or B3 include at least or at least about 5% of the level or degree of binding of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, 10% of the level or degree of binding of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, 15% of the level or degree of binding of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, 20% of the level or degree of binding of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, 25% of the level or degree of binding of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, 30% of the level or degree of binding of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, 35% of the level or degree of binding of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52,40% of the binding level or degree of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, 45% of the binding level or degree of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, 50% of the binding level or degree of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, 55% of the binding level or degree of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, 60% of the binding level or degree of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, 65% of the binding level or degree of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, 70% of the binding level or degree of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, for example. The binding includes at least or about 75% of the level or degree of binding of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, for example, at least or about 80% of the level or degree of binding of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, for example, at least or about 85% of the level or degree of binding of the corresponding wild-type NIV-G as shown in SEQ ID NO: 18 or 52, for example, at least or about 90% of the level or degree of binding of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52, or for example, at least or about 95% of the level or degree of binding of the corresponding wild-type HeV-G as shown in SEQ ID NO: 18 or 52.

[0228] In some embodiments, the G protein or its biologically active moiety 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 its biologically active moiety 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 its biologically active moiety, for example, the mutant NiV-G protein, exhibits reduced binding to its native binding partner. In some embodiments, the reduced binding to ephrin B2 or ephrin B3 is reduced by more than 5% 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 about 100%.

[0229] In some embodiments, the mutations described herein can improve transduction efficiency. In some embodiments, the mutations described herein enable specific targeting of other desired cell types other than ephrin B2 or ephrin B3. In some embodiments, the mutations described herein at least partially prevent binding to at least one intrinsic receptor, for example, by reducing binding to at least one of ephrin B2 or ephrin B3. In some embodiments, the mutations described herein interfere with intrinsic receptor recognition.

[0230] In some embodiments, the G protein contains one or more amino acid substitutions in residues involved in interactions with one or both of ephrin B2 and ephrin B3. In some embodiments, the amino acid substitutions correspond to mutations E501A, W504A, Q530A, and E533A, with reference to the numbering shown in Sequence ID No. 28.

[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 shown in SEQ ID NO: 28, and its biologically active portion contains an N-terminal cleavage. In some embodiments, the mutant NiV-G protein or its biologically active portion is cleaved, with up to 5 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), 6 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), 7 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), 8 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), 9 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), up to 10 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), and at the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28) 11 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), 12 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), 13 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), 14 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), up to 15 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), 16 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), 17 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), 18 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28),19 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), up to 20 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), 21 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), 22 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), 23 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28) 24 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), up to 25 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), 26 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), 27 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), at the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28) 28 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), 29 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), up to 30 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), up to 31 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), 32 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), 33 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), 34 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), 35 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), up to 36 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28), up to 37 consecutive amino acid residues at or near the N-terminus of wild-type NiV-G protein (SEQ ID NO: 28),It lacks up to 38 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), up to 39 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28), or up to 40 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 28).

[0232] In some embodiments, the mutant NiV-G protein has an amino acid sequence that is 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% of sequence identity with SEQ ID NO: 16 or 51. In certain embodiments, the G protein has an amino acid sequence that is at least 99% or about 99% of the sequence shown in SEQ ID NO: 16 or 51.

[0233] In some embodiments, the targeted envelope protein contains a G protein or a functionally active variant or a biologically active moiety and an sdAb variable domain, and the targeted envelope protein exhibits increased binding to a molecule different from the native binding partner of the wild-type G protein. In some embodiments, the molecule may be a protein expressed on the surface of a desired target cell. In some embodiments, the increased binding to the other molecule increases by 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 about 100%. In certain embodiments, the binding confers a retargeted binding compared to the binding of the wild-type G protein, in which case a new or different binding activity is conferred.

[0234] 2. Joint Domain In some embodiments, the binding domain may be any substance that binds to a cell surface molecule on a target cell. In some embodiments, the binding domain may be an antibody or an antibody moiety or fragment.

[0235] The binding domain can be modified to have different binding strengths. For example, scFv and antibodies with varying binding strengths can be used to modify the fusion activity of chimeric binding proteins to cells showing large or small amounts of the target antigen. For example, DARPin with different affinities can be used to modify the fusion activity to cells showing large or small amounts of the target antigen. The binding domain can also be modified to target different regions on the target ligand, which affects the rate of fusion with cells presenting the target.

[0236] The binding domains include: humanized antibody molecules, intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®); antibody fragments (e.g., Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof); single-stranded Fvs; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, e.g., IgNAR or fragments thereof); camel-like antibodies; masked antibodies (e.g., Probodies®); small module immunotherapies ("SMIPs™"); single-stranded or tandem diaphragms. This may include Bodies (TandAb(registered trademark)); VHH; Anticalins(registered trademark); Nanobodies(registered trademark); Minibodies; BiTE(registered trademark); Ankyrin Repeat Protein or DARPIN(registered trademark); Avimer(registered trademark); DART; TCR-like antibodies; Adnectin(registered trademark); Affilin(registered trademark); Trans-bodies(registered trademark); Affibodies(registered trademark); TrimerX(registered trademark); MicroProteins; Fynomers(registered trademark), Centyrins(registered trademark); and KALBITOR(registered trademark). Targeting sites may also include antibodies or their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, disulfide-linked Fv(sdFv), Fd fragment consisting of VH and CH1 domains, linear antibodies, single-domain antibodies, e.g., sdAb(either VL or VH), nanobodies, or camelid VHH domains), antigen-binding fibronectin type III (Fn3) skeletons, e.g., fibronectin polypeptide minibodies, ligands, cytokines, chemokines, or T cell receptors (TCRs).

[0237] In some embodiments, the binding domain is a single-stranded molecule. In some embodiments, the binding domain is a single-domain antibody. In some embodiments, the binding domain is a single-stranded variable fragment. In certain embodiments, the binding domain contains a human or humanized antibody variable sequence(s).

[0238] In some embodiments, the binding domain is a single-domain antibody. In some embodiments, the single-domain antibody may be human or humanized. In some embodiments, the single-domain antibody or its portion is naturally occurring. In some embodiments, the single-domain antibody or its portion is synthetic.

[0239] In some embodiments, a single-domain antibody is an antibody in which the complementarity-determining region is part of a single-domain polypeptide. In some embodiments, a single-domain antibody is an antibody variable domain consisting only of a heavy chain. In some embodiments, a single-domain antibody does not contain a light chain.

[0240] In some embodiments, heavy-chain antibodies lacking a light chain are referred to as VHH. In some embodiments, single-domain antibodies have a molecular weight of 12-15 kDa. In some embodiments, single-domain antibodies include camelid antibodies or shark antibodies. In some embodiments, single-domain antibody molecules are derived from antibodies established in camelid species, such as camels, llamas, dromedary camels, alpacas, vicuñas, and guanacos. In some embodiments, single-domain antibodies are referred to as immunoglobulin neoantigen receptors (IgNARs) and are derived from cartilaginous fish. In some embodiments, single-domain antibodies are produced by splitting the dimeric variable domain of human or mouse IgG into monomers and camelidizing key residues.

[0241] In some embodiments, single-domain antibodies may be generated from a phage display library. In some embodiments, the phage display library is generated from a camelid VHH repertoire 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), and Decanniere et al., Structure, 7, 361-370 (1999). In some embodiments, the phage display library is generated by including antibody fragments from unimmunized camelids. In some embodiments, a single-domain antibody library of human single-domain antibodies is generated synthetically by introducing diversity into one or more scaffolds.

[0242] In some embodiments, the C-terminus of a single-domain antibody is bound to the C-terminus of a G protein or its biologically active portion. In some embodiments, the N-terminus of a single-domain antibody is exposed on the outer surface of a lipid bilayer. In some embodiments, the N-terminus of a single-domain antibody binds to cell surface molecules of a target cell. In some embodiments, the single-domain antibody specifically binds to cell surface molecules present on the target cell. In some embodiments, the cell surface molecules are proteins, glycans, lipids, or low molecular weight molecules.

[0243] In some embodiments, the cell surface molecule of the target cell is an antigen or a part thereof. In some embodiments, the single-domain antibody or a part thereof is an antibody having a single monomeric domain antigen-binding / recognition domain that can selectively bind to a specific antigen. In some embodiments, the single-domain antibody binds to an antigen present on the target cell.

[0244] Exemplary cells include polymorphonuclear cells (PMN, PML, PMNL, or also known as granulocytes), stem cells, embryonic stem cells, neural stem cells, mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), human myogenic stem cells, muscle-derived stem cells (MuStem), embryonic stem cells (ES or ESC), limbal epithelial stem cells, cardiomyogenic stem cells, cardiomyocytes, progenitor cells, immune effector cells, lymphocytes, macrophages, dendritic cells, natural killer cells, T cells, cytotoxic T lymphocytes, allogeneic cells, resident cardiac cells, induced pluripotent stem cells (iPS), adipose-derived or phenotypically modified stem or progenitor cells, CD133+ cells, aldehyde dehydrogenase positive cells (ALDH+), umbilical cord blood (UCB) cells, peripheral blood stem cells (PBSC), neurons, neural progenitor cells, pancreatic beta cells, glial cells, or hepatocytes.

[0245] In some embodiments, the target cell is a cell of a target tissue. The target tissue can include the liver, lung, heart, spleen, pancreas, digestive tract, kidney, testis, ovary, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye.

[0246] In some embodiments, the target cell is a muscle cell (e.g., a skeletal muscle cell), a kidney cell, a liver cell (e.g., a hepatocyte), or a heart cell (e.g., a cardiomyocyte). In some embodiments, the target cell is a heart cell, e.g., a cardiomyocyte (e.g., a quiescent cardiomyocyte), a hepatoblast (e.g., a cholangiolar hepatoblast), an epithelial cell, a T cell (e.g., a naive T cell), a macrophage (e.g., a tumor infiltrating macrophage), or a fibroblast (e.g., a cardiac fibroblast).

[0247] In some embodiments, target cells are tumor-infiltrating lymphocytes, T cells, neoplasms or tumor cells, virus-infected cells, stem cells, central nervous system (CNS) cells, hematopoietic stem cells (HSCs), liver cells, or fully differentiated cells. In some embodiments, target cells are CD3+ T cells, CD4+ T cells, CD8+ T cells, hepatocytes, hematopoietic stem cells, CD34+ hematopoietic stem cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD105+ endothelial cells, B cells, CD20+ B cells, CD19+ B cells, cancer cells, CD133+ cancer cells, EpCAM+ cancer cells, CD19+ cancer cells, Her2 / Neu+ cancer cells, GluA2+ neurons, GluA4+ neurons, NKG2D+ natural killer cells, SLC1A3+ astrocytic cells, SLC7A10+ adipocytes, or CD30+ lung epithelial cells.

