Membraned vesicles and purification methods thereof

Abstract

The present disclosure is directed to membraned vesicles comprising: (a) an envelope membrane; and (b) viral membrane glycoproteins (VMGs), each VMG optionally within a fusion protein; wherein at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one affinity tag, wherein at least one VMG is incorporated into the envelope membrane and comprises a fusogenic membrane glycoprotein (FMG), and wherein at least one affinity tag is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle; and methods of generating membraned vesicles.

Description

MEMBRANED VESICLES AND PURIFICATION METHODS THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 563,181, filed March 8, 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; International Patent Application No. PCT / US2024 / 024516, filed April 12, 2024; U.S. Provisional Patent Application No. 63 / 660,355, filed June 14, 2024; and U.S. Provisional Patent Application No. 63 / 660,217, filed June 14, 2024; the disclosures of which are incorporated herein by reference in their entireties for all purposes.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 157,234 Byte XML (Extensible Markup Language) file named "772578_SequenceListing.xml," created on February 28, 2025.BACKGROUND

[0003] Membraned vesicles are widely used in biomedicine to deliver nucleic acids, proteins, and / or small molecules into target cells. However, purification and concentration from the supernatant of cells in which membraned vesicles are produced remains a challenge and is particularly important where direct in vivo administration is contemplated because impurities may cause toxic side effects. Since some membraned vesicles are produced by budding from mammalian cells which frequently die around the time of maximum membraned vesicle release, the producer cell supernatants from which they are harvested contain numerous impurities, including cell-derived proteins, messenger RNAs, transfer RNAs, chromosomal DNA, and extracellular vesicles, which are a similar size to the membraned vesicles. Further, the concentration of the membraned vesicles in the supernatant is typically insufficient for clinical applications. Existing methods to purify and concentrate membraned vesicles from cell culture supernatant are not easily scalable and / or do not provide optimal results.

[0004] Therefore, there remains a need to develop membraned vesicles that can easily be purified and concentrated from the supernatant of cells in which they are produced. There also remains a need to develop methods of purifying and concentrating such membraned vesicles.BRIEF SUMMARY

[0005] In aspects, the present disclosure provides a membraned vesicle comprising: (a) an envelope membrane; and (b) viral membrane glycoproteins (VMGs), each VMG optionally within a fusion protein; wherein at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one affinity tag, wherein at least one VMG is incorporated into the envelope membrane and comprises a fusogenic membrane glycoprotein (FMG), wherein at least one affinity tag is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle; and wherein the ratio of VMG without an affinity tag to VMG with an affinity tag is from 0: 1 to 5: 1.

[0006] In aspects, the present disclosure provides a membraned vesicle comprising: (a) an envelope membrane; and (b) viral membrane glycoproteins (VMGs), each VMG optionally within a fusion protein; wherein at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one affinity tag, wherein at least one VMG is incorporated into the envelope membrane and comprises a fusogenic membrane glycoprotein (FMG), wherein at least one affinity tag is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle; wherein at least one FMG is a rhabdoviral G glycoprotein engineered to have a mutation to reduce or abolish its natural receptor binding specificity, wherein the rhabdoviral G glycoprotein comprises a mutation at one or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO: 14), and wherein the mutation is a substitution at two or more positions or the mutation is a deletion.

[0007] In aspects, the present disclosure provides a method of generating the membraned vesicle as described herein, the method comprising: (a) transfecting a host cell with: (i) at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one affinity tag; (ii) optionally, at least one packaging plasmid; and (b) collecting membraned vesicles comprising at least one affinity tag displayed at the outer surface of theenvelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

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

[0009] Figure 1A is a diagram showing the experimental protocol of Example 1.

[0010] Figure IB is a western blot showing the binding of lentivirus VSV-G-QQQ (left) and a 1 :3 combination of UCHT1-VSV-G-QQQ and Strep- VSV-G-QQQ (right) against an anti-VSV-G antibody (top), an anti-StrepTag antibody (middle), or an anti-P24 antibody (bottom) in solutions collected at different points in Example 1.

[0011] Figure 2A is a diagram showing the experimental protocol of Example 2.

[0012] Figure 2B is a western blot showing the binding of a 1 :3 combination of lentivirusWT-VSV-G-QQQ and Strep- VSV-G-QQQ against an anti-VSV-G antibody (top), and anti- StrepTag antibody (middle), or an anti-P24 antibody (bottom) in solutions collected at different points in Example 2; bands showing Strep-VSV-G, VSV-G, and P24 are labeled with arrows.

