Rhabdovirus g proteins having one or more cysteine-rich regions and uses thereof

Abstract

In aspects, the present disclosure provides a recombinant protein comprising (a) a rhabdoviral G glycoprotein or a functional fragment or derivative thereof, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is capable of binding to or interacting with low density lipoprotein receptor (LDLR); and (b) a cysteine rich region, wherein the cysteine rich region comprises a cysteine rich domain of LDLR, and wherein the cysteine rich region is capable of binding to or interacting with rhabdoviral G glycoprotein or a functional fragment or derivative thereof; wherein the cysteine rich region is covalently bonded to the rhabdoviral G glycoprotein or functional fragment or derivative thereof.

Description

Leydig 772155 Vyriad P-47-002 1 RHABDOVIRUS G PROTEINS HAVING ONE OR MORE CYSTEINE-RICH REGIONS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Patent Application No.63 / 548,136, filed November 10, 2023; U.S. Provisional Patent Application No. 63 / 625,842, filed January 26, 2024; U.S. Provisional Patent Application No.63 / 626,979, filed January 30, 2024; U.S. Provisional Patent Application No.63 / 560,911, filed March 4, 2024; U.S. Provisional Patent Application No.63 / 633,553, filed April 12, 2024; International Patent Application No. PCT / US2024 / 024511, filed April 12, 2024; and International Patent Application No. PCT / US2024 / 024516, filed April 12, 2024; the disclosures of each of which are incorporated herein by reference in their entireties. INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 894,665 Byte XML (Extensible Markup Language) file named “772155_SequenceListing.xml” created on November 8, 2024. BACKGROUND

[0003] Lentiviruses (LV) have been derived from the retroviridae family of viruses, most commonly the human immunodeficiency virus type 1 (HIV-1), and have been used for research and therapeutic applications. Variant LVs have also been developed using source viruses from different origins such as the simian immunodeficiency virus (SIV), the feline immunodeficiency virus (FIV) or the equine infectious anaemia virus (EIAV). LVs are an effective means of modifying eukaryotic cells by relying on the natural ability of retroviruses to integrate their genetic material to the host cell genome. LVs can be modified to carry up to 10 kb genes and stably transduce a gene of interest, often referred to as the “expression cassette.” The LV genome encodes three structural genes, fulfilling the same function in both wild-type virus and engineered vectors. The three genes are the group-specific-antigen (gag), polymerase (pol) and envelope (env).Leydig 772155 Vyriad P-47-002

[0004] LV vectors can infect both dividing and non-dividing cells and integrate their transgene into the host cell’s genome for sustained gene expression, a favorable trait for gene therapy of many chronic and malignant diseases. The progress of gene therapy has been hampered by the absence of a lentiviral vector that can be robustly and reproducibly functionalized with a cell-homing functionality to mediate specific delivery of therapeutic genes to diseased cells. Existing strategies to reengineer the cell-type specificity of viral vectors have suffered from challenges, including (i) difficulties in achieving a reproducibly high virion incorporation level (for example, when attempting to fuse a cell receptor-binding protein to viral envelope protein, (ii) a lack of in vivo compatibility, and (iii) a very limited repertoire of cell types that can be targeted (e.g., when pseudotyping the lentiviral vectors with envelope proteins from viruses that have a natural receptor binding specificity for the cell type of interest).

[0005] The vesiculovirus glycoprotein (VSV-G) can inhabit the viral surface and serve the role of engaging with cell surface receptors. This interaction facilitates the attachment of virus particles to receptors, subsequently initiating the virus's entry into the cell and the establishment of infection. The VSV-G is responsible for infecting a diverse array of cell types across multiple species. This is achieved by interacting with the relatively widespread low density lipoprotein receptor (LDLR) and related receptor family members. While certain applications may require the targeting of a broad spectrum of cell types, there exists a significant demand to tailor the therapy's cellular tropism to specific types of cells.

[0006] There is a need in the art for components of viruses that can be used in viruses to overcome one or more of these limitations. There is also a need in the art for the resulting viruses that can overcome one or more of these limitations. BRIEF SUMMARY

[0007] In aspects, the present disclosure provides a recombinant protein comprising (a) a rhabdoviral G glycoprotein or a functional fragment or derivative thereof, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is capable of binding to or interacting with low density lipoprotein receptor (LDLR); and (b) a cysteine rich region, wherein the cysteine rich region comprises a cysteine rich domain of LDLR, and wherein the cysteine rich region is capable of binding to or interacting with rhabdoviral G glycoprotein or a functional fragment or derivative thereof; wherein the cysteine rich region is covalently bonded to the rhabdoviral G glycoprotein or functional fragment or derivative thereof.Leydig 772155 Vyriad P-47-002

[0008] Additional aspects of the present disclosure are as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG.1A is a drawing showing the low density lipoprotein receptor (LDLR) domains and a vesiculovirus glycoprotein (VSV-G) interacting with cysteine rich (CR) domain 2 (CR2) and cysteine rich domain 3 (CR3) of LDLR. TM refers to transmembrane domain, sp refers to signal peptide, EGF refers to the epidermal growht factor precursor like domain, and O-link refers to the C-terminal domain containing O-linked oligosaccharides.

[0010] FIG.1B shows a sequence alignment of CR2 (SEQ ID NO: 292) and CR3 (SEQ ID NO: 293) of LDLR. Residues that are conserved in both CR2 (SEQ ID NO: 292) and CR3 (SEQ ID NO: 293) are highlighted in gray and similar residues are outlined in a box. Residues involved in Ca2+ion binding are labeled with 1, 2, 3, and 4. CR residues that interact via polar contact with VSV-G are labeled with triangles for contact established by the main chain and by circles for contact established by the lateral chain. An arrow labels the protruding aromatic residue of CR2 (SEQ ID NO: 292) and CR3 (SEQ ID NO: 293) that establishes hydrophobic interactions with VSV-G.

[0011] FIG.1C is a drawing of constructs of specific aspects of the recombinant protein. SP is signaling peptide (SEQ ID NO: 309), CR2 (SEQ ID NO: 292) is cysteine rich domain 2 of LDLR, CR3 (SEQ ID NO: 293) is cysteine rich domain 3 of LDLR, Link is a linker, specifically (G4S)3 (SEQ ID NO: 36), EGFm123 is a mutant epidermal growth factor (SEQ ID NO: 59), an VSV-G is a vesiculovirus glycoprotein (SEQ ID NO: 21).

[0012] FIGS.2A-2C are western blot images showing the expression levels of VSV-G of the constructs shown in FIG.1C transfected into HEK-293 cells with GAPDH as a control (FIG.2A), of lentivirus virion production measured from cell lysates with GAPDH as a control (FIG.2B), and lentivirus virion production measured from virion particles with P24 as a control (FIG.2C).

[0013] FIG.3A is a set of micrographs showing the transduction of the constructs shown in FIG.1C in K562, K562-EGFR, and K5462-Her2 cells. The boxes around the micrographs of the EGFm123-CR3-VSV-G WT and EGFm123-CRR2-CR3-VSV-G WT constructs indicate the best targetability and specificity among the constructs.

[0014] FIGS.3B-3C are bar graphs showing the number of GFP positive cells for each construct in K562 (left bar), K562-EGFR (middle bar), and K562-Her2 (right bar) cells at 5Leydig 772155 Vyriad P-47-002 MOI. FIG.3B shows all the cells, and FIG.3C shows an enhanced view of the samples below 4,000 cells.

[0015] FIGS.3D-3E are bar graphs showing the number of GFP positive cells for each construct in K562 (left bar, labeled 1), K562-EGFR (middle bar, labeled 1), and K562-Her2 (right bar, labeled 3) cells at 25 MOI. FIG.3D shows all the cells, and FIG.3E shows an enhanced view of the samples with the range between 20,000 and 8,000 cells removed.

[0016] FIGS.4A-4H are overlapping figures showing alternate rhabdoviral G glycoproteins. FIG.4A depicts select glycoproteins of the Vesiculovirus, Sprivivirus, and Perhabdovirus generas. FIG.4B depicts select glycoproteins of the Ledantevirus and Sigmavirus generas. FIG.4C depicts select glycoproteins of the Ephemerovirus, Tibrovirus, and Hapavirus generas. FIG.4D depicts select glycoproteins of the Curiovirus, Caligrhavirus, Tupavirus, Sripuvirus, and Alphanemrhavirus generas. FIG. 4E depicts lines showing links between different rhabdovirus generas. FIG. 4F depicts select glycoproteins of the Lyssavirus, Almendravirus, and Varicosavirus generas. FIG. 4G depicts depicts lines showing links between different rhabdovirus generas. FIG. 4H depicts select glycoproteins of the Cytorhabdovirus, Dichorhavirus, Nucleorhabdovirus, and Novirhabdovirus generas. Arrows indicate nine glycoproteins from nine different rhabdovirus genera (arrows pointing to Ledantevirus: KM205001 Fukuoka virus (FIG. 4B); Sigmavirus: GQ375258 Drosophila melanogaster sigmavirus (FIG. 4B); Ephemerovirus: AF234533 bovine ephemeral fever virus (FIG. 4C); Tibrovirus: JX297815 Bas-Congo virus (FIG. 4C); Hapavirus: KM205002 Flanders virus (FIG. 4C); Curiovirus: KM204994 Curionopolis virus (FIG. 4D); Tupavirus: AY840978 tupaia rhabdovirus (FIG. 4D); Sripuvirus: KC585008 Niakha virus (FIG. 4D); Almendravirus: KF543749 Puerto Almendras virus (FIG. 4F)). DETAILED DESCRIPTION

[0017] In aspects, the present disclosure provides a recombinant protein comprising, consisting essentially of, or consisting of (a) a rhabdoviral G glycoprotein or a functional fragment or derivative thereof, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is capable of binding to or interacting with low density lipoprotein receptor (LDLR); and (b) a cysteine rich region, wherein the cysteine rich region comprises, consists essentially of, or consists of a cysteine rich domain of LDLR, and wherein the cysteine rich region is capable of binding to or interacting with rhabdoviral G glycoprotein orLeydig 772155 Vyriad P-47-002 a functional fragment or derivative thereof; wherein the cysteine rich region is covalently bonded to the rhabdoviral G glycoprotein or functional fragment or derivative thereof.

[0018] Rhabdoviruses are viruses within the family Rhabdoviridae. The viruses encode proteins denoted N (nucleoprotein), P (phosphoprotein), M (matrix protein), G (glycoprotein), and L (large protein, which is a polymerase), and the viruses appear to have a shape of a bullet when observed using electron microscopy. Dimensions of rhabdovirus virions can range from 100 nm to 430 nm in length and can range from 45 nm to 100 nm in diameter. Exemplary rhabdoviruses, without limitation, that are contemplated herein are listed in FIGS.4A-4H.

[0019] Rhabdoviral G glycoprotein mediates binding of rhabdovirus to a receptor on a cell, which binding then mediates entry of the rhabdovirus into the cell and infection of the cell. A rhabdoviral G glycoprotein or functional fragment or derivative thereof as described herein, including recombinant proteins comprising the rhabdoviral G glycoprotein or functional fragment or derivative thereof, can be used to pseudotype a type of virus that is not the native / natural virus of the rhabdoviral G glycoprotein.

[0020] Low-density lipoprotein receptor (LDLR) is the natural receptor of vesicular stomatitis virus (VSV), which is a rhabdovirus. LDLR possesses 7 cysteine rich (CR) domains. As used herein, a “cysteine rich domain” of LDLR refers to a distinct domain or repeat that contains a large number of cysteines. Nikolic et al., Nat. Comm., 9(1029): 1-12 (2018), which is incorporated herein by reference in its entirety, discloses that the G glycoprotein of the rhabdovirus vesicular stomatitis virus (VSV-G) binds to the cysteine rich domains CR2 (SEQ ID NO: 292) and CR3 (SEQ ID NO: 293) of LDLR, as shown in FIG. 1A. The cysteine rich domain 4 (CR4) (SEQ ID NO: 294) of LDLR may also bind to VSV-G. Zhai et al., Nat. Comm., 15(622): 1-16 (2024), which is incorporated herein by reference in its entirety, discloses that the glycoprotein of the alphavirus Getah virus binds to the cysteine rich domains CR4 (SEQ ID NO: 294) and CR5 (SEQ ID NO: 295) of LDLR to mediate virus entry. Without wishing to be bound by theory, this suggests that viruses that have LDLR as a natural receptor bind to or interact with a target cell via an interaction between the viral glycoprotein and the CR domains of LDLR, which binding mediates virus entry into the cell and infection of the cell. As used herein, the terms “interacts with” and “interacting with” refer to any direct or indirect physical, chemical, or biological association between the proteins. This association may include covalent or non-covalent binding; and / or modulation or influence of activity, structure, or function in a way that has a measurable or observableLeydig 772155 Vyriad P-47-002 effect within a system. In aspects, the cysteine rich region comprises, consists essentially of, or consists of a cysteine rich domain of LDLR, e.g., CR1 (SEQ ID NO: 291), CR2 (SEQ ID NO: 292), CR3 (SEQ ID NO: 293), CR4 (SEQ ID NO: 294), CR5 (SEQ ID NO: 295), CR6 (SEQ ID NO: 296), or CR7 (SEQ ID NO: 297).