[0248] In some embodiments, the target cells are antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, atypical antigen-presenting cells, macrophages, dendritic cells, bone marrow dendritic cells, plasmacytoid dendritic cells, CD11c+ cells, CD11b+ cells, spleen cells, B cells, hepatocytes, endothelial cells, or non-cancerous cells.

[0249] In some embodiments, the cell surface molecule is one of the following: CD8, CD4, asialoglycoprotein receptor 2 (ASGR2), transmembrane 4 L6 family member 5 (TM4SF5), low-density lipoprotein receptor (LDLR), or asialoglycoprotein 1 (ASGR1).

[0250] In some embodiments, the G protein or a functionally active variant or biologically active moiety is directly ligated to the sdAb variable domain. In some embodiments, the targeted envelope protein is a fusion protein having the following structure: (N'-single-domain antibody-C')-(C'-G protein-N').

[0251] In some embodiments, a G protein or a functionally active variant or biologically active moiety thereof is indirectly linked to the sdAb variable domain via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a chemical linker.

[0252] In some embodiments, the linker is a peptide linker, and the targeted envelope protein is a fusion protein containing a G protein or a functionally active variant or biologically active moiety linked to the sdAb variable domain via the peptide linker. In some embodiments, the targeted envelope protein is a fusion protein having the following structure: (N'-single-domain antibody-C')-linker-(C'-G protein-N').

[0253] In some embodiments, the peptide linker has a length of up to 65 amino acids. In some embodiments, the peptide linker is approximately 2-65 amino acids, 2-60 amino acids, 2-56 amino acids, 2-52 amino acids, 2-48 amino acids, 2-44 amino acids, 2-40 amino acids, 2-36 amino acids, 2-32 amino acids, 2-28 amino acids, 2-24 amino acids, 2-20 amino acids, 2-18 amino acids, 2-14 amino acids, 2-12 amino acids, 2-10 amino acids, 2-8 amino acids, 2-6 amino acids, 6-65 amino acids, 6-60 amino acids, 6-56 amino acids, 6-52 amino acids, 6-48 amino acids, 6-44 amino acids, 6- 40 amino acids, 6-36 amino acids, 6-32 amino acids, 6-28 amino acids, 6-24 amino acids, 6-20 amino acids, 6-18 amino acids, 6-14 amino acids, 6-12 amino acids, 6-10 amino acids, 6-8 amino acids, 8-65 amino acids, 8-60 amino acids, 8-56 amino acids, 8-52 amino acids, 8-48 amino acids, 8-44 amino acids, 8-40 amino acids, 8-36 amino acids, 8-32 amino acids, 8-28 amino acids, 8-24 amino acids, 8-20 amino acids, 8-18 amino acids, 8-14 amino acids, 8-12 amino acids, 8-10 amino acids, 10-65 amino acids, 10-60 amino acids, 10-56 amino acids, 10-52 amino acids, 10-48 amino acids, 10-44 amino acids, 10-40 amino acids, 10-36 amino acids, 10-32 amino acids, 10-28 amino acids, 10-24 amino acids, 10-20 amino acids, 10-18 amino acids, 10-14 amino acids, 10-12 amino acids, 12-65 amino acids, 12-60 amino acids, 12-56 amino acids, 12-52 amino acids, 12-48 amino acids, 12-44 amino acids, 12-40 amino acids, 12-36 amino acids, 12-32 amino acids, 12-28 amino acids, 12-24 amino acids, 12-20 amino acids, 12-18 amino acids, 12-14 amino acids, 14-65 amino acids, 14-60 amino acids, 14-56 amino acids, 14-52 amino acids, 14-48 amino acids, 14-44 amino acids, 14-40 amino acids, 14-36 amino acids, 14-32 amino acids, 14-28 amino acids, 14-24 amino acids, 14-20 amino acids, 14-18 amino acids, 18-65 amino acids, 18-60 amino acids, 18-56 amino acids, 18-52 amino acids, 18-48 amino acids, 18-44 amino acids,18-40 amino acids, 18-36 amino acids, 18-32 amino acids, 18-28 amino acids, 18-24 amino acids, 18-20 amino acids, 20-65 amino acids, 20-60 amino acids, 20-56 amino acids, 20-52 amino acids, 20-48 amino acids, 20-44 amino acids, 20-40 amino acids, 20-36 amino acids, 20-32 amino acids, 20-28 amino acids, 20-26 amino acids, 20-24 amino acids, 24-65 amino acids, 24-60 amino acids, 2 4-56 amino acids, 24-52 amino acids, 24-48 amino acids, 24-44 amino acids, 24-40 amino acids, 24-36 amino acids, 24-32 amino acids, 24-30 amino acids, 24-28 amino acids, 28-65 amino acids, 28-60 amino acids, 28-56 amino acids, 28-52 amino acids, 28-48 amino acids, 28-44 amino acids, 28-40 amino acids, 28-36 amino acids, 28-34 amino acids, 28-32 amino acids, 32-65 amino acids, 32- 60 amino acids, 32-56 amino acids, 32-52 amino acids, 32-48 amino acids, 32-44 amino acids, 32-40 amino acids, 32-38 amino acids, 32-36 amino acids, 36-65 amino acids, 36-60 amino acids, 36-56 amino acids, 36-52 amino acids, 36-48 amino acids, 36-44 amino acids, 36-40 amino acids, 40-65 amino acids, 40-60 amino acids, 40-56 amino acids, 40-52 amino acids, 40-48 amino acids, 40-4 Contains 4 amino acids, 44-65 amino acids, 44-60 amino acids, 44-56 amino acids, 44-52 amino acids, 44-48 amino acids, 48-65 amino acids, 48-60 amino acids, 48-56 amino acids, 48-52 amino acids, 50-65 amino acids, 50-60 amino acids, 50-56 amino acids, 50-52 amino acids, 54-65 amino acids, 54-60 amino acids, 54-56 amino acids, 58-65 amino acids, 58-60 amino acids, or 60-65 amino acids. In some embodiments, the peptide linker has lengths of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,It is a polypeptide consisting of 62, 63, 64, or 65 amino acids.

[0254] In certain embodiments, the linker is a flexible peptide linker. In some such embodiments, the linker is 1 to 20 amino acids, for example, 1 to 20 amino acids consisting mainly of glycine. In some embodiments, the linker is 1 to 20 amino acids, for example, 1 to 20 amino acids consisting mainly of glycine and serine. In some embodiments, the linker is a flexible peptide linker containing the amino acids glycine and serine, referred to as a GS-linker. In some embodiments, the peptide linker includes the sequences GS, GGS, GGGGS (SEQ ID NO: 43), GGGGGS (SEQ ID NO: 41), or combinations thereof. In some embodiments, the polypeptide linker has the sequence (GGS)n (n is 1 to 10). In some embodiments, the polypeptide linker has the sequence (GGGGS)n (SEQ ID NO: 42) (n is 1 to 10). In some embodiments, the polypeptide linker has the sequence (GGGGGS)n (SEQ ID NO: 27) (n is 1 to 6).

[0255] 3. Polynucleotides This specification provides polynucleotides comprising nucleic acid sequences encoding targeted envelope proteins. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding a G protein or a biologically active portion thereof. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a single-domain antibody (sdAb) variable domain or a biologically active portion thereof. The polynucleotide may include a sequence of nucleotides encoding any of the targeted envelope proteins described above. The polynucleotide may be a synthetic nucleic acid. Expression vectors containing any of the provided polynucleotides are also provided.

[0256] In some of the various embodiments, the expression of native or synthetic nucleic acids is typically achieved by functionally ligating the nucleic acid encoding the gene of interest to a promoter and incorporating the construct into an expression vector. In some embodiments, the vector may be suitable for replication and incorporation in eukaryotes. In some embodiments, the cloning vector contains transcription and translation terminators, start sequences, and promoters useful for the expression of the desired nucleic acid sequence. In some of the various embodiments, the plasmid contains a promoter suitable for expression in cells.

[0257] In some embodiments, a polynucleotide contains at least one promoter functionally linked to control the expression of a targeted envelope protein containing a G protein and a single-domain antibody (sdAb) variable domain. For the expression of the targeted envelope protein, at least one module in each promoter functions to position the start site for RNA synthesis. The most well-known example of this is the TATA box, but in some promoters that lack a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyltransferase gene and the promoter for the SV40 gene, another element overlapping the start site itself helps to fix the location of the start.

[0258] In some embodiments, additional promoter elements, such as enhancers, control the frequency of transcription initiation. In some embodiments, the additional promoter elements are located 30–110 bp upstream of the initiation site, although it has recently been shown that some promoters also contain functional elements downstream of the initiation site. In some embodiments, the spacing between promoter elements is often flexible so that promoter function is maintained even if the elements are inverted or moved relative to one another. In some embodiments, such as with thymidine kinase (TK) promoters, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. In some embodiments, depending on the promoter, individual elements may function either cooperatively or independently to activate transcription.

[0259] A promoter may be naturally associated with a gene or polynucleotide sequence, as can be obtained by isolating a 5′ non-coding sequence located upstream of the coding segment and / or exon. Such a promoter may be referred to as “endogenous.” Similarly, an enhancer may be naturally associated with a polynucleotide sequence located either downstream or upstream of its sequence. Alternatively, a certain advantage may be obtained by positioning a coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter not normally associated with the polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer not normally associated with the polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotes, viruses, or eukaryotic cells, and promoters or enhancers that are not “naturally present,” i.e., those containing different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to synthetically generating promoter and enhancer nucleic acid sequences, the sequences may be generated using nucleic acid amplification techniques, including recombinant cloning and / or PCR, in combination with the compositions disclosed herein (U.S. Patent Nos. 4,683,202 and 5,928,906).

[0260] In some embodiments, a preferred promoter is an early cytomegalovirus (CMV) promoter sequence. In some embodiments, the promoter sequence is a potent constitutive promoter sequence capable of inducing high levels of expression of any polynucleotide sequence functionally ligated thereto. In some embodiments, a preferred promoter is elongation growth factor-la (EF-la). In some embodiments, other constitutive promoter sequences may also be used, including, but are not limited to, human gene promoters such as the monkey virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long-term repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus early promoter, Roussarcoma virus promoter, and human gene promoters, including, but are not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0261] In some embodiments, the promoter is an inductive promoter. In some embodiments, the inductive promoter provides a molecular switch that can activate the expression of a functionally linked polynucleotide sequence when such expression is desired, or deactivate the expression when expression is not desired. In some embodiments, the inductive promoter includes a metallothione promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0262] In some embodiments, exogenously controlled inducible promoters may be used to control the expression of G proteins and single-domain antibody (sdAb) variable domains. For example, radiation-inducible promoters, heat-inducible promoters, and / or drug-inducible promoters may be used, for example, to selectively induce transgene expression in a targeted region. In such embodiments, the location, duration, and level of transgene expression may be controlled by administration of an exogenous source of induction.