[0013] Figure 3 A is a diagram showing the experimental protocol of Example 3.

[0014] Figure 3B is a western blot showing the binding of lentivirus 1 :3 combination ofWT-VSV-G and Strep-VSV-G-QQQ and 1 :3 combination of UCHT1 -VSV-G-QQQ and Strep- VS V-GQQQ against an anti-VSV-G antibody (top), an anti-StrepTag antibody (middle), or an anti-P24 antibody (bottom) in solutions collected at different points in Example 3. Left uses Strep-Tactin® XT 4Flow® affinity column and right uses Strep- Tactin®XT 4Flow® high capacity affinity column.

[0015] Figure 4A is a diagram showing the experimental protocol of Example 4.

[0016] Figure 4B is a western blot showing the binding of lentivirus 1 :3 combination ofUCHT1 -VSV-G-QQQ and Strep-VSV-G-QQQ (left) and 1 :3 combination of UCHT1-VSV- G-QQQ and Strep-(G4S)3 -VSV-G-QQQ (right) against an anti-VSV-G antibody (top) and an anti-PD24 antibody (bottom) in solutions collected at different points in Example 4.

[0017] Figure 5 A is a diagram showing the experimental protocol of Example 5.

[0018] Figure 5B is a western blot showing the binding of lentivirus 1 :3 combination ofUCHT1-VSV-G-QQQ and Strep-(G4S)3 -VSV-G-QQQ against an anti-VSV-G antibody (top), an anti-Strep antibody (middle), and an anti-P24 antibody (bottom) in solutions collected at different points in Example 5.

[0019] Figure 5C is a set of micrograph images showing Jurkat cells 72 hours post transduction with lentiviruses supernatants (F0), 50x concentrated lentiviruses supernatants (F0 (5 Ox)), 5 Ox concentrated eluate (Eluate (5 Ox)), or a no virus control.

[0020] Figure 6A is a diagram showing the experimental protocol of Example 6.

[0021] Figure 6B is a western blot showing the binding of lentivirus 1 : 1 :2 combination ofUCHT1-VSVG-QQQ, VSV-G-QQQ, and Strep-(G4S)3-VSV-G-QQQ against an anti-VSV- G antibody (top) and an anti-P24 antibody (bottom) in solutions collected at different points in Example 6.

[0022] Figure 7A is a diagram showing the experimental protocol of Example 7.

[0023] Figure 7B is a western blot showing the binding of the stated ratios of lentivirusUCHT1 -VSV-G-QQQ, VSV-G-QQQ, and Strep-(G4S)3-VSV-G-QQQ against an anti-VSV- G antibody (top), an anti-Strep antibody (middle), and an anti-P24 antibody (bottom) in solutions collected at different points in Example 7.

[0024] Figure 8A is a diagram showing the experimental protocol of Example 8.

[0025] Figure 8B is a western blot showing the binding of lentivirus 1 : 1.5: 1.5 combination of UCHT1- VSV-G-QQQ, VSV-G-QQQ, and Strep-(G4S)3 -VSV-G-QQQ against an anti-VSV-G antibody (top) and an anti-P24 antibody (bottom) in solutions collected at different points in Example 8 using a Strep-Tactin®XT 4Flow® affinity column.

[0026] Figure 8C is a western blot showing the binding of lentivirus 1 : 1.5: 1.5 combination of UCHT1- VSV-G-QQQ, VSV-G-QQQ, and Strep-(G4S)3 -VSV-G-QQQ against an anti-VSV-G antibody (top) and an anti-P24 antibody (bottom) in solutions collected at different points in Example 8 using a Strep-Tactin®XT 4Flow® high capacity affinity column.

[0027] Figure 8D is a western blot showing the binding of lentivirus 1 : 1.5: 1.5 combination of UCHT1- VSV-G-QQQ, VSV-G-QQQ, and Strep-(G4S)3 -VSV-G-QQQ against an anti-VSV-G antibody (top) and an anti-P24 antibody (bottom) in solutions collected at different points in Example 8 using a Strep-Tactin®XT 4Flow® high capacity affinity column with 30 minutes of incubation.

[0028] Figure 9A is a diagram showing the experimental protocol of Example 9.

[0029] Figure 9B is a western blot showing the binding of lentivirus 1 : 1 :2 combination ofUCHT1 -VSV-G-QQQ, VSV-G-QQQ and Strep-(G4S)3 -VSV-G-QQQ against an anti-VSV- G antibody (top) and an anti-P24 antibody (bottom) in solutions collected at different points in Example 9.