[0021] As used herein, a “pseudotype” of a virus, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. means a virus, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. comprising a molecule, e.g., a rhabdoviral G glycoprotein or functional fragment or derivative thereof (including a recombinant protein comprising the rhabdoviral G glycoprotein or functional fragment or derivative thereof), that is not typically found in the virus, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. Such a molecule can have a mutation (e.g., a substitution or deletion) that impacts the tropism of the virus, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. The impact on tropism can be, e.g., to contribute to, direct, redirect, or completely change, or any combination thereof, the tropism of the virus, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. when compared to the virus, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. without the molecule and / or compared to the wild-type virus, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. The impact on tropism can be, e.g., to target a cell that is different from the cell normally targeted by the virus, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. and / or to not target a cell that is normally targeted by the virus, membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc.

[0022] As used herein, “functional fragment” of a rhabdoviral G glycoprotein means a rhabdoviral G glycoprotein that is not a full length rhabdoviral G glycoprotein but is a portion of a parent rhabdoviral G glycoprotein (e.g., a truncated form of a full length rhabdoviral G glycoprotein), where the portion retains the fusion function of the parent full length rhabdoviral G glycoprotein. As used herein, “functional derivative” or “functional variant” of a rhabdoviral G glycoprotein means a rhabdoviral G glycoprotein that has been modified, e.g., by conservative amino acid substitution, where the rhabdoviral G glycoprotein retains the fusion function of the parent full length rhabdoviral G glycoprotein. As used herein, “functional fragment or derivative” and “functional fragment or variant” encompass theLeydig 772155 Vyriad P-47-002 meanings of both “functional fragment” and “functional derivative” / “functional variant.” The “fusion function” of a rhabdoviral G glycoprotein means that a rhabdoviral G glycoprotein, when part of a virus, can initiate fusion of the virus with a target cell, e.g., such that the virus can infect the target cell. Such fusion can be due to the interaction of the rhabdoviral G glycoprotein with its natural receptor or the interaction of the rhabdoviral G glycoprotein with a different receptor (e.g., the rhabdoviral G glycoprotein is blinded to its natural receptor, such that the rhabdoviral G glycoprotein is engineered to reduce or abolish its natural receptor binding specificity, and the rhabdoviral G glycoprotein is re-targeted to a new receptor). A functional fragment and / or functional derivative / functional variant of a rhabdoviral G glycoprotein can be within a recombinant protein comprising a rhabdoviral G glycoprotein.

[0023] In aspects, the cysteine rich region is covalently bonded to the rhabdoviral G glycoprotein or functional fragment or derivative thereof. The covalent bonding between the cysteine rich region and the rhabdoviral G glycoprotein or functional fragment or derivative thereof can be through any means of covalent association. In aspects, the covalent association between the cysteine rich region and the rhabdoviral G glycoprotein can be through linkage, conjugation, or the like, e.g., attaching a first polypeptide tag / catcher to the cysteine rich region capable of binding a second polypeptide catcher / tag attached to the rhabdoviral G glycoprotein. Any suitable polypeptide tags are contemplated herein, e.g., Isopeptag-Pilin C, SpyTag-SpyCatcher, SnoopTag-SnoopCatcher, DogTag-DogCatcher, and SdyTag- SdyCatcher. In aspects, the covalent association between the cysteine rich region and the rhabdoviral G glycoprotein can be via a fusion protein. As used herein, the term “fusion protein” refers to a single polypeptide comprising within it two or more polypeptides, e.g., formed from the translation of a nucleic acid sequence comprising two or more genes originally encoding distinct polypeptides, where the genes have been joined together. For example, when the nucleic acid sequence of the joined genes is expressed, a single polypeptide is formed comprising the distinct polypeptides joined via the polypeptide backbone as a fusion protein. In aspects, the fusion protein of the cysteine rich region and the rhabdoviral G glycoprotein can include one or more linkers between the cysteine rich region and the rhabdoviral G glycoprotein. Any suitable linker is contemplated.

[0024] In aspects, the cysteine rich region comprises, consists essentially of, or consists of (i) cysteine rich domain 2 (CR2) of LDLR, (ii) cysteine rich domain 3 (CR3) of LDLR, (iii) cysteine rich domain 4 (CR4) of LDLR, or (iv) any combination of (i) – (iii). In aspects,Leydig 772155 Vyriad P-47-002 the cysteine rich region comprises, consists essentially of, or consists of CR2 and CR3 of LDLR. In aspects, the cysteine rich region comprises, consists essentially of, or consists of the amino acid sequence of (A) SEQ ID NO: 292, (B) SEQ ID NO: 293, or (C) both (A) and (B). In aspects, the cysteine rich region comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 292. In aspects, the cysteine rich region comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 293. In aspects, the cysteine rich region comprises, consists essentially of, or consists of the amino acid sequences SEQ ID NO: 292 and SEQ ID NO: 293.

[0025] In aspects, the recombinant protein comprises an additional cysteine rich region, wherein the additional cysteine rich region comprises, consists essentially of, or consists of (a) cysteine rich domain 2 (CR2) of LDLR, (b) cysteine rich domain 3 (CR3) of LDLR, (c) cysteine rich domain 4 (CR4) of LDLR, or (d) any combination of (a) – (c). In aspects, the cysteine rich region and the additional cysteine rich region are contiguous. In aspects, the cysteine rich region and the additional cysteine rich region are not contiguous. In aspects, the additional cysteine rich region comprises, consists essentially of, or consists of CR2 and CR3 of LDLR. In aspects, the additional cysteine rich region comprises, consists essentially of, or consists of the amino acid sequence of (A) SEQ ID NO: 292, (B) SEQ ID NO: 293, or (C) both (A) and (B).

[0026] In aspects, binding of the recombinant protein is reduced or abolished for a cysteine rich domain of LDLR that is separate from the cysteine rich region of the recombinant protein.

[0027] In aspects, the recombinant protein comprises a targeting molecule. As used herein, the term “targeting molecule” refers to a binding moiety (that can bind or that can be bound), such as a natural ligand, antibody, multispecific binding molecule, or others known in the art. In aspects, the targeting molecule is N-terminal to the glycoprotein, optionally N- terminal to the cystein rich region. In aspects, the cysteine rich region is N-terminal to the glycoprotein. In aspects, the targeting molecule is is N-terminal to the glycoprotein and N- terminal to the cysteine rich region.

[0028] In aspects, the targeting molecule is a ligand. In aspects, the ligand is epidermal growth factor (EGF) (SEQ ID NO: 298), a mutant EGF (EFGm123) (SEQ ID NO: 59), stem cell factor (SCF) (e.g., SEQ ID NO: 299), thrombopoietin (TPO) (SEQ ID NO: 300), human hepatocyte growth factor (HGF) (SEQ ID NO: 56), erythropoietin (EPO), or type 1 insulin-Leydig 772155 Vyriad P-47-002 like growth factor (IGF1) (SEQ ID NO: 58). In aspects, the EPO is human EPO (SEQ ID NO: 301).

[0029] Without wishing to be bound by theory, the efficacy observed in transducing cells may be in part attributed to the direct linkage of G protein with the targeting ligand. Rhabdoviral G glycoproteins are known to be efficiently transported to the cell surface, which efficiency can be used to achieve high density packing. Thus, with the direct attachment, the ligand too is efficiently transported to the surface and densely packed. Buchholz et al., J. Virol., 70(6): 3716-3723 (1996), which is incorporated herein by reference in its entirety, discloses that the distance between a viral membrane and an endosome may be important for optimizing ligand binding and fusion. The distance between the viral membrane and the targeting ligand, located at the far terminus of the G protein, may provide a conducive distance.

[0030] In aspects, the targeting molecule is a single chain variable fragment (scFv). In aspects, the scFv has the VL N-terminal to VH. In aspects, the scFv has the VH N-terminal to VL. In aspects, the scFv binds epidermal growth factor receptor (EGFR) (SEQ ID NO: 49), human epidermal growth factor receptor 2 (Her2) (SEQ ID NO: 51), cluster of differentiation 3 (CD3), cluster of differentiation 4 (CD4), cluster of differentiation 8 (CD8), cluster of differentiation 7 (CD7), cluster of differentiation 117 (cKit), mucin-16 (MUC16), B cell maturation antigen (BCMA), or Nectin4. In aspects, the scFv is agonistic to CD3. In aspects, the scFv agonistic to CD3 is UCHT1, HuM291, OKT3, or TR66. In aspects, the scFv is a humanized UCHT1. In aspects, the scFv comprises a variable heavy chain (VH) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 46 and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 45. In aspects, the VH and VL are separated by a flexible linker. In aspects, the flexible linker is SEQ ID NO: 36. In aspects, the scFv is TR66 and wherein the TR66 is codon optimized for expression in human and wherein the scFv comprises a variable heavy chain (VH) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 44 and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 43. In aspects, the scFv is TR66opt and wherein the scFv comprises a variable heavy chain (VH) comprising, consisting essentially of, or consisting of the amino acid sequences of a complementarity determining region 1 (CDR1) of SEQ ID NO: 247, a complementarity determining region 2 (CDR2) of SEQ ID NO: 248, and a complementarity determiningLeydig 772155 Vyriad P-47-002 region 3 (CDR3) of SEQ ID NO: 249, and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequences of a CDR1 of SEQ ID NO: 250, a CDR2 of SEQ ID NO: 251, and a CDR3 of SEQ ID NO: 252. In aspects, the scFv is UCHT1 wherein the scFv comprises a variable heavy chain (VH) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 46 and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 45. In aspects, the scFv is UCHT1 and wherein the scFv comprises a variable heavy chain (VH) comprising, consisting essentially of, or consisting of the amino acid sequences of a CDR1 of SEQ ID NO: 223, a CDR2 of SEQ ID NO: 224, and a CDR3 of SEQ ID NO: 225, and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequences of a CDR1 of SEQ ID NO: 226, a CDR2 of SEQ ID NO: 227, and a CDR3 of SEQ ID NO: 228. In aspects, the scFv is HuM291 wherein the scFv comprises a variable heavy chain (VH) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 38 and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 37. In aspects, the scFv is Hum291 and wherein the scFv comprises a variable heavy chain (VH) comprising, consisting essentially of, or consisting of the amino acid sequences of a CDR1 of SEQ ID NO: 235, a CDR2 of SEQ ID NO: 236, and a CDR3 of SEQ ID NO: 237, and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequences of a CDR1 of SEQ ID NO: 238, a CDR2 of SEQ ID NO: 239, and a CDR3 of SEQ ID NO: 240. In aspects, the scFv is OKT3 wherein the scFv comprises a variable heavy chain (VH) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 40 and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 39. In aspects, the scFv is OKT3 and wherein the scFv comprises a variable heavy chain (VH) comprising, consisting essentially of, or consisting of the amino acid sequences of a CDR1 of SEQ ID NO: 241, a CDR2 of SEQ ID NO: 242, and a CDR3 of SEQ ID NO: 243, and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequences of a CDR1 of SEQ ID NO: 244, a CDR2 of SEQ ID NO: 245, and a CDR3 of SEQ ID NO: 246. In aspects, the scFv is TR66 wherein the scFv comprises a variable heavy chain (VH) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 42 and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 41. In aspects, the scFv is TR66 and wherein the scFv comprises aLeydig 772155 Vyriad P-47-002 variable heavy chain (VH) comprising, consisting essentially of, or consisting of the amino acid sequences of a CDR1 of SEQ ID NO: 229, a CDR2 of SEQ ID NO: 230, and a CDR3 of SEQ ID NO: 231, and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequences of a CDR1 of SEQ ID NO: 232, a CDR2 of SEQ ID NO: 233, and a CDR3 of SEQ ID NO: 234.

[0031] In aspects, the targeting molecule is an anti-cKit binding domain or a functional fragment or derivative thereof capable of selectively binding to the c-KIT receptor. In aspects, the anti-cKit binding domain comprises, consists essentially of, or consists of an anti- cKit 2D1 scFv or a functional fragment or derivative thereof wherein the scFv comprises a variable heavy chain (VH) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 195 and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 196.

[0032] Also contemplated herein as targeting molecules are monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti- Id) antibodies (including, e.g., anti-Id antibodies to antigen specific TCR), Ig-DARTS, designed ankyrin repeat proteins (DARPins), small peptides, superantigens, epitope-binding fragments of any of the above, and what is described in US Patent Publication No. 2007 / 0004909 and US Patent Publication No.2009 / 0060910, each of which is incorporated herein by reference in their entireties. Also contemplated are immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGI , IgG2, IgG3, IgG4, IgAI and IgA2) or subclass. In aspects, the targeting molecule is an antibody comprising a variable domain that binds a cell surface protein on a target cell and a heavy chain constant domain. In aspects, the targeting molecule is an antibody comprising a variable domain that binds a cell surface protein on a target cell and an IgG heavy chain constant domain. In aspects, the targeting molecule is an antibody comprising a variable domain that binds a cell surface protein on a target cell and an IgG heavy chain constant domain.