[0263] In some embodiments, the expression of a targeted envelope protein containing a G protein and a single-domain antibody (sdAb) variable domain is controlled using a drug-inducible promoter. For example, in some cases, the promoter, enhancer, or transactivator includes a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence, a doxycycline operator sequence, a rapamycin operator sequence, a tamoxifen operator sequence, or a hormone-reactive operator sequence, or analogs thereof. In some examples, the inducible promoter includes a tetracycline reactant (TRE). In some embodiments, the inducible promoter includes an estrogen reactant (ERE) that can activate gene expression in the presence of tamoxifen. In some examples, the drug-inducible element, e.g., the TRE, may be combined with a promoter selected to enhance transcription in the presence of a drug, e.g., doxycycline. In some embodiments, the drug-inducible promoter is a small molecule-inducible promoter.

[0264] Any of the provided polynucleotides may be modified to remove CpG motifs and / or optimize codons for translation in specific species, such as humans, dogs, cats, horses, sheep, and cattle. In some embodiments, the polynucleotides are optimized for human codon use frequency (i.e., human codon optimized). In some embodiments, the polynucleotides are modified to remove CpG motifs. In other embodiments, the provided polynucleotides are modified to remove CpG motifs and codon optimized, e.g., human codon optimized. Methods for codon optimization and CpG motif detection and modification are well known. Typically, polynucleotide optimization improves transgene expression, increases transgene stability, and preserves the amino acid sequence of the encoded polypeptide.

[0265] To evaluate the expression of targeted envelope proteins, the expression vector introduced into cells may also contain either or both a selection marker gene or a reporter gene to facilitate the identification and selection of the particles to be expressed, such as viral particles. In other embodiments, the selection marker may be loaded onto a DNA isolation fragment and used in a cotransfection procedure. Both the selection marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selection markers are known in the art and include, for example, antibiotic resistance genes, such as neo.

[0266] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Reporter genes encoding readily assayable proteins are well known in the art. Typically, a reporter gene is a gene that encodes a protein that is not present or expressed in the recipient organism or tissue, and whose expression is indicated by several readily detectable characteristics, such as enzymatic activity. Reporter gene expression is assayed at a suitable time after the DNA has been introduced into the recipient cells.

[0267] Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein gene (see, for example, Ui-Tei et al., 2000, FEBS Lett. 479:79-82). Suitable expression systems are well known and can be prepared using well-known techniques or obtained commercially. Internal deletion constructs can be generated using unique internal restriction sites or by partial digestion of non-unique restriction sites. The construct can then be transfected into cells that exhibit high levels of the desired polynucleotide and / or polypeptide expression. Usually, a construct having a minimum 5′ flanking region that shows the highest level of expression of the reporter gene is identified as a promoter. Such promoter regions can be linked to a reporter gene and used to evaluate agents for their ability to regulate promoter-induced transcription.

[0268] [[ID=,3]] B. Fusogen (e.g., henipavirus F protein) In some embodiments, the targeted lipid particles contain one or more fusogens. In some embodiments, the targeted lipid particles contain an exogenous or overexpressed fusogen. In some embodiments, the fusogen is disposed in the lipid bilayer. In some embodiments, the fusogen facilitates fusion of the targeted lipid particles to the membrane. In some embodiments, the membrane is the plasma cell membrane.

[0269] In some embodiments, the fusogens include protein-based, lipid-based, and chemical-based fusogens. In some embodiments, the targeted lipid particles contain a first fusogen that includes a protein fusogen and a second fusogen that includes a lipid fusogen or a chemical fusogen. In some embodiments, the fusogen binds to a fusogen-binding partner on the surface of the target cell.

[0270] In some embodiments, Fusogen comprises a protein having a hydrophobic fusion peptide domain. In some embodiments, Fusogen comprises a henipavirus F protein molecule or a biologically active portion thereof. In some embodiments, the henipavirus F protein is the hendra (Hev) virus F protein, the nipah (NiV) virus F protein, the cedar (CedPV) virus F protein, the Mojiang virus F protein, or the bat paramyxovirus F protein or a biologically active portion thereof.

[0271] Table 4 provides non-limiting examples of F proteins. In some embodiments, the N-terminal hydrophobic fusion peptide domain of the F protein molecule or its biologically active portion is exposed outside the lipid bilayer.

[0272] The henipavirus F protein is encoded as an F0 precursor containing a signal peptide (e.g., corresponding to amino acid residues 1-26 of SEQ ID NO: 1). After cleavage of the signal peptide, mature F0 (e.g., SEQ ID NO: 2) is transported to the cell surface, then taken up, and cleaved by cathepsin L (between amino acids 109-110 of SEQ ID NO: 1) into mature fusion subunits F1 (e.g., corresponding to amino acids 110-546 of SEQ ID NO: 1, shown in SEQ ID NO: 4) and F2 (e.g., corresponding to amino acid residues 27-109 of SEQ ID NO: 1, shown in SEQ ID NO: 3). The F1 and F2 subunits associate via disulfide bonds and are returned to the cell surface for recycling. The F1 subunit contains a fusion peptide domain located at the N-terminus of the F1 subunit (e.g., corresponding to amino acids 110-129 of SEQ ID NO: 1), which can be inserted into the cell membrane to induce fusion. In certain cases, fusion activity is blocked by the association of the F protein and the G protein until G engages with the target molecule, leading to exposure of the fusion peptide to mediate its dissociation from F and membrane fusion.

[0273] The sequence and activity of F proteins 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 in the provided retargeted lipid particles, the F protein is heterogeneous 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 embodiments, the F protein may be a chimeric F protein containing regions of F proteins from different henipavirus species. In some embodiments, exchanging amino acid residue regions of the F protein from one henipavirus species to another may result in fusion to a G protein of the species, including amino acid insertions. (Brandel-Tretheway et al. 2019). In some cases, chimeric F proteins contain an extracellular domain from one henipavirus species and a transmembrane and / or cytoplasmic domain from a different henipavirus species. For example, an F protein contains an extracellular domain from a hendravirus and a transmembrane / cytoplasmic domain from a nipahvirus. The F protein sequences disclosed herein are primarily disclosed as expressed sequences containing an N-terminal signal sequence. Since such N-terminal signal sequences are generally cleaved in-translation or post-translation, the mature protein sequences for all F protein sequences disclosed herein are also intended to lack an N-terminal signal sequence.

[0274] (Table 4) Henipavirus protein F sequence clusters Column 1: The Genbank ID contains the Genbank ID for the entire viral genome sequence, which is the centroid sequence of the cluster. Column 2: The nucleotides in CDS provide the nucleotides corresponding to the CDS of genes throughout the genome. Column 3: Complete gene name, providing the full name of the gene including Genbank ID, virus species, strain, and protein name. The Nipah virus F protein is over 80% identical to that of Hendra virus and is found within the same sequence cluster. Column 4: A sequence, which provides the amino acid sequence of a gene. Column 5: Number of arrays / cluster, which provides the number of centroid arrays and the number of arrays to cluster. Column 6: Provides the array index for the sequence described. TIFF2026067851000008.tif100165TIFF2026067851000009.tif154165TIFF2026067851000010.tif197165TIFF2026067851000011.tif123165

[0275] In some embodiments, the F protein is encoded by a nucleotide sequence encoding the sequence represented by any one of SEQ ID NOs: 1, 2, 17, 24, 25, 26 or 57-60, or is a functionally active variant or biologically active moiety 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 NOs: 1, 2, 17, 24, 25, 26 or 57-60. In certain embodiments, the F protein or a functionally active variant or biologically active moiety thereof retains fusion activity when combined with a henipavirus G protein, e.g., the G protein shown in Section IA (e.g., NiV-G or HeV-G). The fusion activity includes the activity of the F protein combined with the henipavirus G protein to promote or facilitate the fusion of two membrane lumens, e.g., the lumen of a targeted lipid particle having henipavirus F and G proteins embedded in its lipid bilayer, and the cytoplasm of a target cell, e.g., a cell containing a surface receptor or molecule recognized by or bound to a targeted envelope protein. In some embodiments, the F protein and G protein are from the same henipavirus species (e.g., NiV-G and NiV-F). In some embodiments, the F protein and G protein are from different henipavirus species (e.g., NiV-G and HeV-F). In certain embodiments, the F protein or its functionally active variant or biologically active moiety retains a cleavage site that is cleaved by cathepsin L (for example, corresponding to the cleavage site between amino acids 109-110 of SEQ ID NO: 1).

[0276] In certain embodiments, the F protein has the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 57, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60, or is a functionally active variant thereof or a biologically active moiety thereof that retains fusion activity. In some embodiments, the functionally active variant comprises an amino acid sequence having 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%, 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 with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 57, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60, and retains fusion activity in combination with henipavirus G proteins (e.g., NiV-G or HeV-G). In some embodiments, the biologically active portion has an amino acid sequence having 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%, 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 with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 57, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60, and retains fusion activity in combination with henipavirus G protein (e.g., NiV-G or HeV-G).

[0277] References to retaining fusion activity include a level or degree of binding of the corresponding wild-type F protein between 10% or about 10% and 150% or about 150%, or more, as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 57, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60, for example, at least or at least about 10% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least or at least about 15% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least or at least about 20% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least or at least about 25% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least or at least about 30% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least or at least about 35% of the level or degree of fusion activity of the corresponding wild-type F protein At least or about 40% of the fusion activity level or degree of the corresponding wild-type F protein, for example, at least or about 45% of the fusion activity level or degree of the corresponding wild-type F protein, for example, at least or about 50% of the fusion activity level or degree of the corresponding wild-type F protein, for example, at least or about 55% of the fusion activity level or degree of the corresponding wild-type f protein, for example, at least or about 60% of the fusion activity level or degree of the corresponding wild-type F protein, for example, at least or about 65% of the fusion activity level or degree of the corresponding wild-type F protein, for example, at least or about 70% of the fusion activity level or degree of the corresponding wild-type F protein, for example, at least or about 75% of the fusion activity level or degree of the corresponding wild-type F protein, for example, at least or about 80% of the fusion activity level or degree of the corresponding wild-type F protein, for example, at least or about 85% of the fusion activity level or degree of the corresponding wild-type F protein, for example,The activity (combined with henipavirus G protein) includes at least or about 90% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least or about 95%, for example, at least or about 100%, or for example, at least or about 120% of the level or degree of fusion activity of the corresponding wild-type F protein.

[0278] In some embodiments, the F protein is a mutant F protein which is a functionally active fragment or a biologically active moiety containing one or more amino acid mutations, e.g., one or more amino acid insertions, deletions, substitutions, or cleavages. In some embodiments, the mutations described herein relate to amino acid insertions, deletions, substitutions, or cleavages of amino acids compared to a reference F protein sequence. In some embodiments, the reference F protein sequence is the wild-type sequence of the F protein or its biologically active moiety. In some embodiments, the mutant F protein or its biologically active moiety is a variant of the 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 one of the following: SEQ ID NOs: 1, 2, 17, 24, 25, 26, or 57–60.