[0030] Figures 9C and 9D are bar graphs showing the transduction ratio in Jurkat cells (FIG. 9C) and Nalm6 cells (FIG. 9D) as percent of GFP positive cells (GFP+ Cells %) 72 hours post transduction with lentiviruses supernatants (F0), 40x concentrated lentiviruses supernatants (F0 X40), 40x concentrated Flow through 1 (Fl X40), 40x concentrated Flow through 2 (F2 X40), or 40x concentrated eluate (Eluate (X40).

[0031] Figure 9E is a bar graph showing the transduction ratio in Jurkat cells as shown in FIG. 9C normalized to the transduction ration of FO X40.

[0032] Figure 9F is a set of micrograph images showing Jurkat cells and Nalm6 cells 72 hours post transduction with lentiviruses supernatants (F0), 40x concentrated lentiviruses supernatants (F0 X40), 40x concentrated Flow through 1 (Fl X40), 40x concentrated Flow through 2 (F2 X40), or 40x concentrated eluate (Eluate (X40)

[0033] Figure 10A is a diagram showing the experimental protocol of Example 10.

[0034] Figure 10B is a western blot showing the binding of lentivirus 1 :3 combination ofUCHT1-VSV-G-QQQ and Strep-(G4S)5-TEV site-(G4S)5-VSV-G-QQQ (left) and 1 :3 combination of UCHT1-VSV-G-QQQ and Strep-(G4S)3-TEV site-(G4S)3-VSV-G-QQQ (right) against an anti-VSV-G antibody (top), an anti-Strep antibody (middle), and an anti- P24 antibody (bottom) in solutions collected at different points in Example 10.

[0035] Figure 11 A is a diagram showing the experimental protocol of Example 11.

[0036] Figure 1 IB is a western blot showing the binding of lentivirus 1 :3 combination ofUCHT1-VSV-G-QQQ and Strep-(G4S)5-TEV site-VSV-G-QQQ against an anti-VSV-G antibody (top), anti-Strep antibody (middle) and anti-P24 antibody (bottom) in solutions collected at different points in Example 11.

[0037] Figure 12A is a diagram showing the experimental protocol of Example 12.

[0038] Figure 12B is a western blot showing the binding of lentivirus 1 :3 combination ofUCHT1-VSV-G-QQQ and Strep-(G4S)5-TEV site-VSV-G-QQQ against an anti-VSV-G antibody (top), an anti-Strep antibody (middle) and an anti-P24 antibody (bottom) in solutions collected at different points in Example 12.DETAILED DESCRIPTION

[0039] In aspects, the present disclosure provides a membraned vesicle comprising: (a) an envelope membrane; and (b) viral membrane glycoproteins (VMGs), each VMG optionally within a fusion protein; wherein at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one affinity tag, wherein at leastone VMG is incorporated into the envelope membrane and comprises a fusogenic membrane glycoprotein (FMG), wherein at least one affinity tag is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle; and wherein the ratio of VMG without an affinity tag to VMG with an affinity tag is from 0: 1 to 5: 1.

[0040] 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. A membraned vesicle can be cell-derived and thus be a cell-derived enveloped particle (CDEP).

[0041] 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. As used herein, an “exosome” refers to a lipid bilayer vesicle ranging in size from approximately 30 nm to 150 nm in diameter, secreted by eukaryotic cells via the endosomal pathway, and containing biologically active molecules such as proteins, lipids, RNA, or DNA. An exosome typically originates from multivesicular bodies (MVBs) and is released into the extracellular environment through exocytosis. Exosomes are known to serve as intercellular communication vehicles and may be utilized for diagnostic, therapeutic, or drug delivery applications. Examples of exosomes are provided in Li et al., European Journal of Medicinal Chemistry 207: 112784 (2020); Yang et al., Adv. Mater. 29: 1605604 (2017); U.S. Pat. No. 10,195,290; and U.S. Patent Application Publication No. 2018 / 0028600, each of which is incorporated herein by reference in its entirety.

[0042] 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 fusion protein thereof. Exemplary components of a virus include, without limitation, the gag,pol, or env gene or gene product of lentivirus.