[0033] In aspects, a targeting molecule specifically binds a cell surface molecule, e.g., an oligosaccharide, a receptor, cell surface marker, etc., expressed on the surface of a mammalian (e.g., human) eukaryotic cell. In aspects, a targeting molecule binds a (e.g., human) liver cell, a (e.g., human) brain cell, a (e.g., human) T cell, a (e.g., human) kidneyLeydig 772155 Vyriad P-47-002 cell, an (e.g., human) intestinal cell, a (e.g., human) lung cell, a (e.g., human) cancerous cell, or a (e.g., human) cell infected with heterologous pathogen. In aspects, the targeting molecule binds a receptor expressed by a (e.g., human) liver cell, e.g., an asialoglycoprotein receptor, e.g., hASGRl. In aspects, the targeting molecule binds a molecule expressed by a (e.g., human) neuronal cell, e.g., GABA, transferrin, etc. In aspects, the targeting molecule binds a molecule expressed by a (e.g., human) T cell, e.g., CD3, e.g., CD3c. In aspects, the targeting molecule binds CD63. In aspects, the targeting molecule binds a molecule expressed by a (e.g., human) hematopoietic stem cell, e.g., CD34. In aspects, the targeting molecule binds a molecule expressed by a (e.g., human) kidney cell. In aspects, the targeting molecule binds a molecule expressed by a (e.g., human) muscle cell, e.g., an integrin. In aspects, the targeting molecule binds a molecule expressed by a (e.g., human) cancerous cell, e.g., a tumor associated antigen, e.g., adipophilin, AIM-2, ALDHIAI, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTCI, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNKIAI, CTAGI, CTAG2, cyclin DI, Cyclin-Al, dek-can fusion protein, DKKI, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen (“ETA”), ETV6- AML1 fusion protein, EZH2, E6, E7, FGF5, FLT3-ITD, FNI, G250 / MN / CAIX, GAGE- 1,2,8, GAGE- 3,4,5,6,7, GAS7, glypican-3, GnTV, gpl00 / Pmell 7, GPNMB, HAUS3, Hepsin, HER-2 / neu, HERV-K-MEL, HLA-Al 1, HLA-A2, HLA-DOB, hsp70-2, IDOI, IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLCI, KM-HN-1, KMHNI also known as CCDCI 10, LAGE-I, LDLR- fucosyltransferaseAS fusion protein, Lengsin, M-CSF, MAGE-Al, MAGE-AIO, MAGE-Al2, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-Cl, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MCIR, MCSP, mdm-2, MEI, Melan-A / MART- 1, Meloe, Midkine, MMP-2, MMP-7, MUCI, MUC5AC, mucin, MUM-I, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88- A, neo-PAP, NFYC, NY-BR-I, NY-ESO-l / LAGE-2, OAI, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin (“PEM”), PPPIR3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-l, RAGE-I, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secemin 1, SIRT2, SNRPDI, SOXI0, Spl7, SPA17, SSX-2, SSX-4, STEAPI, survivin, SYT-SSXI or - SSX2 fusion protein, TAG-I, TAG-2, Telomerase, TGF-betaRII, TPBG, TRAG-3, Triosephosphate isomerase, TRP-l / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinaseLeydig 772155 Vyriad P-47-002 (“TYR”), VEGF, WTI, XAGE-lb / GAGED2a, Kras, NYESOI, HPV E2, HPV E6, HPV E7, WT-I antigen (in lymphoma and other solid tumors), ErbB receptors, Melan A [MARTI], gp 100, tyrosinase, TRP-1 / gp 75, and TRP-2 (in melanoma); MAGE-I and MAGE-3 (in bladder, head and neck, and non-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer); Mucin [MUC-1] (in breast, pancreas, colon, and prostate cancers); prostate-specific antigen [PSA] (in prostate cancer); carcinoembryonic antigen [CEA] (in colon, breast, and gastrointestinal cancers), and such shared tumor-specific antigens as MAGE-2, MAGE-4, MAGE-6, MAGE-IO, MAGE-12, BAGE-1, CAGE-1,2,8, CAGE-3 TO 7, LAGE-I, NY- ESO-1 / LAGE-2, NA-88, GnTV, TRP2-INT2, etc. In aspects, the targeting molecule binds E6 and / or E7. In aspects, the targeting molecule binds Her2. In aspects, the targeting molecule binds CD63. In aspects, the targeting molecule binds human glucagon receptor (hGCGR). In aspects, the targeting molecule binds human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3).

[0034] The targeting molecule can target any suitable cell type. Non-limiting examples of cell types include hematopoietic stem cells (HSC), T cells, monocytes, and NK cells. For HSC, non-limiting examples of markers that can be targeted by the targeting molecule include, e.g., one or more of CD34, CD133, and CD90 (Thy-1). For T cells, non-limiting examples of markers that can be targeted by the targeting molecule include, e.g., one or more of CD3, CD4, and CD8. For monocytes, non-limiting examples of markers that can be targeted by the targeting molecule include, e.g., one or both of CD14 and CD11b. For NK cells, non-limiting examples of markers that can be targeted by the targeting molecule include, e.g., one or both of CD56 and NKG2D.

[0035] In aspects, the recombinant protein comprises a signal peptide, optionally N- terminal to the rhabdoviral G glycoprotein or functional fragment or derivative thereof. As used herein, a “signal peptide” refers to a peptide involved in targeting a glycoprotein to the secretory pathway. In aspects, the recombinant protein comprises a signal peptide that is N- terminal to the rhabdoviral G glycoprotein or functional fragment or derivative thereof. In aspects, different signal peptides can be selected to improve glycoprotein targeting to the secretory pathway. In aspects, the signal peptide may be the naturally occurring signal peptide for the rhabdoviral G glycoprotein. In aspects, the signal peptide comprises, consists essentially of, or consists of the amino acid sequence of a signal peptide as described herein. In aspects, the signal peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 60, 121, 122, 123, 253, 256, 257, 265, or 309. In aspects, the signalLeydig 772155 Vyriad P-47-002 peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 60. In aspects, the signal peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 309. In aspects, the recombinant protein comprises a mature form of a rhabdoviral G glycoprotein or functional fragment or derivative thereof, where the signal peptide is not present in the recombinant protein. In aspects, the recombinant protein comprising a rhabdoviral G glycoprotein or functional fragment or derivative thereof does not comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids of the N-terminus of the mature form of the wild-type rhabdoviral G glycoprotein. In aspects, the recombinant protein comprising a rhabdoviral G glycoprotein or functional fragment or derivative thereof does not comprise a signal peptide and also does not comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids of the N-terminus of the mature form of the wild-type rhabdoviral G glycoprotein.

[0036] In aspects, the the cysteine rich region is N-terminal to the rhabdoviral G glycoprotein or functional fragment or derivative thereof.

[0037] In aspects, the recombinant protein is a fusion protein comprising, consisting essentially of, or consisting of the rhabdoviral G glycoprotein or functional fragment or derivative thereof and the cysteine rich region.

[0038] In aspects, the recombinant protein comprises a linker between the cysteine rich region and the rhabdoviral G glycoprotein or functional fragment or derivative thereof. Any suitable linker is contemplated, e.g., as disclosed in Chen et al., Adv. Drug. Deliv. Rev., 65(10: 1357-1369 (2013), which is incorporated herein by reference in its entirety. In aspects, the linker is flexible. Exemplary flexible linkers include, but are not limited to, e.g., AAASGGSGGGGSGGGGSGP (SEQ ID NO: 130), AAASGGSGGGGSGGGGS (SEQ ID NO: 131), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 132), GGGGSGGGGSGGGGS (SEQ ID NO: 36), GGGGSGGGGS (SEQ ID NO: 133), GGGGS (SEQ ID NO: 134), GGGGGGGG (SEQ ID NO: 135), GGGGGG (SEQ ID NO: 136), GSAGSAAGSGEF (SEQ ID NO: 137), and VPGVGVPGVG (SEQ ID NO: 138). In aspects, the linker is rigid. Exemplary rigid linkers include, but are not limited to, e.g.,Leydig 772155 Vyriad P-47-002 PAPAP (SEQ ID NO: 139), EAAAKEAAAKEAAAK (SEQ ID NO: 140), EAAAKEAAAK (SEQ ID NO: 141), EAAAK (SEQ ID NO: 142), AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO: 143), AEAAAKEAAAKA (SEQ ID NO: 144), ESKYGPPCPPCP (SEQ ID NO: 145), CPPCPAPELLGGPSVF (SEQ ID NO: 146), and alanine-proline (AP) repeated for a total of 10 to 34 amino acids (SEQ ID NO: 147).

[0039] In aspects, the recombinant protein comprises a rhabdoviral G glycoprotein or functional fragment or derivative thereof that is of a Flanders virus glycoprotein (FLAV-G) (SEQ ID NO: 13), a Chandipura virus glycoprotein (CHPV-G) (SEQ ID NO: 14), a Perinet virus glycoprotein (PERV-G) (SEQ ID NO: 15), a Piry virus glycoprotein (PIRYV-G) (SEQ ID NO: 16), a Fukuoka virus glycoprotein (FUKV-G) (SEQ ID NO: 17), a Joinjakaka virus glycoprotein (JOIV-G) (SEQ ID NO: 18), a Kumasi virus glycoprotein (KRV-G) (SEQ ID NO: 19), a Keuraliba virus glycoprotein (KEUV-G) (SEQ ID NO: 20), an Isfahan glycoprotein (ISFV-G), a Jurona glycoprotein (JURV-G), a Mediterranean Bat glycoprotein (MBV-G), a Malpais Spring glycoprotein (MSPV-G), a Radi glycoprotein (RADV-G), a Rhinolophus affinis-G, a Yug Bugdanavoc glycoprotein (YBV-G), a Yinshui Bat glycoprotein (YSBV-G), a Kimberley glycoprotein (KIMV-G), a Kanyawara glycoprotein (KYAV-G), a La Joya glycoprotein (LJV-G), a Mosquiero glycoprotein (MQOV-G), a Parry Creek glycoprotein (PCV-G), a Bas Congo glycoprotein (BASV-G), a Bovine Ephemeral fever glycoprotein (BEFV-G), a Curionopolis glycoprotein (CURV-G), a Drosophila melanogaster sigmavirus glycoprotein (DMelSV-G), a Niakha glycoprotein (NIAV-G), a Puerto almandras glycoprotein (PTAMV-G), or a Tupaia rhabdovirus glycoprotein (TUPTV- G). In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus glycoprotein or a functional fragment or derivative thereof. In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus indiana (e.g., SEQ ID NO: 21, 209, 210, or 258), Vesiculovirus newjersey (e.g., SEQ ID NO: 22,129, 213, or 265), Vesiculovirus carajas (e.g., SEQ ID NO: 23 or 214), Vesiculovirus alagoas (e.g., SEQ ID NO: 24 or 215), Vesiculovirus cocal (e.g., SEQ ID NO: 128 or 216), Vesiculovirus marraba (e.g., SEQ ID NO: 211 or 212), Vesiculovirus morreton (e.g., SEQ IDLeydig 772155 Vyriad P-47-002 NO: 217 or 218), or any other rhabdoviral G glycoprotein or functional fragment or derivative thereof as provided herein. In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus indiana (e.g., SEQ ID NO: 21, 209, 210, or 258), Vesiculovirus newjersey (e.g., SEQ ID NO: 22, 129, 213, or 265), Vesiculovirus carajas (e.g., SEQ ID NO: 23 or 214), or Vesiculovirus alagoas (e.g., SEQ ID NO: 24 or 215). In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus indiana (e.g., SEQ ID NO: 21, 209, 210, or 258). In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus newjersey (e.g., SEQ ID NO: 22, 129, 213, or 265). In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus carajas (e.g., SEQ ID NO: 23 or 214). In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus alagoas (e.g., SEQ ID NO: 24 or 215). Shown in FIGs.4A-4H are additional exemplary rhabdoviral G glycoproteins, without limitation, that are contemplated herein.

[0040] In aspects, the recombinant protein comprises a rhabdoviral G glycoprotein or functional fragment or derivative thereof that has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% 75%, 70%, 65%, or 60% sequence identity compared to a rhabdoviral G glycoprotein or functional fragment or derivative thereof described herein. In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof contains one or more conservative amino acid substitutions that do not interfere with the fusion function of the rhabdoviral G glycoprotein or functional fragment or derivative thereof.

[0041] In aspects, the recombinant protein comprises a rhabdoviral G glycoprotein that is substantially intact. As used herein, a “substantially intact” rhabdoviral G glycoprotein means a rhabdoviral G glycoprotein that is a functional fragment of the rhabdoviral G glycoprotein, where the rhabdoviral G glycoprotein has each of the domains of a rhabdoviral G glycoprotein, the domains as defined in Roche et al., Cell. Mol. Life Sci., 65: 1716-1728 (2008), which is incorporated herein by reference in its entirety.

[0042] In aspects, the recombinant protein comprises a rhabdoviral G glycoprotein that is a functional fragment or derivative thereof. In aspects, the cytoplasmic tail of the glycoprotein is truncated, deleted, or replaced with another sequence. Previous work has shown that such truncation or deletion can enhance fusion activity in rhabdoviral G glycoprotein. In aspects, the truncations from the C terminus can be, for example, 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 20 amino acids, 30 amino acids, 40Leydig 772155 Vyriad P-47-002 amino acids, 50 amino acids, 60 amino acids, up to 10 amino acids, up to 20 amino acids, up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, up to 60 amino acids, or more than 60 amino acids. In aspects, the cytoplasmic tail is replaced with another sequence.