[0279] In some embodiments, the mutant F protein is the biologically active portion of the wild-type F protein, which is a fragment cleaved at the N-terminus and / or C-terminus. In some embodiments, the mutant F protein, or the biologically active portion of the wild-type F protein, 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 ability. Exemplary mutations include any of those described herein; see, for example, Khetawat and Broder 2010 Virology Journal 7:312, Witting et al. 2013 Gene Therapy 20:997-1005, Published International, Patent Application No. WO / 2013 / 148327.

[0280] In some embodiments, the mutant F protein is a cleaved and biologically active portion of the wild-type F protein, such as the wild-type F protein, lacking up to 20 consecutive amino acid residues at or near the C-terminus, encoded by the nucleotide sequence encoding the F protein shown in any one of SEQ ID NOs: 1, 17, 24, 25, or 26. In some embodiments, the mutant F protein is cleaved 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.

[0281] In some embodiments, the F protein or its functionally active variant or biologically active moiety includes an F1 subunit or its fusion moiety. In some embodiments, the F1 subunit is a proteolytically cleaved portion of the 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 produces 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.

[0282] In some embodiments, the F protein is the wild-type Nipah virus F (NiV-F) protein, or a functionally active variant or biologically active moiety thereof. In some embodiments, the F0 precursor is encoded by a sequence of nucleotides encoding the sequence shown in SEQ ID NO: 1. The encoding nucleic acid may encode a signal peptide sequence having the sequence MVVILDKRCY CNLLILILMI SECSVG (SEQ ID NO: 34). In some embodiments, the F protein has the sequence shown in SEQ ID NO: 2. In some examples, the F protein is cleaved into an F1 subunit containing the sequence shown in SEQ ID NO: 4 and an F2 subunit containing the sequence shown in SEQ ID NO: 3.

[0283] In some embodiments, the F protein is a NiV-F protein encoded by a sequence of nucleotides encoding the sequence shown in SEQ ID NO: 1, or 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 less It is a functionally active variant or biologically active moiety having an amino acid sequence having sequence identity of at least 89% or approximately 89%, at least 90% or approximately 90%, at least 91% or approximately 91%, at least 92% or approximately 92%, at least 93% or approximately 93%, at least 94% or approximately 94%, at least 95% or approximately 95%, 96% or approximately 96%, at least 97% or approximately 97%, at least 98% or approximately 98%, or at least 99% or approximately 99%. In some embodiments, the NiV-F protein is a functionally active variant or biologically active moiety having an amino acid sequence 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%, 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 with SEQ ID NO: 2.In certain embodiments, the F protein or its functionally active variant or biologically active moiety retains a cleavage site that is cleaved by cathepsin L (for example, corresponding to the cleavage site between amino acids 109-110 of SEQ ID NO: 1).

[0284] In some embodiments, the F protein or its functionally active variant or biologically active moiety includes an F1 subunit having an amino acid sequence 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%, 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 with SEQ ID NO: 4.

[0285] In some embodiments, the F protein or its functionally active variant or biologically active moiety includes an F2 subunit having an amino acid sequence 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%, 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 with SEQ ID NO: 3.

[0286] In some embodiments, the F protein is a mutant NiV-F protein which is a cleaved and biologically active portion of the wild-type NiV-F protein (e.g., SEQ ID NO: 2) lacking up to 20 consecutive amino acid residues at or near its C-terminus. In some embodiments, the mutant NiV-F protein contains the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the mutant NiV-F protein has a sequence that has 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 with SEQ ID NO: 5. In some embodiments, the mutant F protein contains an F1 protein having the sequence shown in SEQ ID NO: 6. In some embodiments, the mutant F protein has a sequence that has 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 with SEQ ID NO: 6.

[0287] In some embodiments, the F protein is a mutant NiV-F protein which is the biologically active portion of the wild-type NiV-F protein (SEQ ID NO: 2) having a 20-amino acid cleavage at or near its C-terminus; and a point mutation at the N-linked glycosylation site. In some embodiments, the mutant NiV-F protein contains the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the mutant NiV-F protein has a sequence that has 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 with SEQ ID NO: 7.

[0288] In some embodiments, the F protein is a mutant NiV-F protein, which is the biologically active portion of the wild-type NiV-F protein (SEQ ID NO: 2) having a 22-amino acid cleavage at or near its C-terminus. In some embodiments, the NiV-F protein is encoded by a nucleotide sequence encoding the sequence shown in SEQ ID NO: 8. In some embodiments, the NiV-F protein is encoded by a nucleotide sequence encoding a sequence having 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 with SEQ ID NO: 8. In certain embodiments, the variant F protein is a mutant NiV-F protein having the amino acid sequence shown in SEQ ID NO: 23. In some embodiments, the NiV-F protein is encoded by a sequence having sequence identity with SEQ ID NO: 23 of 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%.

[0289] C. Lipid bilayer In some embodiments, the targeted lipid particle includes a naturally occurring bilayer of amphiphilic lipids surrounding a lumen or cavity. In some embodiments, the targeted lipid particle includes a lipid bilayer as its outermost surface. In some embodiments, the lipid bilayer surrounds a lumen. In some embodiments, the lumen is aqueous. In some embodiments, the lumen is in contact with hydrophilic head groups inside the lipid bilayer. In some embodiments, the lumen is cytosol. In some embodiments, the cytosol contains cellular components present in the source cell. In some embodiments, the cytosol does not contain components present in the source cell. In some embodiments, the lumen is a cavity. In some embodiments, the cavity contains an aqueous environment. In some embodiments, the cavity does not contain an aqueous environment.

[0290] In some embodiments, the lipid bilayer is induced from the source cell during the process of generating lipid-containing particles. Exemplary methods for generating lipid-containing particles are provided in Section IE. In some embodiments, the lipid bilayer includes membrane components of the cell from which the lipid bilayer is generated, e.g., phospholipids, membrane proteins, etc. In some embodiments, the lipid bilayer includes cytosol containing components found in the cell from which the microvesicles are generated, e.g., solutes, proteins, nucleic acids, etc., but not all of the cellular components, for example, they lack a nucleus. In some embodiments, the lipid bilayer is considered exosome-like. The lipid bilayer can vary in size, having diameters ranging from 30 to 300 nm, such as 30 to 150 nm, including 40 to 100 nm in some examples.

[0291] In some embodiments, the lipid bilayer is the viral envelope. In some embodiments, the viral envelope is obtained from a source cell. In some embodiments, the viral envelope is obtained by a viral capsid from the cell membrane of the source cell. In some embodiments, the lipid bilayer is obtained from a membrane other than the cell membrane of the host cell. In some embodiments, the viral envelope lipid bilayer is embedded together with viral proteins, including viral glycoproteins.

[0292] In other embodiments, the lipid bilayer includes synthetic lipid complexes. In some embodiments, the synthetic lipid complex is a liposome. In some embodiments, the lipid bilayer is a vesicle structure characterized by a phospholipid bilayer membrane and an inner aqueous medium. In some embodiments, the lipid bilayer has multiple lipid layers separated by an aqueous medium. In some embodiments, the lipid bilayer spontaneously forms when phospholipids are suspended in an excess aqueous solution. In some examples, the lipid components undergo self-reorganization before the formation of adjacent structures, trapping water and dissolved solutes between the lipid bilayers.

[0293] In some embodiments, targeted envelope proteins and fusogens, such as those described above, including those exogenously or overexpressed in the source cell, are arranged in the lipid bilayer.

[0294] In some embodiments, the targeted lipid particles contain several different types of lipids. In some embodiments, the lipids are amphiphilic lipids. In some embodiments, the amphiphilic lipids are phospholipids. In some embodiments, the phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine. In some embodiments, the lipids include phospholipids such as phosphocholine and phosphoinositol. In some embodiments, the lipids include DMPC, DOPC, and DSPC.

[0295] In some embodiments, the bilayer is composed of one or more lipids of the same or different types. In some embodiments, the source cells include cells selected from 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.

[0296] D. Exogenous substances In some embodiments, the targeted lipid particle, such as a lentiviral vector, further comprises an exogenous substance (hereinafter also referred to as "cargo" or "payload") relative to the source cell. In some embodiments, the exogenous substance is a protein or nucleic acid (e.g., DNA, chromosome (e.g., human artificial chromosome), RNA, e.g., mRNA or miRNA). In some embodiments, the exogenous substance is a nucleic acid encoding a protein. The protein can be any protein desired for targeted delivery to the target cell. In some embodiments, the protein is a therapeutic or diagnostic agent. In some embodiments, the protein is an antigen receptor, e.g., a chimeric antigen receptor (CAR) or T cell receptor (TCR), for targeting cells expressed by or associated with a disease or pathological condition. References to the coding sequence of a nucleic acid encoding a protein are also referred to herein as the payload gene. In some embodiments, the exogenous substance or the nucleic acid encoding the exogenous substance resides in the lumen of the non-cellular particle.

[0297] In some embodiments, the exogenous substance or cargo comprises or encodes a cytosolic protein. In some embodiments, the exogenous substance or cargo comprises or encodes a membrane protein. In some embodiments, the exogenous substance or cargo comprises or encodes a therapeutic agent. In some embodiments, the therapeutic agent is selected from one or more of the following: proteins, e.g., enzymes, transmembrane proteins, receptors, antibodies; nucleic acids, e.g., DNA, chromosomes (e.g., human artificial chromosomes), RNA, mRNA, siRNA, miRNA, or small molecules.

[0298] In the embodiment, the exogenous substance is at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,00 It exists with 0 copies, or 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies or less. In the embodiments, the targeted lipid particles have modified, for example, increased or decreased levels of one or more endogenous molecules, such as proteins or nucleic acids (for example, endogenous to the source cell in some embodiments, and endogenous to the target cell in some embodiments), by treatment of the source cell, such as a mammalian source cell, with siRNA or a gene editing enzyme. In the embodiment, the endogenous molecules are at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,00 0 copies, or 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies or less. In embodiments, endogenous molecules (e.g., RNA or protein) are present at concentrations at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 500, 10 3 , 5.0×103 , 10 4 , 5.0×10 4 , 10 5 , 5.0×10 5 , 10 6 , 5.0×10 6 , 1.0 × 10 7 , 5.0×10 7 , or 1.0 × 10 8 It is present in high concentrations. In embodiments, endogenous molecules (e.g., RNA or protein) are present in concentrations at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 500, 10 times higher than their concentration in the source cell. 3 , 5.0×10 3 , 10 4 , 5.0×10 4 , 10 5 , 5.0×10 5 , 10 6 , 5.0×10 6 , 1.0 × 10 7 , 5.0×10 7 , or 1.0 × 10 8 It exists at low concentrations.