[0043] A “virus-like particle” as used herein means an enveloped viral particle that cannot produce additional virus-like particles (i.e., is replication-incompetent). Virus-like particles (VLPs) are non-infectious and mimic the conformation of their parental viruses but lack the viral genetic material required for replication. A VLP can contain genetic material / nucleic acid not associated with self-replication. VLPs can preserves the high packaging efficiency and target-specificity of certain viruses while greatly minimizing therisk of host genome integration. Because VLPs do not replicate or integrate into the host genome, they are often associated with lower cytotoxicity and reduced immunogenic risk compared to replication-competent viral systems.

[0044] 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.”

[0045] An “enveloped delivery vehicle” (EDV) can encapsulate a cargo or payload. Any suitable cargo or payload of an EDV is contemplated herein, including, but not limited to, e.g., a protein, a lipid, a nucleic acid, a small molecule, a therapeutic agent, a gene editing system, such as CRISPR Cas, and any combination thereof.

[0046] A “membrane-anchored glycoprotein” (MAG) is a glycoprotein associated with a lipid bilayer membrane. Association with a lipid bilayer can be covalently attached to the membrane. A MAG can be an integral protein, such as a transmembrane protein, or a peripheral protein associated with a surface of a membrane (inner surface or outer surface). In aspects, several peripheral MAGs can, e.g., coat a surface of a membrane. In aspects, several peripheral MAGs can coat a surface of a membrane with several separate MAGs coating the other surface of the membrane. A “viral membrane glycoprotein” (VMG) is a MAG that is from a virus or derived from a virus. A “fusogenic membrane glycoprotein” (FMG) is a MAG that comprises a glycoprotein capable of fusing lipid bilayers. A MAG can comprise an FMG, and a VMG can comprise an FMG. A MAG can comprise a cytoplasmic domain incapable of incorporation into certain membraned vesicles, e.g., EDVs. A MAG comprising a cytoplasmic domain incapable of incorporation can, but does not necessarily, prevent a membraned vesicle from encapsulating cargo / payload, rendering the membraned vesicle incapable of delivery of cargo / payload.

[0047] In aspects, at least one MAG comprises a cytoplasmic domain incapable of incorporation into an EDV. Any suitable cytoplasmic domain can be used. In aspects, the cytoplasmic domain comprises green fluorescent protein (GFP) or epidermal growth factor receptor (EGFR), or any components thereof.

[0048] In aspects, the EDV into which the cytoplasmic domain is incapable of being incorporated is a lentiviral vector particle. In aspects, the lentiviral vector particle, has a diameter of 80 nm to 100 nm, e.g., 80 nm, 81 nm, 82 nm, 83 nm, 84 nm, 85 nm, 86 nm, 87nm, 88 nm, 89 nm, 90 nm, 91 nm, 92 nm, 93 nm, 94 nm, 95 nm, 96 nm, 97 nm, 98 nm, 99 nm, 100 nm, or a diameter defined by a range of any two of the foregoing values.

[0049] In aspects, each VMG is optionally within a fusion protein. Whether one VMG is within a fusion protein is independent of whether another VMG is within a fusion protein, e.g., a membraned vesicle can comprise at least one VMG that is within a fusion protein and at least one VMG that is not within a fusion protein. In aspects, at least one VMG of a membraned vesicle is within a fusion protein. In aspects, about half of the VMGs of a membraned vesicle are within a fusion protein. In aspects, all VMGs of a membraned vesicle are within a fusion protein.

[0050] In aspects, at least one VMG is within a fusion protein comprising at least one affinity tag. Whether one VMG is within a fusion protein comprising at least one affinity tag is independent of whether another VMG is within fusion protein comprising at least one affinity tag. In aspects, at least one VMG is within a fusion protein comprising more than one affinity tag, e.g., two affinity tags, three affinity tags, four affinity tags, or more affinity tags. In aspects where a VMG is within a fusion protein comprising more than one affinity tags, the more than one affinity tags can be the same or different affinity tags. In aspects, whether one VMG is within a fusion protein comprising one affinity tag is independent of whether another VMG is within a fusion protein comprising more than one affinity tag. In aspects, at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one affinity tag, and wherein at least one affinity tag is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

[0051] In aspects, the ratio of VMG without an affinity tag to VMG with an affinity tag is from 0: 1 to 5 : 1. The ratio of VMG without an affinity tag to VMG with an affinity tag can be of any amount from 0: 1 to 5: 1, e.g. a ratio of 0:1, 1 :1, 2: 1, 3: 1, 4: 1, 5: 1, or any range between these ratios. In aspects, the ratio of VMG with an affinity tag to VMG without an affinity tag is from 1 : 1 to 3 : 1. In aspects, the ratio of VMG with an affinity tag to VMG without an affinity tag is 3: 1. In aspects, the collected membraned vesicles comprise at least one VMG with an affinity tag and no VMG without an affinity tag.