[0043] In aspects, the recombinant protein comprises a rhabdoviral G glycoprotein or functional fragment or derivative thereof that is engineered to reduce or abolish its natural receptor binding specificity. Reduction in binding specificity can be of any amount, e.g., reduced by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, or any range between these percentages. Vesicular stomatitis virus (VSV) is a rhabdovirus, having a natural receptor of the low-density lipoprotein receptor (LDLR) or very low density lipoprotein receptor (VLDLR), which are expressed on the cell membrane of many types of cells. In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is engineered to have a mutation to reduce or abolish its natural receptor binding specificity to LDLR or VLDLR, or other receptors which exhibit cross-reactivity to these receptors. In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof comprises a mutation at one or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus indiana glycoprotein (SEQ ID NO: 21) In aspects, the mutation is a substitution of a wild-type amino acid to another amino acid. In aspects, the substitution is with a Q. In aspects, the mutation is a substitution at three or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus indiana glycoprotein (SEQ ID NO: 21). In aspects, the mutation is associated with the amino acid sequence of SEQ ID NO: 26, 71, 72, 73, 267, 269, 275, 279, 282, or 286. The US Patent Publication No.2020 / 0216502 is incorporated herein by reference in its entirety. In aspects, the mutation is a deletion of a wild-type amino acid. In aspects, the mutation is deletion of one or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus indiana glycoprotein (SEQ ID NO: 21), wherein each deleted amino acid is not present within the amino acid sequence of the glycoprotein. In aspects, the mutation is a single deletion at K47, wherein the K47 amino acid is not present within the amino acid sequence of the glycoprotein. In aspects, the mutation is associated with the amino acid sequence of SEQ ID NO: 27, 29, 268, 270, 276, 277, 280, 283, 284, or 287.

[0044] In aspects, preferred full length rhabdoviral G glycoproteins, ectodomains, signal peptides, and engineered mutations of the ectodomains to reduce or abolish its natural receptor binding affinity are shown in Table 1. VSIV indicates Vesiculovirus indiana G glycoprotein, VSNJV indicates Vesiculovirus newjersey G glycoprotein, VSCV indicatesLeydig 772155 Vyriad P-47-002 Vesiculovirus carajas G glycoprotein, VSAV indicates Vesiculovirus alagoas G glycoprotein, VSCOV indicates Vesiculovirus cocal G glycoprotein, WT indicates wild type, 41J indicates residue 41 can be an I or L, K1R indicates a R substitution at residue 1 corresponding to SEQ ID NO: 21, G115A indicates an A substitution at residue 115 corresponding to SEQ ID NO: 21, Delta K47 indicates a deletion at residue 47 corresponding to SEQ ID NO: 21, and K47Q+Y209Q+R354Q indicates Q substitutions at residues 47, 209, and 354 corresponding to SEQ ID NO: 21. Table 1

[0045] It has been found that a Vesiculovirus indiana rhabdoviral G glycoprotein having a deletion of K47 reduces or abolishes the natural receptor binding specificity of the rhabdoviral G glycoprotein when the rhabdoviral G glycoprotein while unexpected retaining full function of the G protein in other respects. In particular, this feature is seen when incorporated into a lentiviral vector. However, when used in a VSV vector, there is second site mutation (F405I) generated in the VSV-G protein during the virus amplification. This mutation leads to the loss of detargeting effect caused by the deletion on K47 residue. Without wishing to be bound by theory, this phenomenon may be due to VSV being a replicating virus, whereas lentivirus is a non-replicating virus.

[0046] Vesiculovirus indiana glycoprotein having H8 and / or K47 deletions, when incorporated into a lentiviral system, can support production of functional pseudotyped lentivirus and successful cell transduction. The H8 and / or K47 deletions, but not Y209 or R354 deletions or Y209 / R354 deletion combinations, demonstrated generation and rescue of pseudotyped lentivirus with low to no LDLR background binding.Leydig 772155 Vyriad P-47-002

[0047] In aspects, the recombinant protein comprises a rhabdoviral G glycoprotein or functional fragment or derivative thereof that comprises one or more viral titer increasing mutations. In aspects, the one or more viral titer increasing mutations is one or both of M184T and F250L in SEQ ID NO: 21 (or the positions corresponding to M184T and F250L). Other exemplary mutations, without limitation, for increasing viral titer include those described in US Patent Publication No.2022 / 0162266, which is incorporated herein by reference in its entirety. The mutations include H22N and S422I in the ectodomain of Vesiculovirus indiana G glycoprotein (SEQ ID NO: 21) (or substitutions in positions corresponding to these in other rhabdoviral G glycoproteins).

[0048] Amino acid positions of other rhabdoviral G glycoproteins or functional fragments or derivatives thereof that are “positions corresponding to” the amino acids discussed above can be determined using a global sequence alignment algorithm (see, e.g., Madeira et al., Nuc. Acids Res., 50(W1): W276-W279 (2022), which is incorporated herein by reference in its entirety) comparing the base rhabdoviral G glycoprotein to another rhabdoviral G glycoprotein.

[0049] In aspects, the recombinant protein comprises a tag for western detection or purification. In aspects, the tag is a tag for western detection. In aspects, the tag is a tag for purification. In aspects, the tag is a FLAG tag, GFP, or others.

[0050] An advantage of the technology of the present disclosure is that it provides for finely tuning stoichiometry of ligand-bound rhabdoviral G glycoprotein to free rhabdoviral G glycoprotein. In aspects, the present disclosure provides methods of assessing the stoichiometry of ligand-bound rhabdoviral G glycoprotein to free rhabdoviral G glycoprotein. For example, in aspects, a single antibody on a western blot can indicate the ratio of ligand- bound protein to free protein.

[0051] In aspects, the present disclosure provides a membraned vesicle comprising a recombinant protein as described herein. As used herein, a “membraned vesicle” is a vesicle bound (delimited) by a lipid bilayer. In aspects, the membraned vesicle is either naturally- derived or engineered. In aspects, the vesicle is a gesicle or an exosome. A gesicle can be as described in Mangeot et al., Mol. Ther., 19: 1656–1666 (2011), which is incorporated herein by reference in its entirety, where overexpression of glycoprotein of VSV-G in human cells induced release of fusogenic vesicles. A membraned vesicle can be cell-derived and thus be a cell-derived enveloped particle (CDEP).Leydig 772155 Vyriad P-47-002

[0052] In aspects, membraned vesicles, cell-derived enveloped particles, enveloped viral particles, and virus-like particles can encapsulate proteins, lipids, nucleic acids, etc. for delivery, such that membraned vesicles, cell-derived enveloped particles, enveloped viral particles, and virus-like particles can be considered “enveloped delivery vehicles.”

[0053] In aspects, the recombinant protein comprising, consisting essentially of, or consisting of a rhabdoviral G glycoprotein or a functional fragment or derivative thereof can be put into any system comprising at least two membrane-bound entities where fusion between the two is desired. Exemplary systems include, but are not limited to, enveloped vector platforms, e.g., engineered viruses with lipid bilayer envelopes or enveloped lipid particle platforms, used to precisely deliver biologically active payloads to targeted cells in the context of gene and cell therapies, e.g. chimeric antigen receptor T cell (CAR-T) therapy, as well as in diagnostics, regenerative medicine, synthetic biology, and other medical, research, and industrial applications. Engineered viruses with lipid bilayer envelopes include Lentiviral Vectors (LVs), Gamma-Retroviral Vectors, Herpes Simplex Virus (HSV) Vectors, Vesicular Stomatitis Virus (VSV) Vectors, Vaccinia Virus Vectors, Sendai Virus Vectors, Measles Virus Vectors, and Alphavirus Vectors. Enveloped lipid particle platforms include Lipid Nanoparticles (LNPs), Solid Lipid Nanoparticles (SLNs), Nanostructured Lipid Carriers (NLCs), Liposomes, Cationic Lipid Particles, Transferosomes, Cubosomes, Niosomes, Exosomes (Lipid-Based Extracellular Vesicles), and Archaeosomes.

[0054] In aspects, the membraned vesicle comprises a mixed trimer, wherein the mixed trimer comprises (a) at least one recombinant protein comprising a cysteine rich region, and (b) at least one rhabdoviral G glycoprotein or functional fragment or derivative thereof that does not comprise a cysteine rich region. Without being bound by theory, decreasing the number of units in a recombinant rhabdoviral G glycoprotein trimer that are decorated with a covalently bound targeting molecule, particularly a large targeting molecule, better allows the transformation of the trimer from a prefusion to a fusion conformation. In aspects, the membraned vesicle comprises an unmixed trimer, wherein the unmixed trimer comprises only rhabdoviral G glycoprotein or a functional fragment or derivative thereof that does not comprise a cysteine rich region.

[0055] In aspects, the membraned vesicle comprises the maximum number of the recombinant proteins comprising a cysteine rich region possible to be accommodated within the membrane of the membraned vesicle. In aspects, a membraned vesicle comprises less than the maximum number of the recombinant proteins comprising a cysteine rich regionLeydig 772155 Vyriad P-47-002 possible to be accommodated within the membrane of the membraned vesicle. Less than the maximum amount can be of any amount less than 100%, e.g., 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, or 1%, or any range between these percentages. In aspects, any enveloped delivery vehicle as described herein comprises the maximum number of the recombinant proteins possible to be accommodated within the membrane.

[0056] In aspects, the present disclosure provides an enveloped viral particle comprising a recombinant protein as described herein. As used herein, an “enveloped viral particle” is a vesicle bound (delimited) by a lipid bilayer and has the ability to infect a cell and produce additional enveloped viral particles. An enveloped viral particle can include one or more components of a virus, in addition to a rhabdoviral G glycoprotein or functional fragment or derivative thereof or recombinant protein thereof. Exemplary components of a virus include, without limitation, the gag, pol, or env gene or gene product of lentivirus. A “virus-like particle” as used herein means an enveloped viral particle that cannot produce additional virus-like particles (e.g., is replication-incompetent).

[0057] In aspects, an enveloped viral particle comprises a mixed trimer, wherein the mixed trimer comprises (a) at least one recombinant protein comprising a cysteine rich region, and (b) at least one rhabdoviral G glycoprotein or functional fragment or derivative thereof that does not comprise a cysteine rich region. Without being bound by theory, decreasing the number of units in a recombinant rhabdoviral G glycoprotein trimer that are decorated with a covalently bound targeting molecule, particularly a large targeting molecule, better allows the transformation of the trimer from a prefusion to a fusion conformation. In aspects, an enveloped delivery vehicle described herein comprises a mixed trimer. In aspects, the enveloped viral particle comprises an unmixed trimer, wherein the unmixed trimer comprises only rhabdoviral G glycoprotein or a functional fragment or derivative thereof that does not comprise a cysteine rich region.

[0058] In aspects, the enveloped viral particle comprises the maximum number of the recombinant proteins comprising a cysteine rich region possible to be accommodated within the membrane of the enveloped viral particle. In aspects, the enveloped viral particle comprises less than the maximum number of the recombinant proteins comprising a cysteine rich region possible to be accommodated within the membrane of the enveloped particle. Less than the maximum amount can be of any amount less than 100%, e.g., 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, or 1%, or any range between these percentages.Leydig 772155 Vyriad P-47-002

[0059] In aspects, the present disclosure provides a recombinant viral vector comprising a nucleotide encapsulated by an enveloped delivery vehicle as described herein, a membraned vesicle as described herein, or an enveloped viral particle as described herein.

[0060] In aspects, the present disclosure provides a composition comprising, consisting essentially of, or consisting of a pharmaceutically acceptable carrier and an enveloped delivery vehicle as described herein, a membraned vesicle as described herein, an enveloped viral particle as described herein, or a recombinant viral vector as described herein. Pharmaceutically acceptable carriers are well-known in the art.

[0061] In aspects, the present disclosure provides a method of delivering a payload to a target cell, the method comprising, consisting essentially of, or consisting of contacting the target cell with a membraned vesicle as described herein, an enveloped viral particle as described herein, an enveloped delivery vehicle as described herein, a recombinant viral vector as described herein, or a composition as described herein. In aspects, the target cell is a T cell. In aspects, the target cell is in vitro or ex vivo. In aspects, the target cell is in vivo. In aspects, the payload is a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising, consisting essentially of, or consisting of an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In aspects, the CAR has antigenic specificity for CD19 (e.g., SEQ ID NO: 165) or BCMA (e.g., SEQ ID NOs: 166- 170). In aspects, the CAR comprises a hinge domain, wherein the hinge domain is the hingedomain of CD28 or CD8 (SEQ ID NO: 149). In aspects, the transmembrane domain is thetransmembrane domain of CD28 or CD8 (SEQ ID NO: 150). In aspects, the intracellular signaling domain comprises a costimulatory domain and an activating domain. In aspects, thecostimulatory domain is 4 1BB (SEQ ID NO: 151). In aspects, the activating domain isCD3zeta (SEQ ID NO: 152).