[0299] In some embodiments, targeted lipid particles deliver at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the cargo (e.g., therapeutic agent, e.g., exogenous therapeutic agent) contained in fusosomes to target cells. In some embodiments, targeted lipid particles fusing with target cells(s) deliver at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% on average of the cargo (e.g., therapeutic agent, e.g., exogenous therapeutic agent) contained in the lipid particles fusing with target cells(s). In some embodiments, the targeted lipid particle composition delivers at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the cargo (e.g., therapeutic agent, e.g., exogenous therapeutic agent) contained in the targeted lipid particle composition to the target tissue.

[0300] In some embodiments, the exogenous substance or cargo is not spontaneously expressed in the cells from which the targeted lipid particles originate. In some embodiments, the exogenous substance or cargo is spontaneously expressed in the cells from which the targeted lipid particles originate. In some embodiments, the exogenous substance or cargo is loaded onto the targeted lipid particles via expression in the cells from which the lipid particles originate (e.g., expression from DNA or mRNA introduced via transfection, transduction, or electroporation). In some embodiments, the exogenous substance or cargo is expressed from DNA integrated into the genome or maintained episomatically. In some embodiments, the expression of the exogenous substance or cargo is constitutive. In some embodiments, the expression of the exogenous substance or cargo is induced. In some embodiments, the expression of the exogenous substance or cargo is induced immediately before the generation of the targeted lipid particles. In some embodiments, the expression of the exogenous substance or cargo is induced concurrently with the expression of the fusogen.

[0301] In some embodiments, the exogenous substance or cargo is loaded onto the lipid particles via electroporation into the lipid particles themselves or into the cells from which the fusosomes originated. In some embodiments, the exogenous substance or cargo is loaded onto the lipid particles via transfection (e.g., of DNA or mRNA encoding the cargo) into the lipid particles themselves or into the cells from which the lipid particles originated.

[0302] In some embodiments, the exogenous substance or cargo may include one or more nucleic acid sequences, one or more polypeptides, a combination of nucleic acid sequences and / or polypeptides, one or more organelles, and any combination thereof. In some embodiments, the exogenous substance or cargo may include one or more cellular components. In some embodiments, the exogenous substance or cargo may include one or more cytosolic and / or nuclear components.

[0303] In some embodiments, the exogenous substance or cargo may include nucleic acids such as DNA, nDNA (nuclear DNA), mtDNA (mitochondrial DNA), protein-coding DNA, genes, operons, chromosomes, genomes, transposons, retrotransposons, viral genomes, introns, exons, modified DNA, mRNA (messenger RNA), tRNA (transfer RNA), modified RNA, microRNA, siRNA (small interfering molecule), tmRNA (transfer messenger RNA), rRNA (ribosomal RNA), mtRNA (mitochondrial RNA), snRNA (micronuclear RNA), micronucleolar RNA (snoRNA), SmY RNA (mRNA trans-splicing RNA), gRNA (guide RNA), TERC (telomerase RNA component), aRNA (antisense RNA), cis-NAT (cis-natural antisense transcript), and CRISPR This includes RNA (crRNA), IncRNA (long non-coding RNA), piRNA (piwi-interacting RNA), shRNA (short hairpin RNA), tasiRNA (transacting siRNA), eRNA (enhancer RNA), satellite RNA, pcRNA (protein-coding RNA), dsRNA (double-stranded RNA), RNAi (interfering RNA), circRNA (circular RNA), reprogramming RNA, aptamers, and any combination thereof. In some embodiments, the nucleic acid is wild-type nucleic acid. In some embodiments, the protein is mutant nucleic acid. In some embodiments, the nucleic acid is a fusion or chimera of multiple nucleic acid sequences.

[0304] In some embodiments, the exogenous substance or cargo may include nucleic acids. For example, the exogenous substance or cargo may include RNA to enhance the expression of an endogenous protein, or siRNA or miRNA to inhibit the protein expression of an endogenous protein. For example, the endogenous protein may have a modulated structure or function in the target cell. In some embodiments, the cargo may include nucleic acids encoding an engineered protein that modulates structure or function in the target cell. In some embodiments, the exogenous substance or cargo is a nucleic acid that targets a transcription activator that modulates structure or function in the target cell.

[0305] In some embodiments, the exogenous substance or cargo is or encodes polypeptides, e.g., enzymes, structural polypeptides, signaling polypeptides, regulatory polypeptides, transport polypeptides, sensory polypeptides, motor polypeptides, defensive polypeptides, storage polypeptides, transcription factors, antibodies, cytokines, hormones, catabolic polypeptides, anabolic polypeptides, proteolytic polypeptides, metabolic polypeptides, kinases, transferases, hydrolases, lyases, isomerases, ligases, enzyme regulator polypeptides, protein-binding polypeptides, lipid-binding polypeptides, membrane fusion polypeptides, cell differentiation polypeptides, epigenetic polypeptides, cell death polypeptides, nuclear transport polypeptides, nucleic acid fusion polypeptides, reprogramming polypeptides, DNA editing polypeptides, DNA repair polypeptides, DNA recombinant polypeptides, transposase polypeptides, DNA integration polypeptides, targeted endonucleases (e.g., zinc finger nucleases, transcription activator-like nucleases (TALENs), Cas9 and its homologs), recombinases, and any combination thereof. In some embodiments, the protein targets a protein in the cell for degradation. In some embodiments, the protein targets a protein in the cell for degradation by localizing the protein to the proteasome. In some embodiments, the protein is a wild-type protein. In some embodiments, the protein is a mutant protein. In some embodiments, the protein is a fusion or chimeric protein.

[0306] In some embodiments, the exogenous substance or cargo is a small molecule, for example, an ion (e.g., Ca 2+ , Cl-, Fe 2+These include carbohydrates, lipids, reactive oxygen species, reactive nitrogen species, isoprenoids, signaling molecules, heme, polypeptide cofactors, electron-withdrawing compounds, electron-donating compounds, metabolites, ligands, and any combination thereof. In some embodiments, the small molecule is a drug that interacts with a target in a cell. In some embodiments, the small molecule targets a protein in a cell for degradation. In some embodiments, the small molecule targets a protein in a cell for degradation by localizing the protein to the proteasome. In some embodiments, the small molecule is a proteolytically targeted chimeric molecule (PROTAC).

[0307] In some embodiments, the exogenous substance or cargo includes a mixture of proteins, nucleic acids, or metabolites, e.g., multiple polypeptides, multiple nucleic acids, multiple small molecules; combinations of nucleic acids, polypeptides, and small molecules; ribonucleoprotein complexes (e.g., Cas9-gRNA...

Claims

1. Targeted lipid particles, (a) A lipid bilayer surrounding the lumen, (b) Henipavirus F protein molecule or its biologically active portion, (c) A targeted envelope protein comprising (i) a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active moiety and (ii) a single-domain antibody (sdAb) variable domain, wherein the sdAb variable domain is bound to the C-terminus of the G protein or its biologically active moiety, and / or the sdAb is bound to the G protein or its biologically active moiety via a peptide linker, and the sdAb binds to a cell surface molecule of a target cell. The targeted lipid particle comprises the F protein molecule or its biologically active portion and the targeted envelope protein, wherein the F protein molecule or its biologically active portion and the targeted envelope protein are embedded in a lipid bilayer.

2. The targeted lipid particle according to claim 1, wherein the cell surface molecule is a protein, glycan, lipid, or low molecular weight molecule.

3. The targeted lipid particle according to claim 1 or 2, wherein the target cell is selected from the group consisting of tumor-infiltrating lymphocytes, T cells, neoplasms or tumor cells, virus-infected cells, stem cells, central nervous system (CNS) cells, hematopoietic stem cells (HSCs), liver cells, or fully differentiated cells.

4. Targeted lipid particles according to any one of claims 1 to 3, wherein the target cells are selected from the group consisting of CD3+ T cells, CD4+ T cells, CD8+ T cells, hepatocytes, hematopoietic stem cells, CD34+ hematopoietic stem cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD105+ endothelial cells, B cells, CD20+ B cells, CD19+ B cells, cancer cells, CD133+ cancer cells, EpCAM+ cancer cells, CD19+ cancer cells, Her2 / Neu+ cancer cells, GluA2+ neurons, GluA4+ neurons, NKG2D+ natural killer cells, SLC1A3+ astrocytic cells, SLC7A10+ adipocytes, or CD30+ lung epithelial cells.

5. The targeted lipid particle according to any one of claims 1 to 4, wherein the single-domain antibody binds to an antigen or a portion thereof present on hepatocytes.

6. The targeted lipid particle according to any one of claims 1 to 5, wherein the cell surface molecule or antigen is selected from the group consisting of ASGR1, ASGR2, and TM4SF.

7. The targeted lipid particle according to any one of claims 1 to 4, wherein the single-domain antibody binds to an antigen or a portion thereof present on a T cell.

8. The targeted lipid particle according to any one of claims 1 to 4 and 7, wherein the cell surface molecule or antigen is CD8 or CD4.

9. The targeted lipid particle according to any one of claims 1 to 4, wherein the cell surface molecule or antigen is LDL-R.

10. Targeted lipid particles, (a) A lipid bilayer surrounding the lumen, (b) Henipavirus F protein molecule or its biologically active portion, (c) A targeted envelope protein comprising (i) a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active moiety thereof and (ii) a binding domain, wherein the binding domain is bound to the C-terminus of the G protein or its biologically active moiety, and the binding domain binds to a cell surface molecule selected from the group consisting of ASGR1, ASGR2, TM4SF5, CD8, CD4 and LDL-R. A targeted lipid particle comprising the F protein molecule or its biologically active portion and the targeted envelope protein, wherein the F protein molecule or its biologically active portion and the targeted envelope protein are embedded in the lipid bilayer.

11. The targeted lipid particle according to claim 10, wherein the binding domain is bound to the G protein via a linker.

12. The targeted lipid particle according to claim 11, wherein the linker is a peptide linker.

13. A lentiviral vector, the targeted lipid particle according to any one of claims 1 to 12.

14. (a) Henipavirus F protein molecule or its biologically active portion, (b) A targeted envelope protein comprising (i) a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active moiety and (ii) a binding domain, wherein the binding domain is bound to the C-terminus of the G protein or its biologically active moiety, and the binding domain binds to CD4, (c) A cargo comprising a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR is (i) an extracellular antigen-binding domain that binds to CD19, wherein the extracellular antigen-binding domain is optionally scFv, (ii) Transmembrane domain, and (iii) Intracellular signaling region containing the CD3 zeta signaling domain Including the cargo and A lentiviral vector containing...

15. The lentiviral vector according to claim 14, wherein the intracellular signaling region of the CAR further comprises a 4-1BB costimulatory signaling domain.

16. The lentiviral vector according to claim 14 or claim 15, wherein the lentiviral vector is capable of delivering the nucleic acid encoding the CAR to a T cell, and optionally the T cell is in vivo in the subject.