[0052] In aspects, at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one targeting polypeptide, and wherein at least one targeting polypeptide is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle. Whether one VMG isincorporated into the envelope membrane and is within a fusion protein comprising at least one targeting polypeptide is independent of whether another VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one targeting polypeptide. In aspects, at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising more than one targeting polypeptide, e.g., two targeting polypeptides, three targeting polypeptides, four targeting polypeptides, or more targeting polypeptides. Whether one VMG is within a fusion protein comprising one targeting polypeptide is independent of whether another VMG is within a fusion protein comprising more than one targeting polypeptide. In aspects where a VMG is within a fusion protein comprising more than one targeting polypeptide, the more than one targeting polypeptides can be the same or different targeting polypeptides.

[0053] Any suitable targeting polypeptide can be used. “Targeting polypeptide” and “targeting molecule” are used interchangeably herein. As used herein the term “targeting molecule” refers to a binding moiety, such as a natural ligand, antibody, multispecific binding molecule, or others known in the art. In aspects, the targeting molecule is an antibody or portion thereof. In aspects, the portion thereof is an antigen-binding fragment. In aspects, the antibody or portion thereof is a single chain fragment variable (scFv), an affibody, a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a single heavy chain antibody. Also contemplated herein are monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (dsFv), intrabodies, and anti -idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antigen specific TCR), Ig-DARTS, 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 antibodycomprising a variable domain that binds a cell surface protein on a target cell and an IgG heavy chain constant domain.

[0054] 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) kidney cell, a (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, gplOO / Pmell 7, GPNMB, HAUS3, Hepsin, HER-2 / neu, HERV-K-MEL, HLA-A1 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-A1, MAGE-AIO, MAGE-A12, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-CI, 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-1, RAGE-I, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secemin 1, SIRT2, SNRPDI, SOXIO, 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, tyrosinase (“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-l / 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-10, MAGE-12, BAGE-1, CAGE-1,2,8, CAGE-3 TO 7, LAGE-I, NY- ESO-l / 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).

[0055] In aspects, the targeting molecule is a polypeptide antibody construct. In aspects, the polypeptide antibody construct is agonistic to CD3. In aspects, the polypeptide antibody construct comprises 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 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: 30 and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 29. In aspects, the VH and VL are separated by a flexible linker. In aspects, the flexible linker is SEQ ID NO: 58. 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: 28 and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 27. In aspects, the scFv is TR66opt and whereinthe 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: 110, a complementarity determining region 2 (CDR2) of SEQ ID NO: 111, and a complementarity determining region 3 (CDR3) of SEQ ID NO: 112, and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequences of a CDR1 of SEQ ID NO: 113, a CDR2 of SEQ ID NO: 114, and a CDR3 of SEQ ID NO: 115. 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: 30 and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 29. 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: 86, a CDR2 of SEQ ID NO: 87, and a CDR3 of SEQ ID NO: 88, and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequences of a CDR1 of SEQ ID NO: 89, a CDR2 of SEQ ID NO: 90, and a CDR3 of SEQ ID NO: 91. 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: 22 and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 21. 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: 98, a CDR2 of SEQ ID NO: 99, and a CDR3 of SEQ ID NO: 100, and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequences of a CDR1 of SEQ ID NO: 101, a CDR2 of SEQ ID NO: 102, and a CDR3 of SEQ ID NO: 103. 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: 24 and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 23. 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: 104, a CDR2 of SEQ ID NO: 105, and a CDR3 of SEQ ID NO: 106, and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequences of a CDR1 of SEQ ID NO: 107, a CDR2 of SEQ ID NO: 108, and a CDR3 of SEQ ID NO: 109. In aspects, the scFv is TR66 wherein the scFv comprises a variable heavychain (VH) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 26 and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 25. In aspects, the scFv is TR66 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: 92, a CDR2 of SEQ ID NO: 93, and a CDR3 of SEQ ID NO: 94, and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequences of a CDR1 of SEQ ID NO: 95, a CDR2 of SEQ ID NO: 96, and a CDR3 of SEQ ID NO: 97.

[0056] 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 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: 74 and a variable light chain (VL) comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 75.