[0062] In aspects, a virus, a membraned vesicle, an enveloped delivery vehicle, an enveloped viral particle, or a recombinant viral vector, etc. as described herein comprises a T cell costimulatory molecule or a nucleotide encoding a T cell costimulatory molecule. A nucleotide encoding a T cell costimulatory molecule can be encoded within the membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. as described herein, e.g., encoded by a nucleotide encoding a chimeric antigen receptor (CAR) that comprises the T cell costimulatory molecule. The costimulatory molecule can be presented at the surface of the membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. as described herein. Presentation at the surfaceLeydig 772155 Vyriad P-47-002 can be, e.g., by anchoring a T cell costimulatory molecule to the envelope membrane, by expressing a T cell costimulatory molecule that is a transmembrane protein, by expressing a T cell costimulatory molecule as part of a fusion protein that is a transmembrane protein, etc. The costimulatory molecule can be a full molecule or a portion of a larger molecule, e.g., a costimulatory domain. Non-limiting examples include, e.g., CD80, CD83, CD86, 4- 1BB / CD137, CD28, and others as known in the art. Without wishing to be bound by theory, the costimulatory molecule is thought to provide signal 2 for activation of T cells.

[0063] In aspects, a virus, a membraned vesicle, an enveloped delivery vehicle, an enveloped viral particle, or a recombinant viral vector, etc. as described herein does not comprise one or more T cell costimulatory molecules or nucleotides encoding one or more T cell costimulatory molecules. In aspects, a virus, a membraned vesicle, an enveloped delivery vehicle, an enveloped viral particle, or a recombinant viral vector, etc. as described herein does not comprise any T cell costimulatory molecules or any nucleotides encoding a T cell costimulatory molecule. In aspects, a virus, a membraned vesicle, an enveloped delivery vehicle, an enveloped viral particle, or a recombinant viral vector, etc. as described herein comprises one or more T cell costimulatory molecules or nucleotides encoding one or more T cell costimulatory molecules but does not comprise one or more other T cell costimulatory molecules or nucleotides encoding one or more other T cell costimulatory molecules. For example, the membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. as described herein does not comprise one or more nucleotides encoding one or more T cell costimulatory molecules within the membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. as described herein, e.g., not encoded by a nucleotide encoding a CAR; wherein the membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. as described herein comprises one or more nucleotides encoding one or more other T cell costimulatory molecules within the membraned vesicle, enveloped delivery vehicle, enveloped viral particle, or recombinant viral vector, etc. as described herein, e.g., encoded by one or more nucleotides encoding one or more CARs. In aspects, a virus, a membraned vesicle, an enveloped delivery vehicle, an enveloped viral particle, or a recombinant viral vector, etc. as described herein does not comprise CD80 but does comprise one or more other T cell costimulatory molecules, e.g., CD83, CD86, 4-1BB / CD137, CD28, or others as known in the art. In aspects, a virus, a membraned vesicle, an enveloped delivery vehicle, an enveloped viral particle, or a recombinant viral vector, etc. as described herein does notLeydig 772155 Vyriad P-47-002 comprise CD83 but does comprise one or more other T cell costimulatory molecules, e.g., CD80, CD86, 4-1BB / CD137, CD28, or others as known in the art. In aspects, a virus, a membraned vesicle, an enveloped delivery vehicle, an enveloped viral particle, or a recombinant viral vector, etc. as described herein does not comprise CD86 but does comprise one or more other T cell costimulatory molecules, e.g., CD80, CD83, 4-1BB / CD137, CD28, or others as known in the art. In aspects, a virus, a membraned vesicle, an enveloped delivery vehicle, an enveloped viral particle, or a recombinant viral vector, etc. as described herein does not comprise 4-1BB / CD137 but does comprise one or more other T cell costimulatory molecules, e.g., CD80, CD83, CD86, CD28, or others as known in the art. In aspects, a virus, a membraned vesicle, an enveloped delivery vehicle, an enveloped viral particle, or a recombinant viral vector, etc. as described herein does not comprise CD28 but does comprise one or more other T cell costimulatory molecules, e.g., CD80, CD83, CD86, 4-1BB / CD137, or others as known in the art.

[0064] Without wishing to be bound by theory, CD19 acts as a co-receptor for B-cell receptor (BCR) signaling and plays a role in B-cell activation, development, and differentiation. CD19 is expressed on B cells from the earliest recognizable B-lineage cells during development to B-cell precursors. It is present on many B-cell malignancies, including, e.g., acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL).

[0065] Without wishing to be bound by theory, BCMA is involved in the regulation of plasma cell survival and proliferation. BCMA is expressed on mature B cells and plasma cells. BCMA is present on, e.g., multiple myeloma, as it is a disease characterized by malignant plasma cell proliferation.

[0066] In aspects, the payload is a nucleotide sequence encoding a transgene or gene editing system. In aspects, the gene editing system comprises one or more nucleases, such as CRISPR-Cas9, CasMINI, TALENs, or zinc finger nucleases, as well as guide RNAs or other molecules that direct the nucleases to specific genomic loci.

[0067] In aspects, the present disclosure provides a retroviral vector expression system comprising one or more nucleotide sequences encoding a recombinant protein as described herein. In aspects, the retroviral vector expression system comprises a vector construct and helper constructs that are each on separate plasmids. In aspects, the retroviral vector expression system is a lentiviral vector expression system, a foamy vector expression system, or a respiratory syncytial virus (RSV). A rhabdoviral G glycoprotein or functional fragmentLeydig 772155 Vyriad P-47-002 or derivative thereof as described herein can be expressed within a retroviral vector expression system to pseudotype the vector and alter the tropism of the vector.

[0068] As used herein, a “vector construct” comprises a nucleic acid of interest, e.g., encoding a rhabdoviral G glycoprotein or functional fragment or derivative thereof, or recombinant protein thereof, as described herein, and “helper constructs” comprise proteins useful for the formation of, e.g., virions. Such helper constructs can encode, e.g., structural proteins, and can be packaging vectors that are, e.g., plasmids.

[0069] In aspects, the retroviral vector expression system comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising, consisting essentially of, or consisting of an antigen binding domain, a transmembrane domain, and an intracellular signaling domain. In aspects, the CAR has antigenic specificity for CD19 (e.g., SEQ ID NO: 165) or BCMA (e.g., SEQ ID NOs: 166-170). In aspects, the CAR comprises a hinge domain,wherein the hinge domain is the hinge domain of CD28 or CD8 (SEQ ID NO: 149). Inaspects, the transmembrane domain is the transmembrane domain of CD28 or CD8 (SEQ ID NO: 150). In aspects, the intracellular signaling domain comprises a costimulatory domainand an activating domain. In aspects, the costimulatory domain is 4 1BB (SEQ ID NO: 151).In aspects, the activating domain is CD3zeta (SEQ ID NO: 152).

[0070] In aspects, the retroviral vector expression system comprises a nucleotide sequence encoding a transgene or gene editing system. In aspects, the gene editing system comprises one or more nucleases, such as CRISPR-Cas9, CasMINI, TALENs, or zinc finger nucleases, as well as guide RNAs or other molecules that direct the nucleases to specific genomic loci.

[0071] In aspects, the present disclosure provides a method of making a membraned vesicle, an enveloped viral particle, an enveloped delivery vehicle, or a recombinant viral vector, the method comprising, consisting essentially of, or consisting of: a) transfecting or transducing a packaging host cell with a retroviral vector expression system as described herein; and b) recovering the membraned vesicle, enveloped viral particle, enveloped delivery vehicle, or recombinant viral vector produced by the transfected or transduced packaging host cell.

[0072] “Transfecting” and “transfection” as used herein means non-viral means of introducing nucleic acid into a cell. “Transducing” and “transduction” as used herein means use of viral means of introducing nucleic acid into a cell.Leydig 772155 Vyriad P-47-002

[0073] In aspects, any method of making an enveloped delivery vehicle, lentivirus, retroviral vector expression system, recombinant viral vector, etc. as described herein can comprise transduction in a medium comprising a poloxamer-based chemical adjuvant. In aspects, the poloxamer-based chemical adjuvant is selected from vectofusin-1, poloxamer F108, and LentiboostTM. In aspects, the method of making comprises a spinoculation step at the beginning of transduction. In aspects, the packaging host cell is HEK 293T or a 293-cell suspension line.

[0074] In aspects, the present disclosure provides a plasmid comprising, consisting essentially of, or consisting of one or more nucleotide sequences encoding a recombinant protein as described herein.

[0075] In aspects, the present disclosure provides a composition as described herein or a retroviral vector expression system as described herein for use in treating a disease in a mammal.

[0076] The mammal may be any suitable mammal. Mammals include, but are not limited to, the order Rodentia, such as mice, and the order Lagomorpha, such as rabbits. The mammal can be from the order Carnivora, including Felines (cats) and Canines (dogs). The mammal can be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perissodactyla, including Equines (horses). The mammal can be of the order Primates, Cebids, or Simioids (monkeys) or of the order Anthropoids (humans and apes). In aspects, the mammal is human.

[0077] In aspects, the disease is a genetic disease. In aspects, the disease is a cancer. In aspects, the disease is beta-thalassemia, anemia, sickle cell disease, Gaucher disease, Parkinson disease, immunologic deficiency syndromes, mucopolysaccharidosis III, severe combined immunodeficiency disease, hepatitis C, chronic hepatitis C, pancytopenia, X-linked combined immunodeficiency diseases, epidermolysis bullosa, solid tumors, melanoma, Fanconi anemia, adenosine deaminase deficiency, granulomatous disease, chronic cystic fibrosis, HIV infections, HIV seropositivity, Hodgkin’s lymphoma, Bruton type agammaglobulinemia, Hemophilia A, arthritis, or choroidal neovascularization. In other aspects, the disease is any disease or condition that can be treated by delivery of a transgene. In other aspects, the disease is any disease or condition that can be treated by delivery of a gene editing system.

[0078] As used herein, “treatment” or “treat” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun toLeydig 772155 Vyriad P-47-002 develop, and also includes addressing a medical condition or disease with the objective of improving or stabilizing an outcome in a mammal being treated. “Treating,” “treat,” and “treatment” have grammatically corresponding meanings. In the context of cancer, the terms “treat” or “treating” or “treatment” can, among other things, refer to inducing apoptosis of cancerous cells, reducing the size of a cancerous tumor, delaying the growth of tumors, or inducing or enhancing an immune response against one or more cancerous cells, where the immune response has the effect of inducing apoptosis, reducing the size of a tumor, or the like. The terms “treat,” “treating,” “treatment,” “therapeutically effective,” “prevention,” etc. used herein do not necessarily imply 100% or complete treatment / prevention / etc. Rather, there are varying degrees, which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the membraned vesicles, enveloped viral particles, recombinant viral vectors, etc. as described herein and methods as described herein can provide any amount of any level of treatment. Furthermore, the treatment provided by the disclosed method can include the treatment of one or more conditions or symptoms of the disease or condition being treated.

[0079] In aspects, a method of treating comprises administering a composition as described herein or a retroviral vector expression system as described herein, comprising a rhabdoviral G glycoprotein or functional fragment or derivative thereof or recombinant protein thereof, and subsequently administering a composition as described herein or a retroviral vector expression system as described herein, comprising a different rhabdoviral G glycoprotein or functional fragment or derivative thereof or recombinant protein thereof. The subsequent administration may be within hours, days, months, or years of the first administration, with scheduling to be determined by patient needs. Multiple rounds of subsequent administrations may be performed, using the same or different rhabdoviral G glycoproteins as determined by the patient needs. Without wishing to be bound by theory, it is believed that administering a different rhabdoviral G glycoprotein or functional fragment or derivative thereof reduces the likelihood of decreased effectiveness due to any immune response that is directed against the first rhabdoviral G glycoprotein or functional fragment or derivative thereof. A rhabdoviral G glycoprotein or functional fragment or derivative thereof or recombinant protein thereof described herein can be the first administered, and a rhabdoviral G glycoprotein or functional fragment or derivative thereof or recombinant protein thereof as described herein that has a different rhabdoviral G glycoprotein than the first rhabdoviral G glycoprotein or functional fragment or derivative thereof or recombinantLeydig 772155 Vyriad P-47-002 protein thereof can be the subsequently administered rhabdoviral G glycoprotein or functional fragment or derivative thereof or recombinant protein thereof. This sequence of events can be termed “re-dosing”.

[0080] In aspects, the composition is administered intravenously. In aspects, the composition is administered intraperitoneally.

[0081] Rhabdoviral G glycoproteins can associate into trimers at the surface of a native virus, an enveloped delivery vehicle as described herein, a membraned vesicle as described herein, an enveloped viral particle as described herein, or a recombinant viral vector as described herein. As used herein, a “mixed rhabdoviral G glycoprotein trimer” comprises, consists essentially of, or consists of three rhabdoviral G glycoproteins or functional fragments or derivatives thereof (wherein each is optionally within a recombinant protein), wherein (a) at least one of the rhabdoviral G glycoproteins or functional fragments or derivatives thereof in the trimer is within a recombinant protein comprising, consisting essentially of, or consisting of a rhabdoviral G glycoprotein or a functional fragment or derivative thereof and a cysteine rich region, and (b) at least one of the rhabdoviral G glycoproteins or functional fragments or derivatives thereof in the trimer is not within a recombinant protein and does not have a cysteine rich region.