17. (a) Henipavirus F protein molecule or its biologically active portion, (b) A targeted envelope protein comprising (i) a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active moiety and (ii) a binding domain, wherein the binding domain is attached to the C-terminus of the G protein or its biologically active moiety, and the binding domain binds to a cell surface molecule selected from the group consisting of ASGR1, ASGR2 and TM4SF5. A lentiviral vector containing...

18. The lentiviral vector according to claim 17, which is capable of targeting liver cells.

19. A lentiviral vector according to any one of claims 17 and 18, further comprising an exogenous substance for delivery to hepatocytes, and / or the lentiviral vector is capable of delivering the exogenous substance to hepatocytes, and optionally the hepatocytes are in vivo in the subject.

20. The lentiviral vector according to any one of claims 14 to 19, wherein the binding domain is bound to the G protein via a linker, and the linker is optionally a peptide linker.

21. The targeted lipid particle according to any one of claims 10 to 13 or the lentiviral vector according to any one of claims 14 to 20, wherein the binding domain is a single-domain antibody or a single-stranded variable fragment (scFv).

22. The targeted lipid particle according to any one of claims 1 to 9, 12, and 13, or the lentiviral vector according to claim 20 or claim 21, wherein the peptide linker comprises a length of up to 65 amino acids, and optionally 2 to 65 amino acids.

23. The peptide linker is a flexible linker comprising GS, GGS, GGGGS (SEQ ID NO: 43), GGGGGS (SEQ ID NO: 41), or a combination thereof, or the peptide linker comprises (GGS)n where n is 1 to 10, (GGGGGS)n where n is 1 to 10 (SEQ ID NO: 42), or (GGGGGS)n where n is 1 to 6 (SEQ ID NO: 27), as described in any one of claims 1 to 9, 12, 13, and 22, or the lentiviral vector as described in any one of claims 20 to 22.

24. The targeted lipid particle according to any one of claims 1 to 13, 22, and 23, or the lentiviral vector according to any one of claims 14 to 23, wherein the G protein or its biologically active portion is wild-type Nipah virus G (NiV-G) protein or Hendra virus G protein, or a functionally active variant or biologically active portion thereof.

25. The targeted lipid particle according to any one of claims 1 to 13 and 22 to 24, wherein the G protein or its biologically active portion is a wild-type NiV-G protein or a functionally active variant or biologically active portion, or the lentiviral vector according to any one of claims 14 to 24.

26. The NiV-G protein or its functionally active variant or biologically active moiety 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%, and at least 88%. A targeted lipid particle or lentiviral vector according to claim 24 or claim 25, comprising an amino acid sequence having sequence identity of approximately 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%.

27. The NiV-G protein is A cleaved and biologically active moiety lacking up to 40 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44). The targeted lipid particles or lentiviral particles according to any one of claims 24 to 26.

28. The NiV-G protein is a biologically active portion cleaved at the N-terminus of wild-type NiV-G, and is a sequence shown in any of SEQ ID NOs: 10-15, 35-40, or 45-50, or a sequence 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%, at least 86% or about 86%, at least 87% or about 87%, at least 88% Targeted lipid particles or lentiviral particles according to any one of claims 24 to 27, having an amino acid sequence having sequence identity of % 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%.

29. The NiV-G protein is a biologically active moiety having a cleavage at or near the N-terminus of the wild-type NiV-G protein, selected from the group consisting of a 5-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein, a 10-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein, a 15-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein, a 20-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein, a 25-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein, a 30-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein, or a 34-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein, and optionally the wild-type NiV-G protein is shown in SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44, as described in any one of claims 24 to 28.

30. The targeted lipid particle or lentiviral vector according to any one of claims 24 to 29, wherein the NiV-G protein is a biologically active portion having a 34-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44).

31. The targeted lipid particle according to any one of claims 1 to 13 and 22 to 30, wherein the G protein or its biologically active portion is a mutant NiV-G protein exhibiting reduced binding to ephrin B2 or ephrin B3, or the lentiviral vector according to any one of claims 14 to 30.

32. The aforementioned mutant NiV-G protein, Refer to the numbering shown in Sequence ID No. 28 and select one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A. A targeted lipid particle or lentiviral vector according to claim 31, comprising:

33. The aforementioned mutant NiV-G protein, Refer to the numbering shown in Sequence ID No. 28 for amino acid substitutions E501A, W504A, Q530A, and E533A. A targeted lipid particle or lentiviral vector according to claim 31 or claim 32, comprising:

34. The mutant NiV-G protein or the biologically active portion is 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%, A targeted lipid particle or lentiviral vector according to any one of claims 31 to 33, having an amino acid sequence having sequence identity of 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%.

35. The targeted lipid particle according to any one of claims 1 to 13 and 22 to 34, wherein the F protein or its biologically active portion is wild-type Nipah virus F (NiV-F) protein or Hendra virus F protein, or a functionally active variant or biologically active portion thereof, or the lentiviral vector according to any one of claims 14 to 34.

36. The targeted lipid particle according to any one of claims 1 to 13 and 22 to 35, wherein the F protein or its biologically active portion is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof, or the lentiviral vector according to any one of claims 14 to 35.

37. The NiV-F protein or its functionally active variant or biologically active moiety is the amino acid sequence shown in SEQ ID NO: 2, or 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%. Alternatively, a targeted lipid particle according to any one of claims 1 to 13 and 22 to 36, comprising an amino acid sequence having sequence identity of approximately 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%, or a lentiviral vector according to any one of claims 14 to 36.

38. The NiV-F protein, i) A 20-amino acid cleavage at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 2), and / or ii) Point mutations in N-linked glycosylation sites A targeted lipid particle according to any one of claims 1 to 13 and 22 to 37, or a lentiviral vector according to any one of claims 14 to 37, comprising the biologically active portion thereof.

39. The NiV-F protein or the biologically active portion is also 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%. The targeted lipid particle or lentiviral vector according to claim 38, having an amino acid sequence having sequence identity of approximately 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%.

40. The targeted lipid particle according to any one of claims 1 to 13 and 22 to 37, wherein the NiV-F protein is the biologically active portion of the wild-type NiV-F protein (SEQ ID NO: 2) having a 22-amino acid cleavage at or near its C-terminus, or the lentiviral vector according to any one of claims 14 to 37.

41. The NiV-F protein or the biologically active portion is 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%, and The targeted lipid particle or lentiviral vector according to claim 40, having an amino acid sequence encoded by a sequence of nucleotides encoding a sequence having 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.

42. A targeted lipid particle according to any one of claims 1 to 13 and 22 to 41, wherein the F protein comprises the sequence shown in SEQ ID NO: 23, and the G protein comprises the sequence shown in SEQ ID NO: 16, or a lentiviral vector according to any one of claims 14 to 41.

43. The targeted lipid particle according to any one of claims 1 to 13 and 22 to 42, wherein the lipid bilayer is derived from the membrane of a host cell used to generate a retrovirus or retrovirus-like particle.

44. The targeted lipid particle according to claim 43, wherein the host cell is selected from the group consisting of 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, MRC 5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH 3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells.

45. The targeted lipid particle according to any one of claims 1 to 13 and 22 to 44, wherein the lipid bilayer is a viral envelope or comprises one.

46. Targeted lipid particles according to any one of claims 1 to 13 and 22 to 45, comprising one or more viral components other than the F protein molecule and the G protein.

47. The targeted lipid particle according to claim 46, wherein one or more viral components are derived from a retrovirus.

48. The targeted lipid particle according to any one of claims 43 to 47, wherein the retrovirus is a lentivirus or a lentivirus-like particle.

49. A targeted lipid particle according to any one of claims 1 to 13 and 22 to 48, further comprising an exogenous substance, or a lentiviral vector according to any one of claims 14 to 42.

50. The targeted lipid particle or lentiviral vector according to claim 49, wherein the exogenous substance is present in the lumen.

51. The targeted lipid particle or lentiviral vector according to claim 49 or claim 50, wherein the exogenous substance is a protein or nucleic acid, and optionally the nucleic acid is DNA or RNA.

52. The targeted lipid particle or lentiviral vector according to any one of claims 49 to 51, wherein the exogenous substance is a nucleic acid encoding cargo for delivery to the target cells.

53. The targeted lipid particle or lentiviral vector according to any one of claims 49 to 52, wherein the exogenous substance encodes a therapeutic agent or a diagnostic agent.

54. The targeted lipid particle or lentiviral vector according to any one of claims 49 to 53, wherein the exogenous substance encodes a membrane protein, and optionally the membrane protein is an antigen receptor for targeting cells expressed by or associated with a disease or pathological condition.

55. The targeted lipid particle or lentiviral vector according to claim 53, wherein the membrane protein is a chimeric antigen receptor (CAR).

56. The targeted lipid particle or lentiviral vector according to any one of claims 52 to 55, wherein the target cell is a T cell.

57. The targeted lipid particle or lentiviral vector according to any one of claims 49 to 53, wherein the exogenous substance is a nucleic acid containing a gene defect, optionally a payload gene for correcting the gene defect in the target cell, and optionally the gene defect is related to liver cells or hepatocytes.

58. A polynucleotide comprising a nucleic acid sequence encoding (i) a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion thereof and (ii) a single-domain antibody (sdAb) variable domain, wherein the sdAb variable domain is conjugated to the C-terminus of the G protein or a biologically active portion thereof.

59. The polynucleotide according to claim 58, wherein the single-domain antibody binds to a cell surface molecule present on a target cell.

60. The polynucleotide according to claim 59, wherein the cell surface molecule is a protein, a glycan, a lipid, or a low molecular weight molecule.

61. The polynucleotide according to claim 59 or 60, wherein the target cells are selected from the group consisting of tumor-infiltrating lymphocytes, T cells, neoplasms or tumor cells, virus-infected cells, stem cells, central nervous system (CNS) cells, hematopoietic stem cells (HSCs), liver cells, or fully differentiated cells.

62. The polynucleotide according to any one of claims 59 to 61, wherein the target cells are selected from the group consisting of CD3+ T cells, CD4+ T cells, CD8+ T cells, hepatocytes, hematopoietic stem cells, CD34+ hematopoietic stem cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD105+ endothelial cells, B cells, CD20+ B cells, CD19+ B cells, cancer cells, CD133+ cancer cells, EpCAM+ cancer cells, CD19+ cancer cells, Her2 / Neu+ cancer cells, GluA2+ neurons, GluA4+ neurons, NKG2D+ natural killer cells, SLC1A3+ astrocytic cells, SLC7A10+ adipocytes, or CD30+ lung epithelial cells.

63. The polynucleotide according to any one of claims 59 to 62, wherein the cell surface molecule or antigen is selected from the group consisting of ASGR1, ASGR2, TM4SF5, CD8, CD4, and low-density lipoprotein receptor (LDL-R).

64. A polynucleotide comprising a nucleic acid sequence encoding (i) a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion and (ii) a binding domain that binds to a cell surface molecule selected from the group consisting of ASGR1, ASGR2, TM4SF5, CD4, CD8, and low-density lipoprotein receptor (LDL-R).