[0057] In aspects, the targeting molecule is a ligand. In aspects, the ligand is epidermal growth factor (EGF), a mutant EGF (EFGml23) (SEQ ID NO: 40), stem cell factor (SCF) (e.g., SEQ ID NOs: 33-35), thrombopoietin (TPO) (SEQ ID NO: 36), human hepatocyte growth factor (HGF) (SEQ ID NO: 37 or 38), or type 1 insulin-like growth factor (IGF1) (SEQ ID NO: 39). In aspects, the targeting molecule is a single chain variable fragment (scFv). In aspects, the scFv binds epidermal growth factor receptor (EGFR) (SEQ ID NO: 31), human epidermal growth factor receptor 2 (Her2) (SEQ ID NO: 32), cluster of differentiation 3 (CD3), or cluster of differentiation 117 (cKit), mucin- 16 (MUC16), B cell maturation antigen (BCMA), or Nectin4.

[0058] 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 CD1 lb. 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.

[0059] In aspects, the targeting molecule is SpyTag (SEQ ID NO: 1), SpyTag002 (SEQ ID NO: 2), SpyTagOO3 (SEQ ID NO: 3), Isopeptag (SEQ ID NO: 4), or SnoopTag (SEQ ID NO: 5). In aspects, the polypeptide tag is SpyTag (SEQ ID NO: 1). In aspects the targeting molecule is Spy-Tag.

[0060] Each VMG can be in a fusion protein that comprises an affinity tag, a targeting polypeptide, both, or neither. For example, in aspects, at least one VMG is within a fusion protein comprising at least one affinity tag, wherein the fusion protein does not comprise a targeting polypeptide. In aspects, at least one VMG is within a fusion protein comprising at least one affinity tag and at least one targeting polypeptide. In aspects, at least one VMG is within a fusion protein comprising at least one targeting polypeptide, wherein the fusion protein does not comprise an affinity tag. In aspects, at least one VMG is not within a fusion protein.

[0061] In aspects, the ratio of VMG with a targeting polypeptide to VMG without an affinity tag to VMG with an affinity tag is from 1 :0: 1 to 1 :5: 1. The ratio of the ratio of VMG with a targeting polypeptide to VMG without an affinity tag to VMG with an affinity tag is can be of any amount from 1 :0: 1 to 1 :5: 1, e.g. a ratio of 1 :0: 1, 1 : 1 :1, 1:2: 1, 1 :3: 1, 1 :4: 1, 1 :5: 1, or any range between these ratios. In aspects, the ratio of VMG with a targeting polypeptide to VMG without an affinity tag to VMG with an affinity tag is from 1 : 1 : 1 to 1 :3 : 1. In aspects, the ratio of VMG with a targeting polypeptide to VMG without an affinity tag to VMG with an affinity tag is 1 :3:1.

[0062] In aspects, each VMG can be in a fusion protein comprising a linker between the VMG and polypeptide tag and / or affinity tag. 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: 54), AAASGGSGGGGSGGGGS (SEQ ID NO: 55), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 56) f‘(G4S)5”) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 57) (“(G4S)4)”), GGGGSGGGGSGGGGS (SEQ ID NO: 58) (“(G4S)3)”), GGGGSGGGGS (SEQ ID NO: 59) (“(G4S)2)”), GGGGS (SEQ ID NO: 60), GGGGGGGG (SEQ ID NO: 61),GGGGGG (SEQ ID NO: 62),GSAGSAAGSGEF (SEQ ID NO: 63), and VPGVGVPGVG (SEQ ID NO: 64).In aspects, the linker is rigid. Exemplary rigid linkers include, but are not limited to, e.g., PAPAP (SEQ ID NO: 65), EAAAKEAAAKEAAAK (SEQ ID NO: 66), EAAAKEAAAK (SEQ ID NO: 67), EAAAK (SEQ ID NO: 68), AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO: 69), AEAAAKEAAAKA (SEQ ID NO: 70), ESKYGPPCPPCP (SEQ ID NO: 71), CPPCPAPELLGGPSVF (SEQ ID NO: 72), and alanine-proline (AP) repeated for a total of 10 to 34 amino acids (SEQ ID NO: 73).