[0082] In aspects, the present disclosure provides a method of making a mixed rhabdoviral G glycoprotein trimer, the method comprising, consisting essentially of, or consisting of: a) transfecting or transducing a packaging host cell with a retroviral vector expression system as described herein; and b) recovering the mixed rhabdoviral G glycoprotein trimer. Mixed trimers may be prepared using any suitable method.

[0083] In aspects, the present disclosure provides a method of reducing inactivation of a rhabdoviral G glycoprotein or a functional fragment or derivative thereof by serum, LDL, or vLDL, the method comprising, consisting essentially of, or consisting of producing the rhabdoviral G glycoprotein or functional fragment or derivative thereof as a recombinant protein as described herein, and exposing the recombinant protein to serum, LDL, or vLDL, wherein inactivation by serum, LDL, or vLDL is reduced. In aspects, the recombinant protein is inactivated to a lesser degree by serum, LDL, or vLDL compared to a rhabdoviral G glycoprotein without the cysteine rich region. Reduction in inhibition can be of any amount, e.g., reduced by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, or any range between these percentages. In aspects, producing vectors as described herein in a cellLeydig 772155 Vyriad P-47-002 that expresses CD55 (or overexpresses CD55) can increase resistance to complement inactivation.

[0084] It is contemplated that the recombinant proteins, membraned vesicles, enveloped viral particles, recombinant viral vectors, compositions, and retroviral vector expression systems as described herein can be used in any use as described in U.S. Provisional Patent Application No.63 / 633,553, filed April 12, 2024, which provisional patent application is incorporated by reference herein in its entirety.

[0085] The present invention leverages the efficient yet simple membrane fusion mechanism of rhabdoviral G glycoprotein, combined with specificity rendered by self- blinding to LDLR through binding to or interacting with a cysteine rich domain and a retargeting molecule, resulting in enabling versatile applications in current and future advancements within biomedicine and biotechnology.

[0086] The following are certain aspects of the disclosure.

[0087] 1. A recombinant protein comprising, consisting essentially of, or consisting of: (a) a rhabdoviral G glycoprotein or a functional fragment or derivative thereof, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is capable of binding to or interacting with low density lipoprotein receptor (LDLR); and (b) a cysteine rich region, wherein the cysteine rich region comprises, consists essentially of, or consists of a cysteine rich domain of LDLR, and wherein the cysteine rich region is capable of binding to or interacting with rhabdoviral G glycoprotein or a functional fragment or derivative thereof; wherein the cysteine rich region is covalently bonded to the rhabdoviral G glycoprotein or functional fragment or derivative thereof.

[0088] 2. The recombinant protein of aspect 1, wherein the cysteine rich region comprises, consists essentially of, or consists of (i) cysteine rich domain 2 (CR2) of LDLR, (ii) cysteine rich domain 3 (CR3) of LDLR, (iii) cysteine rich domain 4 (CR4) of LDLR, or (iv) any combination of (i) – (iii).

[0089] 3. The recombinant protein of aspect 2, wherein the cysteine rich region comprises, consists essentially of, or consists of CR2 and CR3 of LDLR.

[0090] 4. The recombinant protein of any one of aspects 1-3, wherein the cysteine rich region comprises, consists essentially of, or consists of the amino acid sequence of (A) SEQ ID NO: 292, (B) SEQ ID NO: 293, or (C) both (A) and (B).Leydig 772155 Vyriad P-47-002

[0091] 5. The recombinant protein of any one of aspects 1-4, wherein the recombinant protein comprises an additional cysteine rich region, wherein the additional cysteine rich region comprises, consists essentially of, or consists of (a) cysteine rich domain 2 (CR2) of LDLR, (b) cysteine rich domain 3 (CR3) of LDLR, (c) cysteine rich domain 4 (CR4) of LDLR, or (d) any combination of (a) – (c).

[0092] 6. The recombinant protein of aspect 5, wherein the cysteine rich region and the additional cysteine rich region are contiguous.

[0093] 7. The recombinant protein of aspect 5, wherein the cysteine rich region and the additional cysteine rich region are not contiguous.

[0094] 8. The recombinant protein of any one of aspects 5-7, wherein the additional cysteine rich region comprises, consists essentially of, or consists of CR2 and CR3 of LDLR.

[0095] 9. The recombinant protein of any one of aspects 5-8, wherein the additional cysteine rich region comprises, consists essentially of, or consists of the amino acid sequence of (A) SEQ ID NO: 292, (B) SEQ ID NO: 293, or (C) both (A) and (B).

[0096] 10. The recombinant protein of any one of aspects 1-9, wherein binding of the recombinant protein is reduced or abolished for a cysteine rich domain of LDLR that is separate from the cysteine rich region of the recombinant protein.

[0097] 11. The recombinant protein of any one of aspects 1-10, wherein the recombinant protein comprises a targeting molecule.

[0098] 12. The recombinant protein of aspect 11, wherein the targeting molecule is N- terminal to the rhabdoviral G glycoprotein or functional fragment or derivative thereof, optionally N-terminal to the cysteine rich region.

[0099] 13. The recombinant protein of aspect 11 or 12, wherein the targeting molecule is a ligand.

[0100] 14. The recombinant protein of aspect 13, wherein the ligand is epidermal growth factor (EGF) (SEQ ID NO: 298), a mutant EGF (EFGm123) (SEQ ID NO: 59), stem cell factor (SCF) (SEQ ID NO: 299), thrombopoietin (TPO) (SEQ ID NO: 300), human hepatocyte growth factor (HGF) (SEQ ID NO: 57), erythropoietin (EPO), or type 1 insulin- like growth factor (IGF1) (SEQ ID NO: 58).

[0101] 15. The recombinant protein of aspect 11 or 12, wherein the targeting molecule is a single chain variable fragment (scFv).

[0102] 16. The recombinant protein of aspect 15, wherein the scFv binds epidermal growth factor (EGF), human epidermal growth factor receptor 2 (Her2), cluster ofLeydig 772155 Vyriad P-47-002 differentiation 3 (CD3), cluster of differentiation 4 (CD4), cluster of differentiation 8 (CD8), cluster of differentiation 7 (CD7), cluster of differentiation 117 (cKit), mucin-16 (MUC16), B cell maturation antigen (BCMA), or Nectin4.

[0103] 17. The recombinant protein of any one of aspects 1-16, wherein the recombinant protein comprises a signal peptide, optionally N-terminal to the rhabdoviral G glycoprotein or functional fragment or derivative thereof.

[0104] 18. The recombinant protein of any one of aspects 1-17, wherein the cysteine rich region is N-terminal to the rhabdoviral G glycoprotein or functional fragment or derivative thereof.

[0105] 19. The recombinant protein of any one of aspects 1-18, wherein the recombinant protein is a fusion protein comprising, consisting essentially of, or consisting of the rhabdoviral G glycoprotein or functional fragment or derivative thereof and the cysteine rich region.

[0106] 20. The recombinant protein of any one of aspects 1-19, wherein the recombinant protein comprises a linker between the cysteine rich region and the rhabdoviral G glycoprotein or functional fragment or derivative thereof.

[0107] 21. The recombinant protein of aspect 20, wherein the linker is flexible.

[0108] 22. The recombinant protein of aspect 21, wherein the linker is AAASGGSGGGGSGGGGSGP (SEQ ID NO: 130), AAASGGSGGGGSGGGGS (SEQ ID NO: 131), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 132), GGGGSGGGGSGGGGS (SEQ ID NO: 36), GGGGSGGGGS (SEQ ID NO: 133), GGGGS (SEQ ID NO: 134), GGGGGGGG (SEQ ID NO: 135), GGGGGG (SEQ ID NO: 136), GSAGSAAGSGEF (SEQ ID NO: 137), and VPGVGVPGVG (SEQ ID NO: 138).

[0109] 23. The recombinant protein of aspect 20, wherein the linker is rigid.

[0110] 24. The recombinant protein of aspect 23, wherein the linker is PAPAP (SEQ ID NO: 139), EAAAKEAAAKEAAAK (SEQ ID NO: 140), EAAAKEAAAK (SEQ ID NO: 141),Leydig 772155 Vyriad P-47-002 EAAAK (SEQ ID NO: 142), AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO: 143), AEAAAKEAAAKA (SEQ ID NO: 144), ESKYGPPCPPCP (SEQ ID NO: 145), CPPCPAPELLGGPSVF (SEQ ID NO: 146), and alanine-proline (AP) repeated for a total of 10 to 34 amino acids (SEQ ID NO: 147).

[0111] 25. The recombinant protein of any one of aspects 1-24, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of a Flanders virus glycoprotein (FLAV-G) (SEQ ID NO: 13), a Chandipura virus glycoprotein (CHPV-G) (SEQ ID NO: 14), a Perinet virus glycoprotein (PERV-G) (SEQ ID NO: 15), a Piry virus glycoprotein (PIRYV-G) (SEQ ID NO: 16), a Fukuoka virus glycoprotein (FUKV-G) (SEQ ID NO: 17), a Joinjakaka virus glycoprotein (JOIV-G) (SEQ ID NO: 18), a Kumasi virus glycoprotein (KRV-G) (SEQ ID NO: 19), a Keuraliba virus glycoprotein (KEUV-G) (SEQ ID NO: 20), an Isfahan glycoprotein (ISFV-G), a Jurona glycoprotein (JURV-G), a Mediterranean Bat glycoprotein (MBV-G), a Malpais Spring glycoprotein (MSPV-G), a Radi glycoprotein (RADV-G), a Rhinolophus affinis-G, a Yug Bugdanavoc glycoprotein (YBV- G), a Yinshui Bat glycoprotein (YSBV-G), a Kimberley glycoprotein (KIMV-G), a Kanyawara glycoprotein (KYAV-G), a La Joya glycoprotein (LJV-G), a Mosquiero glycoprotein (MQOV-G), a Parry Creek glycoprotein (PCV-G), a Bas Congo glycoprotein (BASV-G), a Bovine Ephemeral fever glycoprotein (BEFV-G), a Curionopolis glycoprotein (CURV-G), a Drosophila melanogaster sigmavirus glycoprotein (DMelSV-G), a Niakha glycoprotein (NIAV-G), a Puerto almandras glycoprotein (PTAMV-G), or a Tupaia rhabdovirus (TUPTV-G).

[0112] 26. The recombinant protein of any one of aspects 1-24, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus glycoprotein or a functional fragment or derivative thereof.

[0113] 27. The recombinant protein of any one of aspects 1-24, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus indiana, Vesiculovirus newjersey, Vesiculovirus carajas, or Vesiculovirus alagoas.

[0114] 28. The recombinant protein of aspect 27, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus indiana (SEQ ID NO: 21).Leydig 772155 Vyriad P-47-002

[0115] 29. The recombinant protein of aspect 27, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus newjersey (SEQ ID NO: 22).

[0116] 30. The recombinant protein of any one of aspects 1-29, wherein the rhabdoviral G glycoprotein is substantially intact.

[0117] 31. The recombinant protein of any one of aspects 1-29, wherein the rhabdoviral G glycoprotein is a functional fragment or derivative thereof.

[0118] 32. The recombinant protein of any one of aspects 1-31, wherein the recombinant protein comprises a tag for western detection or purification.

[0119] 33. The recombinant protein of aspect 32, wherein the tag is a tag for western detection.

[0120] 34. The recombinant protein of aspect 32, wherein the tag is a tag for purification.

[0121] 35. A membraned vesicle comprising the recombinant protein of any one of aspects 1-34.

[0122] 36. The membraned vesicle of aspect 35, wherein the vesicle is a gesicle or an exosome.

[0123] 37. The membraned vesicle of aspect 35 or 36, wherein the membraned vesicle comprises a mixed trimer, wherein the mixed trimer comprises (a) at least one recombinant protein comprising a cysteine rich region, and (b) at least one rhabdoviral G glycoprotein or functional fragment or derivative thereof that does not comprise a cysteine rich region.

[0124] 38. The membraned vesicle of aspect 37, wherein the membraned vesicle comprises an unmixed trimer, wherein the unmixed trimer comprises only rhabdoviral G glycoprotein or a functional fragment or derivative thereof that does not comprise a cysteine rich region.

[0125] 39. The membraned vesicle of aspect 37 or 38, wherein the membraned vesicle comprises the maximum number of the recombinant proteins comprising a cysteine rich region possible to be accommodated within the membrane of the membraned vesicle.

[0126] 40. An enveloped viral particle comprising the recombinant protein of any one of aspects 1-34.

[0127] 41. The enveloped viral particle of aspect 40, wherein the enveloped viral particle comprises a mixed trimer, wherein the mixed trimer comprises (a) at least one recombinant protein comprising a cysteine rich region, and (b) at least one rhabdoviral G glycoprotein or functional fragment or derivative thereof that does not comprise a cysteine rich region.Leydig 772155 Vyriad P-47-002

[0128] 42. The enveloped viral particle of aspect 41, wherein the enveloped viral particle comprises an unmixed trimer, wherein the unmixed trimer comprises only rhabdoviral G glycoprotein or a functional fragment or derivative thereof that does not comprise a cysteine rich region.