65. The polynucleotide according to claim 64, wherein the binding domain is a single-domain antibody (sdAb) or a single-stranded variable fragment (scFv).

66. The polynucleotide according to any one of claims 58 to 65, wherein the nucleic acid sequence is a first nucleic acid sequence, and the polynucleotide further comprises a second nucleic acid sequence encoding a henipavirus F protein molecule or a biologically active portion thereof.

67. The polynucleotide according to claim 66, wherein the polynucleotide comprises an IRES or a sequence encoding a linking peptide between the first and second nucleic acid sequences, and optionally the linking peptide is a self-cleaving peptide or a peptide that causes ribosome skipping, optionally a T2A peptide.

68. The polynucleotide according to any one of claims 58 to 67, further comprising at least one promoter functionally linked to control the expression of the nucleic acid, optionally the expression of the first nucleic acid sequence and the second nucleic acid sequence.

69. The polynucleotide according to any one of claims 58 to 68, wherein the sdAb variable domain or the binding domain is bound to the G protein via an encoded peptide linker.

70. The polynucleotide according to claim 69, wherein the encoded peptide linker has a length of up to 65 amino acids, and optionally 2 to 65 amino acids.

71. The polynucleotide according to claim 69 or 70, wherein the encoded peptide linker comprises GS, GGS, GGGGS (SEQ ID NO: 43), GGGGGS (SEQ ID NO: 41) and combinations thereof, or the encoded peptide linker comprises (GGS)n where n is 1 to 10, (GGGGGS)n where n is 1 to 10 (SEQ ID NO: 42), or (GGGGGS)n where n is 1 to 4 (SEQ ID NO: 27).

72. The polynucleotide according to any one of claims 58 to 71, wherein the encoded G protein is wild-type Nipah virus G (NiV-G) protein or Hendra virus G protein, or a functionally active variant or biologically active moiety thereof, wherein the variant is a variant that exhibits reduced binding to its native binding partner.

73. The polynucleotide according to any one of claims 58 to 72, wherein the encoded G protein is a wild-type NiV-G protein or a functionally active variant or biologically active moiety thereof.

74. The NiV-G protein or its functionally active variant or biologically active moiety comprises the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44, or comprises 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%, and The polynucleotide according to claim 72 or claim 73, comprising an amino acid sequence having sequence identity of 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%.

75. The NiV-G protein is A cleaved and biologically active moiety lacking up to 40 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44). The polynucleotide according to any one of claims 72 to 74.

76. The NiV-G protein is a biologically active portion cleaved at the N-terminus of wild-type NiV-G, and 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%, less than The polynucleotide according to any one of claims 72 to 75, comprising an amino acid sequence having sequence identity of 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%.

77. The polynucleotide according to any one of claims 72 to 76, wherein the NiV-G protein is a biologically active moiety having a cleavage at or near the N-terminus of the wild-type NiV-G protein, selected from the group consisting of a 5-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein, a 10-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein, a 15-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein, a 20-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein, a 30-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein, or a 34-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein, and optionally the wild-type NiV-G protein is shown in SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO:

44.

78. The polynucleotide according to any one of claims 72 to 77, wherein the NiV-G protein is a biologically active portion having a 34-amino acid cleavage at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44).

79. The polynucleotide according to any one of claims 58 to 78, wherein the G protein is a mutant NiV-G protein that exhibits reduced binding to ephrin B2 or ephrin B3.

80. The polynucleotide according to claim 79, wherein the mutant NiV-G protein 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 shown in Sequence ID No.

28.

81. The polynucleotide according to claim 79 or claim 80, wherein the mutant NiV-G protein comprises amino acid substitutions E501A, W504A, Q530A and E533A with reference to the numbering shown in Sequence ID No.

28.

82. The polynucleotide according to any one of claims 79 to 81, wherein the mutant NiV-G protein comprises an amino acid sequence 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 with SEQ ID NO:

16.

83. The polynucleotide according to any one of claims 66 to 82, wherein the F protein or its biologically active portion is wild-type Nipah virus F (NiV-F) protein or Hendra virus F protein, or a functionally active variant or biologically active portion thereof.

84. The polynucleotide according to any one of claims 66 to 83, wherein the F protein or its biologically active portion is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.

85. The NiV-F protein or its functionally active variant or biologically active moiety is 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 The polynucleotide according to claim 83 or claim 84, comprising an amino acid sequence having sequence identity of 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%.

86. The NiV-F protein, i) A 20-amino acid cleavage at or near the C-terminus of the wild-type NiV-F protein (SEQ ID NO: 2), and / or ii) Point mutations in N-linked glycosylation sites The polynucleotide according to claim 85 or claim 85, which includes the biologically active portion thereof.

87. The polynucleotide according to claim 86, wherein the NiV-F protein or the biologically active portion has an amino acid sequence having 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 with the sequence shown in SEQ ID NO: 5 or SEQ ID NO:

7.

88. The polynucleotide according to claim 85 or claim 86, wherein the NiV-F protein is the biologically active portion of the wild-type NiV-F protein (SEQ ID NO: 2) having a 22-amino acid cleavage at or near its C-terminus.

89. The NiV-F protein or the biologically active portion is the sequence shown in SEQ ID NO: 8 or SEQ ID NO: 23, or 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%. The polynucleotide according to claim 88, having an amino acid sequence encoded by a sequence of nucleotides encoding a sequence having sequence identity of %, 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%.

90. The polynucleotide according to any one of claims 66 to 89, wherein the F protein comprises the sequence shown in SEQ ID NO: 23, and the G protein comprises the sequence shown in SEQ ID NO:

16.

91. A vector comprising a polynucleotide according to any one of claims 58 to 90.

92. The vector according to claim 91, wherein the vector is a mammalian vector, a viral vector, or an artificial chromosome, and optionally the artificial chromosome is a bacterial artificial chromosome (BAC).

93. A plasmid comprising a polynucleotide according to any one of claims 58 to 90.

94. The plasmid according to claim 93, further comprising one or more nucleic acids encoding proteins for lentivirus generation.

95. A cell comprising a polynucleotide according to any one of claims 58 to 90, or a vector according to claim 91 or 92, or a plasmid according to claim 93 or 94.

96. A method for producing targeted lipid particles comprising a henipavirus F protein molecule or its biologically active portion, and a targeted envelope protein comprising a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion and a single-domain antibody (sdAb) variable domain, a) To provide a cell comprising a nucleic acid encoding a henipavirus F protein molecule or a biologically active portion thereof, and a nucleic acid encoding a targeted envelope protein, wherein the targeted envelope protein comprises a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion thereof and a single-domain antibody (sdAb) variable domain, b) Culturing the cells under conditions that enable the generation of targeted lipid particles, and c) Separating, concentrating, or purifying the targeted lipid particles from the cells, thereby producing the targeted lipid particles. The method, including the method described above.

97. A method for producing a pseudotyped lentiviral vector, a) To provide producer cells comprising lentiviral nucleic acids (multiple may be allowed), a nucleic acid encoding a henipavirus F protein molecule or a biologically active portion thereof, and a nucleic acid encoding a targeted envelope protein, wherein the targeted envelope protein comprises henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion thereof and a single-domain antibody. b) Culturing the cells under conditions that enable the generation of the lentiviral vector, and c) Isolating, concentrating, or purifying the lentiviral vector from the cells, thereby producing the pseudotyped lentiviral vector. The method, including the method described above.

98. A method for producing targeted lipid particles comprising a henipavirus F protein molecule or its biologically active portion, and a targeted envelope protein comprising a henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion and binding domain, a) To provide a cell comprising a nucleic acid encoding a henipavirus F protein molecule or a biologically active portion thereof, and a nucleic acid encoding a targeted envelope protein, wherein the targeted envelope protein comprises a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion and a binding domain, and the binding domain is (i) ASGR1, ASGR2, and TM4SF5, optionally bound to a cell surface molecule selected from the group consisting of human ASGR1, human ASGR2, and human ASGR2, (ii) A cell surface molecule that binds to CD4 or CD8, or is selected from the group consisting of human CD4 or human CD8, or (iii) Low-density lipoprotein receptor (LDL-R), which optionally binds to a cell surface molecule that is human LDL-R. To provide as described above, b) Culturing the cells under conditions that enable the generation of targeted lipid particles, and c) Separating, concentrating, or purifying the targeted lipid particles from the cells, thereby producing the targeted lipid particles. The method, including the method described above.

99. A method for producing a pseudotyped lentiviral vector, a) Providing producer cells comprising lentiviral nucleic acid(s), a nucleic acid encoding a henipavirus F protein molecule or a biologically active portion thereof, and a nucleic acid encoding a targeted envelope protein, wherein the targeted envelope protein comprises a henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion and a binding domain, and the binding domain is (i) ASGR1, ASGR2, and TM4SF5, optionally bound to a cell surface molecule selected from the group consisting of human ASGR1, human ASGR2, and human ASGR2, (ii) A cell surface molecule that binds to CD4 or CD8, or is selected from the group consisting of human CD4 or human CD8, or (iii) Low-density lipoprotein receptor (LDL-R), which optionally binds to a cell surface molecule that is human LDL-R. To provide as described above, b) Culturing the producer cells under conditions that enable the generation of lentiviral vectors, and c) Isolating, concentrating, or purifying the lentiviral vector from the cells, thereby producing the pseudotyped lentiviral vector. The method, including the method described above.

100. The method according to claim 98 or claim 99, wherein the binding domain is a single-domain antibody or a single-stranded variable fragment (scFv).

101. A method for producing targeted lipid particles comprising a henipavirus F protein molecule or its biologically active portion and a targeted envelope protein, a) To provide a cell comprising a polynucleotide according to any one of claims 58 to 90, or a vector according to claim 91 or claim 92, or a plasmid according to claim 93 or claim 94, b) Culturing the cells under conditions that enable the generation of targeted lipid particles, and c) Separating, concentrating, or purifying the targeted lipid particles from the cells, thereby producing the targeted lipid particles. The method, including the method described above.

102. A method for producing a pseudotyped lentiviral vector, a) To provide a producer cell comprising lentiviral nucleic acid (or multiple nucleic acids), a polynucleotide according to any one of claims 58 to 90, or a vector according to claim 91 or claim 92, or a plasmid according to claim 93 or claim 94, b) Culturing the cells under conditions that enable the generation of the lentiviral vector, and c) Isolating, concentrating, or purifying the lentiviral vector from the cells, thereby producing the pseudotyped lentiviral vector. The method, including the method described above.

103. The method according to claim 101 or claim 102, further comprising providing the cells with a henipavirus F protein molecule or a polynucleotide encoding a biologically active portion thereof, prior to step (b).