[0063] In aspects, whether one VMG is incorporated into the envelope membrane is independent of whether another VMG is incorporated into the envelope membrane, e.g., a membraned vesicle comprises at least one VMG that is incorporated into the envelope membrane, and at least one VMG that is not incorporated into the envelope membrane. In aspects, at least one VMG of a membraned vesicle is incorporated into the envelope membrane. In aspects, about half of the VMGs of a membraned vesicle are incorporated into the envelope membrane. In aspects, all of the VMGs of a membraned vesicle are incorporated into the envelope membrane. In aspects, at least one VMG is incorporated into the envelope membrane and is in a fusion protein comprising an affinity tag. In aspects, at least one VMG is incorporated into the envelope membrane and is in a fusion protein comprising a targeting protein. In aspects, at least one VMG is incorporated into the envelope membrane and comprises an FMG.

[0064] In aspects, two or more VMGs hetero-oligomerize to form at least one protomercomplex. In aspects, two or more VMGs hetero-oligomerize to form at least one protomercomplex comprising: (i) at least one VMG within a fusion protein comprising at least one affinity tag, wherein the fusion protein does not comprise a targeting polypeptide; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide. In aspects, two or more VMGs hetero-oligomerize to form at least one protomer-complex comprising: (i) at least one VMG within a fusion protein comprising atleast one targeting polypeptide, wherein the fusion protein does not comprise an affinity tag; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide. In aspects, two or more VMGs hetero-oligomerize to form at least one protomer-complex comprising: (i) at least one VMG within a fusion protein comprising at least one affinity tag and at least one targeting polypeptide; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide.

[0065] The VMGs may be any suitable glycoprotein. For example, the VMGs may be a Type 1 membrane glycoprotein (N-terminus opposite the lumen, C-terminus inside the lumen) or a Type 2 membrane glycoprotein (C-terminus opposite the lumen, N-terminus inside the lumen). In aspects, at least one VMG is a Type I membrane glycoprotein. In aspects, the Type 1 membrane glycoprotein is within a fusion protein. In aspects, the Type 1 membrane glycoprotein is within a fusion protein comprising at least one affinity tag at the N-terminus of the fusion protein, wherein the affinity tag of the fusion protein comprising the Type 1 membrane glycoprotein is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle. In aspects, the Type 1 membrane glycoprotein is within a fusion protein comprising at least one retroviral gag protein at the C-terminus of the fusion protein wherein the retroviral gag protein is inside the lumen of the membraned vesicle. In aspects, the Type 1 membrane glycoprotein is within a fusion protein comprising at least one lentiviral gag protein at the C-terminus of the fusion protein wherein the lentiviral gag protein is inside the lumen of the membraned vesicle.

[0066] In aspects, at least one VMG is a Type 2 membrane glycoprotein. In aspects, the Type 2 membrane glycoprotein is within a fusion protein comprising at least one affinity tag at the C-terminus of the fusion protein, wherein the affinity tag of the fusion protein comprising the Type 2 membrane glycoprotein is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle. In aspects, the Type 2 membrane glycoprotein is within a fusion protein comprising at least one retroviral or lentiviral gag protein at the N-terminus of the fusion protein wherein the retroviral or lentiviral gag protein is inside the lumen of the membraned vesicle.

[0067] Any suitable retroviral or lentiviral gag protein may be used. In aspects, the retroviral or lentiviral gag protein is chimeric. In aspects, the retroviral or lentiviral gag protein is nonchimeric.

[0068] The identity of each VMG is independent of any other VMG, e.g., the type of glycoprotein of one VMG is independent of the type of glycoprotein of any other VMG,although there may be multiple copies of any given VMG. In aspects, at least one Type 2 membrane glycoprotein is within a bipartite protomer-complex comprising at least one Type 1 membrane glycoprotein comprising a fusion protein comprising at least one retroviral or lentiviral gag protein at the C-terminus of the fusion protein, wherein the retroviral or lentiviral gag protein is inside the lumen of the membraned vesicle, and wherein the Type 1 membrane glycoprotein is an FMG.

[0069] In aspects, at least one retroviral or lentiviral gag protein is tethered to the inner surface of the envelope membrane of the membraned vesicle. The retroviral or lentiviral gag protein can be tethered to the inner surface of the envelope membrane of the membraned vesicle in any suitable manner. Whether a retroviral or lentiviral gag protein is tethered to the inner surface of the envelope membrane of the membraned vesicle is independent of whether another retroviral or lentiviral gag protein is tethered to the inner surface of the envelope membrane of the membraned vesicle. In aspects, at least one retroviral or lentiviral gag protein is truncated at its C-terminus. In aspects, at least one retroviral or lentiviral gag protein is truncated at its N-terminus. For example, in aspects, the gag protein is HIV gag and the N-terminal myrisotylation site is removed via truncation.