[0129] 43. The enveloped viral particle of aspect 41 or 42, wherein the enveloped viral particle comprises the maximum number of the recombinant proteins comprising a cysteine rich region possible to be accommodated within the membrane of the enveloped viral particle.

[0130] 44. A recombinant viral vector comprising a nucleotide encapsulated by the membraned vesicle of any one of aspects 35-39 or the enveloped viral particle of any one of aspects 40-43.

[0131] 45. A composition comprising a pharmaceutically acceptable carrier and the membraned vesicle of any one of aspects 35-39, the enveloped viral particle of any one of aspects 40-43, or the recombinant viral vector of aspect 44.

[0132] 46. A method of delivering a payload to a T cell, the method comprising contacting the T cell with the membraned vesicle of any one of aspects 35-39, the enveloped viral particle of any one of aspects 40-43, the recombinant viral vector of aspect 44, or the composition of aspect 45.

[0133] 47. The method of aspect 46, wherein the T cell in in vitro or ex vivo.

[0134] 48. The method of aspect 46, wherein the T cell in in vivo.

[0135] 49. The method of any one of aspects 46-48, wherein the payload is a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

[0136] 50. The method of aspect 49, wherein the CAR has antigenic specificity for CD19 or BCMA.

[0137] 51. The method of aspect 49 or 50, wherein the CAR comprises a hinge domain,wherein the hinge domain is the hinge domain of CD28 or CD8 .

[0138] 52. The method of any one of aspects 49-51, wherein the transmembrane domain is the transmembrane domain of CD28 or CD8.

[0139] 53. The method of any one of aspects 49-52, wherein the intracellular signaling domain comprises a costimulatory domain and an activating domain.

[0140] 54. The method of aspect 53, wherein the costimulatory domain is 4 1BB.

[0141] 55. The method of aspect 53 or 54, wherein the activating domain is CD3zeta.Leydig 772155 Vyriad P-47-002

[0142] 56. The method of any one of aspects 46-48, wherein the payload is a transgene or gene editing system.

[0143] 57. A retroviral vector expression system comprising one or more nucleotide sequences encoding the recombinant protein of any one of aspects 1-34.

[0144] 58. The retroviral vector expression system of aspect 57, wherein the retroviral vector expression system comprises a vector construct and helper constructs that are each on separate plasmids.

[0145] 59. The retroviral vector expression system of aspect 57 or 58, wherein the retroviral vector expression system is a lentiviral vector expression system.

[0146] 60. The retroviral vector expression system of any one of aspects 57-59, comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

[0147] 61. The retroviral vector expression system of aspect 60, wherein the CAR has antigenic specificity for CD19 or BCMA.

[0148] 62. The retroviral vector expression system of aspect 60 or 61, wherein the CARcomprises a hinge domain, wherein the hinge domain is the hinge domain of CD28 orCD8 .

[0149] 63. The retroviral vector expression system of any one of aspects 60-62, wherein the transmembrane domain is the transmembrane domain of CD28 or CD8.

[0150] 64. The retroviral vector expression system of any one of aspects 60-63, wherein the intracellular signaling domain comprises a costimulatory domain and an activating domain.

[0151] 65. The retroviral vector expression system of aspect 64, wherein thecostimulatory domain is 4 1BB.

[0152] 66. The retroviral vector expression system of aspect 64 or 65, wherein the activating domain is CD3zeta.

[0153] 67. The retroviral vector expression system of any one of aspects 57-59, comprising one or more nucleotides sequence encoding a transgene or gene editing system.

[0154] 68. A method of making a membraned vesicle, an enveloped viral particle, or a recombinant viral vector, the method comprising: a) transfecting or transducing a packaging host cell with the retroviral vector expression system of any one of aspects 57-67; andLeydig 772155 Vyriad P-47-002 b) recovering the membraned vesicle, enveloped viral particle, or recombinant viral vector produced by the transfected or transduced packaging host cell.

[0155] 69. A plasmid comprising one or more nucleotide sequences encoding the recombinant protein of any one of aspects 1-34.

[0156] 70. The composition of aspect 45 or the retroviral vector expression system of any one of aspects 57-67 for use in treating a disease in a mammal.

[0157] 71. The composition or the retroviral vector expression system for use according to aspect 70, wherein the mammal is a human.

[0158] 72. The composition or the retroviral vector expression system for use according to aspect 70 or 71, wherein the disease is a genetic disease.

[0159] 73. The composition or the retroviral vector expression system for use according to aspect 70 or 71, wherein the disease is a cancer.

[0160] 74. The composition or the retroviral vector expression system for use according to aspects 70-73, wherein the composition is administered intravenously.

[0161] 75. The composition or the retroviral vector expression system for use according to aspects 70-73, wherein the composition is administered intraperitoneally.

[0162] 76. A method of making a mixed rhabdoviral G glycoprotein trimer, the method comprising: a. transfecting or transducing a packaging host cell with the retroviral vector expression system of any one of aspects 57-67; and b. recovering the mixed rhabdoviral G glycoprotein trimer.

[0163] 77. A method of reducing inactivation of a rhabdoviral G glycoprotein or a functional fragment or derivative thereof by serum, LDL, or vLDL, the method comprising producing the rhabdoviral G glycoprotein or functional fragment or derivative thereof as the recombinant protein of any one of aspects 1-34, and exposing the recombinant protein to serum, LDL, or vLDL, wherein inactivation by serum, LDL, or vLDL is reduced.

[0164] It shall be noted that the preceding are merely examples of aspects of the disclosure. Other exemplary aspects are apparent from the entirety of the description herein. It will also be understood by one of ordinary skill in the art that each of these aspects may be used in various combinations with the other aspects provided herein.

[0165] The following examples further illustrate aspects of the disclosure, but, of course, should not be construed as in any way limiting its scope.Leydig 772155 Vyriad P-47-002 EXAMPLE 1

[0166] This example demonstrates the development of constructs of recombinant proteins of Vesiculovirus glycoprotein and cysteine rich regions of low density lipoprotein receptor (LDLR)

[0167] It has been shown that VSV G (SEQ ID NO: 21) can bind to cysteine rich (CR) regions, either CR2 (SEQ ID NO: 292) or CR3 (SEQ ID NO: 293) or both on the LDLR (FIG.1A). In addition, it has been found that H8, K47, Y209, and R354 are residues in VSV G (SEQ ID NO: 21) that bind to LDLR (FIG.1B). Constructs were developed to test if a tethered CR2 (SEQ ID NO: 292), CR3 (SEQ ID NO: 293), or combination of the two domains combined onto VSV G (SEQ ID NO: 21) with a glycine-4-serine (G4S) three-time repeat linker sequence (SEQ ID NO: 36) could ablate VSV G interaction with LDLR. Additionally, the targeting molecule EGFm123 (SEQ ID NO: 59) was added to these constructs to determine if the recombinant proteins could both blind the interaction of VSV G with LDLR and specifically target a receptor, in this case epidermal growth factor receptor (EGFR). Multiple constructs were designed and placed into pCG-4MC11 expression vectors with various orientations of each of the CR domains and EGFm123 (SEQ ID NO: 59) to determine the best orientation for targeting and specificity (FIG.1C). EXAMPLE 2

[0168] This example demonstrates the construction of targeted pseudotyped lentivirus particles by transfection of cells with constructs for recombinant proteins of Vesiculovirus glycoprotein and cysteine rich regions of LDLR.

[0169] HEK-293T cells were transfected with plasmids for lentivirus production. The plasmids fo lentivirus production were pCG-4MC11 expression vectors encoding VSV-G- WT alone (SEQ ID NO: 96); EGFm123 N-terminal to VSV-G-WT (SEQ ID NO: 302); CR2 N-terminal to EGFm123 N-terminal to VSV-G-WT (SEQ ID NO: 303); CR3 N-terminal to EGFm123 N-terminal to VSV-G-WT (SEQ ID NO: 304); EGFm123 N-terminal to CR2 N- terminal to VSV-G-WT (SEQ ID NO: 305); EGFm123 N-terminal to CR3 N-terminal to VSV-G-WT (SEQ ID NO: 306); CR2 N-terminal to CR3 N-terminal to EGFm123 N-terminal to VSV-G-WT (SEQ ID NO: 307); or EGFm123 N-terminal to CR2 N-terminal to CR3 N- terminal to VSV-G-WT (SEQ ID NO: 306). Three days post transfection, during collection of each lentivirus, cell lysates were harvested through pipette washing with 5 mLs of 1X-DPBSLeydig 772155 Vyriad P-47-002 off a 15-cm plate. One-fifth of the total cells from the 15-cm dish was collected in the Eppendorf tube, spun down in a tabletop centrifuge at 350xg for 4 minutes, PBS was aspirated with a 1 mL pipette, and the remaining cell pellet was frozen at -80°C for at least one day. Sample processing was conducted by thawing the cell pellet and resuspending it in RIPA lysis buffer with protease inhibitor. Upon resuspension, samples were vortexed on power 7, three times for 1 second each time. The samples were then allowed to rest on ice for 10 minutes. After that period of rest, the samples were vortexed again 3 times for 1 second each time. Samples were spun at 15,000xg for 5 minutes at 4°C. The supernatant was transferred to a new 1.5 mL Eppendorf tube and protein concentration was identified through the Thermo Scientific Pierce 660 nm Protein Assay Reagent process on the nanodrop.20 μg or 15 μg of each protein sample was loaded per well into a 4-12% Bis-Tris PAGE gel, ran at 150V for 1 hour and 30 minutes, and then transferred to a PVDF membrane. Membranes were blotted with polyclonal rabbit IgG VSV G and either a mouse monoclonal GAPDH antibody (FIGs.2A-2B) or a P24 antibody (FIG.2C). Proteolytic cleavage appears to occur in some constructs. EXAMPLE 3

[0170] This example demonstrates the specific targeted infection of certain cell populations with pseudotyped lentivirus particles using constructs for recombinant proteins of Vesiculovirus glycoprotein, cysteine rich regions of LDLR, and EGFm123.

[0171] K562, K562-EGFR, and K562-Her2 expressing chronic myelogenous leukemia cells were transduced with lentiviruses containing a GFP expression cassette (SEQ ID NO: 176) displaying different orientations of EGFm123 (SEQ ID NO: 59), CR2 (SEQ ID NO: 292), and CR3 (SEQ ID NO: 293) domains at an MOI of 5 (FIGs.3B-3C) and an MOI of 25 (FIGs.3D-3E). MOI was determined based on P24 ELISA titers shown in Table 2. Four days post transduction, Nikon GFP images of a small portion of each well (FIG.3A) and Celigo readouts of GFP positive cell number per well were captured and quantified (FIGs.3B-3E). The VSV-G WT virus caused broad infection of K562, K562-EGFR, and K562-Her2 cells, while the viruses constructed with EGFm123 (SEQ ID NO: 59), CR2 (SEQ ID NO: 292) and / or CR3 (SEQ ID NO: 293), and VSV-G WT (SEQ ID NO: 21) had less broad infection and were targeted preferentially to K562-EGFR cells. The virus particles produced using the EGFm123-CR2-CR3-VSV-G WT and EGFm123-CR3-VSV-G WT constructs (FIG.3A, see boxed micrographs) showed the greatest targeting and specificity of the tested constructs.Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002 40 Table 2

[0172] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0173] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unlessLeydig 772155 Vyriad P-47-002 otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0174] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.Leydig 772155 Vyriad P-47-002 SEQUENCESLeydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002Leydig 772155 Vyriad P-47-002

Claims

CLAIMS:1 . A recombinant protein comprising:(a) a rhabdoviral G glycoprotein or a functional fragment or derivative thereof, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is capable of binding to or interacting with low density’ lipoprotein receptor (LDLR); and(b) a cysteine rich region, wherein the cysteine rich region comprises a cysteine rich domain of LDLR, and wherein the cysteine rich region is capable of binding to or interacting with rhabdoviral G glycoprotein or a functional fragment or derivative thereof; wherein the cysteine rich region is covalently bonded to the rhabdoviral G glycoprotein or functional fragment or derivative thereof.

2. The recombinant protein of claim 1, wherein the cysteine rich region comprises (i) cysteine rich domain 2 (CR2) of LDLR, (ii) cysteine rich domain 3 (CR3) of LDLR, (iii) cysteine rich domain 4 (CR4) of LDLR, or (iv) any combination of (i) - (iii).

3. The recombinant protein of claim 2, wherein the cysteine rich region comprises CR2 and CR3 of LDLR.

4. Hie recombinant protein of any one of claims 1-3, wherein the cysteine rich region comprises the ammo acid sequence of (A) SEQ ID NO: 292, (B) SEQ ID NO: 293, or (C) both (A) and (B).

5. The recombinant protein of any one of claims 1 -4, wherein the recombinant protein comprises an additional cysteine rich region, wherein the additional cysteine rich region comprises (a) cysteine rich domain 2 (CR2) of LDLR, (b) cysteine rich domain 3 (CR3) of LDLR, (c) cysteine rich domain 4 (CR4) of LDLR, or (d) any combination of (a) -(c).

6. The recombinant protein of claim 5, wherein the cysteine rich region and the additional cysteine rich region are contiguous.