104. The method according to any one of claims 96 to 103, wherein the cells are mammalian cells.

105. The method according to any one of claims 96 to 104, wherein the cell is a producer cell containing viral nucleic acid, optionally retroviral nucleic acid, or lentiviral nucleic acid, and the targeted lipid particle is a viral particle or virus-like particle, optionally retroviral particle or retroviral-like particle, optionally lentiviral particle or lentiviral-like particle.

106. A producer cell comprising a polynucleotide according to any one of claims 58 to 90, or a vector according to claim 91 or claim 92, or a plasmid according to claim 93 or claim 94.

107. The producer cell according to claim 106, further comprising a nucleic acid encoding the henipavirus F protein or a biologically active portion thereof.

108. The producer cell according to claim 106 or claim 107, wherein the cell further comprises viral nucleic acid, and optionally the viral nucleic acid is lentiviral nucleic acid.

109. A producer cell comprising (i) viral nucleic acid(s), (ii) nucleic acid encoding a henipavirus F protein molecule or a biologically active portion thereof, and (iii) nucleic acid encoding a targeted envelope protein including henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion thereof and a single-domain antibody (sdAb) variable domain, wherein the viral nucleic acid(s) is optionally lentiviral nucleic acid.

110. The producer cell according to claim 109, wherein the single-domain antibody binds to a cell surface molecule present on the target cell.

111. The producer cell according to claim 110, wherein the cell surface molecule is a protein, glycan, lipid, or low molecular weight molecule.

112. The producer cell according to claim 110 or claim 111, wherein the target cell is selected from the group consisting of tumor-infiltrating lymphocytes, T cells, neoplasms or tumor cells, virus-infected cells, stem cells, central nervous system (CNS) cells, hematopoietic stem cells (HSCs), liver cells, or fully differentiated cells.

113. The producer cell according to any one of claims 110 to 112, wherein the target cell is selected from the group consisting of CD3+ T cells, CD4+ T cells, CD8+ T cells, hepatocytes, hematopoietic stem cells, CD34+ hematopoietic stem cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD105+ endothelial cells, B cells, CD20+ B cells, CD19+ B cells, cancer cells, CD133+ cancer cells, EpCAM+ cancer cells, CD19+ cancer cells, Her2 / Neu+ cancer cells, GluA2+ neurons, GluA4+ neurons, NKG2D+ natural killer cells, SLC1A3+ astrocytocytes, SLC7A10+ adipocytes, or CD30+ lung epithelial cells.

114. A producer cell comprising (i) viral nucleic acid(s), (ii) nucleic acid encoding a henipavirus F protein molecule or a biologically active portion thereof, and (iii) nucleic acid encoding a targeted envelope protein including henipavirus envelope-binding glycoprotein G (G protein) or a biologically active portion and a binding domain thereof, wherein the binding domain is (i) ASGR1, ASGR2, and TM4SF5, optionally bound to a cell surface molecule selected from the group consisting of human ASGR1, human ASGR2, and human ASGR2, (ii) A cell surface molecule that binds to CD4 or CD8, or is selected from the group consisting of human CD4 or human CD8, or (iii) Low-density lipoprotein receptor (LDL-R), which optionally binds to a cell surface molecule that is human LDL-R, The viral nucleic acid(s) may be any one of the above, and the viral nucleic acid(s) may be a lentiviral nucleic acid. The aforementioned producer cells.

115. The producer cell according to claim 114, wherein the binding domain is a single-domain antibody or a single-stranded variable fragment.

116. The producer cell according to any one of claims 109 to 115, wherein the F protein or its biologically active portion is wild-type Nipah virus F (NiV-F) protein or Hendra virus F protein, or a functionally active variant or biologically active portion thereof.

117. The producer according to any one of claims 109 to 116, wherein the F protein or its biologically active portion is a wild-type NiV-F protein or a functionally active variant or biologically active portion thereof.

118. The henipavirus F protein molecule or its biologically active portion (i) Sequences shown in Sequence ID 2, Sequence ID 5, Sequence ID 7, Sequence ID 8 or Sequence ID 23, (ii) Amino acid sequences having 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%, 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% sequence identity with SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:

23. A producer cell according to any one of claims 109 to 117, including the following:

119. The producer cell according to any one of claims 109 to 118, wherein the encoded G protein is wild-type Nipah virus G (NiV-G) protein or Hendra virus G protein, or a functionally active variant or biologically active moiety thereof, wherein the variant is optionally a variant that exhibits reduced binding to its native binding partner.

120. The producer cell according to any one of claims 109 to 119, wherein the encoded G protein is a wild-type NiV-G protein or a functionally active variant or biologically active portion thereof.

121. The henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion (i) The sequence shown in Sequence ID No. 9, Sequence ID No. 28, or Sequence ID No. 44, (ii) Amino acid sequences having 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%, 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% sequence identity with SEQ ID NO: 9, SEQ ID NO: 28, or SEQ ID NO: 44 A producer cell according to any one of claims 109 to 120, including the following:

122. The henipavirus envelope-binding glycoprotein G (G protein) or its biologically active portion (i) Sequences shown in sequence numbers 10-16, 35-40 or 45-51, (ii) Amino acid sequences having sequence identity of 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%, 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%. A producer cell according to any one of claims 109 to 121, including the following:

123. The producer cells have membrane (e.g., cell membrane) expression of the targeting envelope protein, which is more abundant in the membrane (e.g., cell membrane) compared to reference producer cells in which the same envelope protein is incorporated into the membrane (e.g., cell membrane) but is fused to the alternative targeting site, and optionally the alternative targeting site is a single-stranded variable fragment (scFv), and / or The producer cells express the targeted envelope protein on the membrane (e.g., cell membrane) of the producer cells, wherein the targeted envelope protein is expressed at least 20 proteins per square micron (e.g., at least 50, 100, 200, 500, 1,000, 2,000, 5,000, or 10,000 proteins), and / or the targeted envelope protein constitutes at least 0.1% (e.g., at least 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%) of the total membrane (e.g., cell membrane) protein of the producer cells (e.g., relative to the total protein weight). Producer cells according to any one of claims 109 to 122.

124. Targeted lipid particles or pseudotyped lentiviral vectors generated by the method described in any one of claims 96 to 105 or from producer cells described in any one of claims 106 to 122.

125. The targeted lipid particle according to any one of claims 1 to 13, 21 to 57, and 124, wherein the targeted lipid particle has a higher expression of the targeted envelope protein compared to a reference lipid particle in which the same envelope protein is incorporated into a similar lipid bilayer but is fused to an alternative targeting site, and optionally the alternative targeting site is a single-stranded variable fragment (scFv).

126. The titer in the target cells after transfection is 1×10 ６ transfection units (TU) / mL or more, 2×10 ６ TU / mL or more, 3×10 ６ TU / mL or more, 4×10 ６ TU / mL or more, 5×10 ６ TU / mL or more, 6×10 ６ TU / mL or more, 7×10 ６ TU / mL or more, 8×10 ６ TU / mL or more, 9x10 ６ TU / mL or more, or 1x10 ７ TU / mL or more, and / or The targeting envelope protein is at least about (0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, or 0.5) in number of targeting envelope proteins / nm ２ The density present on the surface of the targeted lipid particles is Targeted lipid particles according to any one of claims 1 to 13, 21 to 57, 124 and 125; lentiviral vector according to any one of claims 13 to 42, 49 to 57 and 124.

127. A composition comprising a plurality of targeted lipid particles or a plurality of lentiviral vectors according to any one of claims 1 to 57 and 124 to 126.

128. The composition according to claim 127, further comprising a pharmaceutically acceptable carrier.

129. In the population of lipid particles or lentiviral vectors in the composition, 50% or more than 50%, 55% or more than 55%, 60% or more than 60%, 65% or more than 65%, 70% or more than 70%, or 75% or more than 75% are surface positive for the targeted envelope protein, and / or The targeting envelope protein is at least about (0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, or 0.5) in number of targeting envelope proteins / nm ２ The average density present on the surface of the targeted lipid particles is The composition according to claim 127 or claim 128.

130. A method for transducing cells, comprising transducing cells with a lentiviral vector according to any one of claims 13-42, 49-57, and 124, or a composition comprising a lentiviral vector according to any one of claims 13-42, 49-57, and 124, or a plurality of lentiviral vectors.

131. The targeted envelope protein of the lentiviral vector or targeted lipid particle targets CD4, and the cell is a CD4+ cell, or The targeted envelope protein of the lentiviral vector targets CD8, and the cell is a CD8+ cell, or The targeted envelope protein of the lentiviral vector targets ASGR1, ASGR2, or TM4SF5, and the cells are hepatocytes. The method according to claim 130.

132. A method for delivering an exogenous substance to a subject (for example, a human subject), comprising administering a targeted lipid particle or lentiviral vector according to any one of claims 49 to 57, 125 and 126 to the subject, wherein the targeted lipid particle or lentiviral vector contains the exogenous substance.

133. A method for delivering an exogenous substance to a subject (for example, a human subject), comprising administering to the subject a composition according to any one of claims 127 to 129, wherein a plurality of targeted lipid particles or lentiviral vectors contain the exogenous substance.

134. A method for delivering a chimeric antigen receptor (CAR) to a cell, comprising contacting the cell with a lentiviral vector according to any one of claims 14-16 and 19-42 or a targeted lipid particle according to claim 55 or claim 56, wherein the lentiviral vector or targeted lipid particle contains a nucleic acid encoding the CAR.

135. A method for delivering a chimeric antigen receptor (CAR) to a cell, comprising contacting the cell with a composition according to any one of claims 127 to 129, wherein a plurality of lentiviral vectors or targeted lipid particles contain nucleic acids encoding the CAR.

136. A method for delivering an exogenous substance to hepatocytes, comprising contacting the cells with a lentiviral vector according to any one of claims 17 to 42 or a targeted lipid particle or lentiviral vector according to claim 57.

137. A method for delivering an exogenous substance to hepatocytes, comprising contacting the cells with a composition according to any one of claims 127 to 129, wherein a plurality of lentiviral vectors or targeted lipid particles contain the exogenous substance for delivery to the hepatocytes.

138. The method according to any one of claims 134 to 137, wherein the contact comprises transduction into the cells with a lentiviral vector or the targeted lipid particles, and optionally the contact is in vivo in the subject.

139. A method for treating a disease or disorder in a subject (for example, a human subject), comprising administering to the subject a targeted lipid particle according to any one of claims 1 to 13, 21 to 57, and 124 to 126, or a lentiviral vector according to any one of claims 13 to 42, 49 to 57, 124, and 126, or a composition according to any one of claims 127 to 129.

140. A method for fusing mammalian cells to targeted lipid particles, comprising administering to a subject the targeted lipid particles according to any one of claims 1 to 13, 21 to 57, and 124 to 126, the lentiviral vector according to any one of claims 13 to 42, 49 to 57, 124, and 126, or the composition according to any one of claims 127 to 129.

141. The method according to claim 140, wherein fusing the mammalian cells to the targeted lipid particles delivers an exogenous substance to a target (for example, a human target).

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