7. The recombinant protein of claim 5, wherein the cysteine rich region and the additional cysteine rich region are not contiguous.

8. The recombinant protein of any one of claims 5-7, wherein the additional cysteine rich region comprises CR2 and CR3 of LDLR.

9. The recombinant protein of any one of claims 5-8, wherein the additional cysteine rich region comprises the amino acid sequence of (A) SEQ ID NO: 292, (B) SEQ ID NO: 293, or (C) both (A) and (B).

10. The recombinant protein of any one of claims I -9, wherein binding of the recombinant protein is reduced or abolished for a cysteine rich domain of LDLR that is separate from the cysteine rich region of the recombinant protein.

11. The recombinant protein of any one of claims 1-10, wherein the recombinant protein comprises a targeting molecule.

12. The recombinant protein of claim 11, wherein the targeting molecule is N- terminal to the rhabdoviral G glycoprotein or functional fragment or derivative thereof, optionally N-terminal to the cysteine rich region.

13. The recombinant protein of claim 11 or 12, wherein the targeting molecule is a ligand.

14. The recombinant protein of claim 13, wherein the ligand is epidermal growth factor (EGF) (SEQ ID NO: 298), a mutant EGF (EFGml23) (SEQ ID NO: 59), stem cell factor (SCF) (SEQ ID NO: 299), thrombopoietin (TPO) (SEQ ID NO: 300), human hepatocyte growth factor (HGF) (SEQ ID NO: 57), erythropoietin (EPO), or type 1 insulinlike growth factor (IGF1) (SEQ ID NO: 58).

15. The recombinant protein of claim 11 or 12, wherein the targeting molecule is a single chain variable fragment (scFv).

16. The recombinant protein of claim 15, wherein the scFv binds epidermal growth factor (EGF), human epidermal growth factor receptor 2 (Her2), cluster of differentiation 3 (CD3), duster of differentiation 4 (CD4), duster of differentiation 8 (CD8), cluster of differentiation 7 (CD7), cluster of differentiation 117 (cKit), mucin-16 (MUC16), B cell maturation antigen (BC MA), orNectind.

17. The recombinant protein of any one of claims 1-16, wherein the recombinant protein comprises a signal peptide, optionally N-termmal to the rhabdoviral G glycoprotein or functional fragment or derivative thereof18. Hie recombinant protein of any one of claims 1-17, wherein the cysteine rich region is N-terminal to the rhabdoviral G glycoprotein or functional fragment or derivative thereof.

19. The recombinant protein of any one of claims 1-18, wherein the recombinant protein is a fusion protein comprising the rhabdoviral G glycoprotein or functional fragment or derivative thereof and the cysteine rich region.

20. The recombinant protein of any one of claims 1-19, wherein the recombinant protein comprises a linker between the cysteine rich region and the rhabdoviral G glycoprotein or functional fragment or derivative thereof.

21. The recombinant protein of claim 20, wherein the linker is flexible.

22. Hie recombinant protein of claim 21, wherein the linker isAAASGGSGGGGSGGGGSGP (SEQ ID NO: 130),AAASGGSGGGGSGGGGS (SEQ ID NO: 131),GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 132),GGGGSGGGGSGGGGS (SEQ ID NO: 36),GGGGSGGGGS (SEQ ID NO: 133),GGGGS (SEQ ID NO: 134),GGGGGGGG (SEQ ID NO: 135),GGGGGG (SEQ ID NO: 136),GSAGSAAGSGEF (SEQ ID NO: 137), andVPGVGVPGVG (SEQ ID NO: 138).

23. The recombinant protein of claim 20, wherein the linker is rigid.

24. The recombinant protein of claim 23, wherein the linker isPAPAP (SEQ ID NO: 139),EAAAKEAAAKEAAAK (SEQ ID NO: 140),EAAAKEAAAK (SEQ ID NO: 141),EAAAK (SEQ ID NO: 142),AEAAAKEAAAKEAAAKEAAAKAI.EAEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO: 143),AEAAAKEAAAKA (SEQ ID NO: 144).ESKYGPPCPPCP (SEQ ID NO: 145),CPPCPAPELLGGPSVF (SEQ ID NO: 146), and alanine-proline (AP) repeated for a total of 10 to 34 amino acids (SEQ ID NO: 147).

25. The recombinant protein of any one of claims I -24, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of a Flanders virus glycoprotein (FLAV-G) (SEQ ID NO: 13), a Chandipura virus glycoprotein (CHPV-G) (SEQ ID NO: 14), a Perinet virus glycoprotein (PERV-G) (SEQ ID NO: 15), a Pity virus glycoprotein (PIRYV- G) (SEQ ID NO: 16), a Fukuoka virus gly coprotein (FUKV -G) (SEQ ID NO: 17), a Joinjakaka virus glycoprotein (JOIV -G) (SEQ ID NO: 18), a Kumasi virus glycoprotein (KRV-G) (SEQ ID NO: 19), a Keuraliba virus glycoprotein (KEUV-G) (SEQ ID NO: 20), an Isfahan glycoprotein (ISFV-G), a Jurona glycoprotein (JURV-G), a Mediterranean Bat glycoprotein (MBV-G), a Malpais Spring glycoprotein (MSPV-G), a Radi glycoprotein (RADV-G), a Rhinolophus affinis-G, a Yug Bugdanavoc glycoprotein (YBV-G), a Yinshui Bat glycoprotein (YSBV-G), a Kimberley glycoprotein (KIMV-G), a Kanyawara glycoprotein (KYAV-G), a La Joy a glycoprotein (LJV -G), a Mosquiero glycoprotein (MQOV-G), a Pany Creek glycoprotein (PCV-G), a Bas Congo glycoprotein (BASV-G), a Bovine Ephemeral fever glycoprotein (BEFV-G), a Curionopolis glycoprotein (CURV-G), aDrosophila melanogaster sigmavirus glycoprotein (DMelSV-G), a Niakha glycoprotein (NIAV-G), a Puerto almandras glycoprotein (PTAMV-G), or a Tupaia rhabdovirus (TUPTV- G).

26. Hie recombinant protein of any one of claims 1-24, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus glycoprotein or a functional fragment or derivative thereof.

27. The recombinant protein of any one of claims 1 -24, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus Indiana, Vesiculovirus newjersey, Vesiculovirus carajas, or Vesiculovirus alagoas.

28. The recombinant protein of claim 27, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus Indiana (SEQ ID NO: 21).

29. The recombinant protein of claim 27, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus newjersey (SEQ ID NO: 22).

30. Hie recombinant protein of any one of claims 1-29, wherein the rhabdoviral G gly coprotein is substantially intact.

31. Tire recombinant protein of any one of claims 1-29, wherein the rhabdoviral G gly coprotein is a functional fragment or derivative thereof.

32. The recombinant protein of any one of claims 1-31, wherein the recombinant protein comprises a tag for western detection or purification.

33. The recombinant protein of claim 32, wherein the tag is a tag for western detection.

34. The recombinant protein of claim 32, wherein the tag is a tag for purification.

35. A membraned vesicle comprising the recombinant protein of any one of claims 1-34.

36. The membraned vesicle of claim 35, wherein the vesicle is a gesicle or an exosome.

37. The membraned vesicle of claim 35 or 36, wherein the membraned vesicle comprises a mixed trimer, wherein the mixed trimer comprises (a) at least one recombinant protein comprising a cysteine rich region, and (b) at least one rhabdoviral G glycoprotein or functional fragment or derivative thereof that does not comprise a cysteine rich region.

38. The membraned vesicle of claim 37, wherein the membraned vesicle comprises an unmixed trimer, wherein the unmixed trimer comprises only rhabdoviral G glycoprotein or a functional fragment or derivative thereof that does not comprise a cysteine rich region.

39. The membraned vesicle of claim 37 or 38, wherein the membraned vesicle comprises the maximum number of the recombinant proteins comprising a cysteine rich region possible to be accommodated within the membrane of the membraned vesicle.

40. An enveloped viral particle comprising the recombinant protein of any one of claims 1-34.

41. The enveloped viral particle of claim 40, wherein the enveloped viral particle comprises a mixed trimer, wherein the mixed trimer comprises (a) at least one recombinant protein comprising a cysteine rich region, and (b) at least one rhabdoviral G glycoprotein or functional fragment or derivative thereof that does not comprise a cysteine rich region.

42. The enveloped viral particle of claim 41, wherein the enveloped viral particle comprises an unmixed trimer, wherein the unmixed trimer comprises only rhabdoviral G glycoprotein or a functional fragment or derivative thereof that does not comprise a cysteine rich region.

43. The enveloped viral particle of claim 41 or 42, wherein the enveloped viral particle comprises the maximum number of the recombinant proteins comprising a cysteine rich region possible to be accommodated within the membrane of the enveloped viral particle.

44. A recombinant viral vector comprising a nucleotide encapsulated by the membraned vesicle of any one of claims 35-39 or the enveloped viral particle of any one of claims 40-43.

45. A composition comprising a pharmaceutically acceptable carrier and the membraned vesicle of any one of claims 35-39, the enveloped viral particle of any one of claims 40-43, or the recombinant viral vector of claim 44.

46. A method of delivering a payload to a T cell, the method comprising contacting the T cell with the membraned vesicle of any one of claims 35-39, the enveloped viral particle of any one of claims 40-43, the recombinant viral vector of claim 44, or the composition of claim 45.

47. The method of claim 46, wherein the T cell in in vitro or ex vivo.

48. The method of claim 46, wherein the T cell in in vivo.

49. The method of any one of claims 46-48, wherein the payload is a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

50. The method of claim 49, wherein the CAR has antigenic specificity for CD19 or BCMA.

51. The method of claim 49 or 50, wherein the CAR comprises a hinge domain, wherein the hinge domain is the hinge domain of CD28a or CD8a.

52. The method of any one of claims 49-51, wherein the transmembrane domain is the transmembrane domain of CD28 or CD8.

53. The method of any one of claims 49-52, wherein the intracellular signaling domain comprises a costimulatory domain and an activating domain.

54. The method of claim 53, wherein the costimulatory domain is 4-1BB.

55. The method of claim 53 or 54, wherein the activating domain is CD3zeta.

56. The method of any one of claims 46-48, wherein the payload is a transgene or gene editing system.

57. A retroviral vector expression system comprising one or more nucleotide sequences encoding the recombinant protein of any one of claims 1-34.

58. The retroviral vector expression system of claim 57, wherein the retroviral vector expression system comprises a vector construct and helper constructs that are each on separate plasmids.

59. Hie retroviral vector expression system of claim 57 or 58, wherein the retroviral vector expression system is a lentival vector expression system.

60. The retroviral vector expression system of any one of claims 57-59, comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

61. The retroviral vector expression system of claim 60, wherein the CAR has antigenic specificity for CD 19 or BCMA.

62. The retroviral vector expression system of claim 60 or 61, wherein the CAR comprises a hinge domain, wherein the hinge domain is the hinge domain of CD28a or CD8a.

63. Hie retroviral vector expression system of any one of claims 60-62, wherein the transmembrane domain is the transmembrane domain of CD28 or CDS.

64. The retroviral vector expression system of any one of claims 60-63, wherein the intracellular signaling domain comprises a costimulatory domain and an activating domain.

65. The retroviral vector expression system of claim 64, wherein the costimulatory domain is 4-1BB.

66. The retroviral vector expression system of claim 64 or 65, wherein the activating domain is CD3zeta.

67. Hie retroviral vector expression system of any one of claims 57-59, comprising one or more nucleotides sequence encoding a transgene or gene editing system.

68. A method of making a membraned vesicle, an enveloped viral particle, or a recombinant viral vector, the method comprising:a) transfecting or transducing a packaging host cell with the retroviral vector expression system of any one of claims 57-67; and b) recovering the membraned vesicle, enveloped viral particle, or recombinant viral vector produced by the transfected or transduced packaging host celi.

69. A plasmid comprising one or more nucleotide sequences encoding the recombinant protein of any one of claims 1-34.

70. The composition of claim 45 or the retroviral vector expression system of any one of claims 57-67 for use in treating a disease in a mammal.

71. The composition or the retroviral vector expression system for use according to claim 70, wherein the mammal is a human.

72. The composition or the retroviral vector expression system for use according to claim 70 or 71, wherein the disease is a genetic disease.

73. The composition or the retroviral vector expression system for use according to claim 70 or 71, wherein the disease is a cancer,74. The composition or the retroviral vector expression system for use according to claims 70-73, wherein the composition is administered intravenously.

75. Hie composition or the retroviral vector expression system for use according to claims 70-73, wherein the composition is administered intraperitoneally.

76. A method of making a mixed rhabdoviral G glycoprotein trimer, the method comprising: a. transfecting or transducing a packaging host cell with the retroviral vector expression system of any one of claims 57-67; and b. recovering the mixed rhabdoviral G glycoprotein trimer.

77. A method of reducing inactivation of a rhabdoviral G glycoprotein or a functional fragment or derivative thereof by serum, LDL, or vLDL, the method comprising producing the rhabdoviral G glycoprotein or functional fragment or derivative thereof as therecombinant protein of any one of claims 1-34, and exposing the recombinant protein to serum, LDL, or vLDL, wherein inactivation by serum, LDL, or vLDL is reduced.

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