[0070] In aspects, more than one retroviral or lentiviral gag protein form a polyhedral core structure within the membraned vesicle. In aspects, the gag proteins comprising the polyhedral core structure comprise both nonchimeric gag proteins and gag proteins fused to the C-terminus of a membrane anchored Type 1 membrane glycoprotein, which is optionally an FMG. In aspects, the membraned vesicle comprises a least one VMG within a fusion protein comprising more than one retroviral or lentiviral gag protein, that form a polyhedral core structure, and an affinity tag.

[0071] In aspects, at least one VMG is a Type 2 membrane glycoprotein comprising Paramyxoviridae canine distemper virus H protein and at least one VMG is a Type 1 membrane glycoprotein comprising Paramyxoviridae canine distemper virus F protein. In aspects, at least one VMG is within a fusion protein comprising a Type 2 membrane glycoprotein comprising Paramyxoviridae canine distemper virus H protein and at least one affinity tag at the C-terminus of the fusion protein, and wherein at least one VMG is within a fusion protein comprising a Type 1 membrane glycoprotein comprising Paramyxoviridae canine distemper virus F protein and at least one retroviral gag protein at the C-terminus of the fusion protein.

[0072] In aspects, at least one VMG is incorporated in the envelope membrane and comprises a fusogenic membrane glycoprotein (FMG). The FMG can be any suitable FMG. In aspects, the membrane vesicle comprises at least one FMG, at least two FMGs, at least three FMGs, at least four FMGs, or more FMGs. In aspects, at least two FMGs, at least three FMGs, at least four FMGs, or more FMGs are not the same. For example, in aspects where the membraned vesicle comprises at least two FMGs, at least one FMG is a rhabdoviral G glycoprotein or functional fragment or derivative thereof and at least one is a different rhabdoviral G glycoprotein or functional fragment or derivative thereof. For example, in aspects where the membraned vesicle comprises at least two FMGs at least one is a rhabdoviral G glycoprotein or functional fragment or derivative thereof of Vesiculovirus Indiana (e.g., SEQ ID NO: 14, 76, 77, or 117) and at least one is a rhabdoviral G glycoprotein or functional fragment or derivative thereof of Vesiculovirus newjersey (e.g., SEQ ID NO: 53 or 80).

[0073] In aspects, at least one VMG comprises an FMG that is engineered to reduce or eliminate binding to its natural receptor. In aspects, the FMG comprises a receptor binding glycoprotein (RBG). In aspects, at least one VMG comprises an FMG that is engineered to reduce or eliminate binding to its natural receptor.

[0074] In aspects, the at least one FMG is a rhabdoviral G glycoprotein or functional fragment thereof. 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. 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.

[0075] In aspects the rhabdoviral G glycoprotein is a functional fragment or derivative of a glycoprotein. In aspects the rhabdoviral G glycoprotein or functional fragment or derivative thereof is a vesicular stomatitis virus glycoprotein (VSV-G), a Flanders virus glycoprotein (FLAV-G) (SEQ ID NO: 6), a Chandipura virus glycoprotein (CHPV-G) (SEQ ID NO: 7), a Perinet virus glycoprotein (PERV-G) (SEQ ID NO: 8), a Piry virus glycoprotein (PIRYV-G) (SEQ ID NO: 9), a Fukuoka virus glycoprotein (FUKV-G) (SEQ ID NO: 10), a Joinjakaka virus glycoprotein (JOIV-G) (SEQ ID NO: 11), a Kumasi virus glycoprotein (KRV-G) (SEQID NO: 12), a Keuraliba virus glycoprotein (KEUV-G) (SEQ ID NO: 13), 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). In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of a 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: 14, 76, 77, or 117), Vesiculovirus newjersey (e.g., SEQ ID NO: 53 or 80), Vesiculovirus carajas (e.g., SEQ ID NO: 16 or 81), Vesiculovirus alagoas (e.g., SEQ ID NO: 17 or 82), Vesiculovirus cocal (e.g., SEQ ID NO: 52 or 83), Vesiculovirus marraba (e.g., SEQ ID NO: 78 or 79), Vesiculovirus morreton (e.g., SEQ ID NO: 84 or 85), 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: 14 or 77). In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus newjersey (e.g., SEQ ID NO: 53 or 80). In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus newjersey (SEQ ID NO: 15 or 18). In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus alagoas (e.g., SEQ ID NO: 48, 49, 134, orl37). In aspects, the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus carajas (e.g., SEQ ID NO: 50, 51, 127, or 130).

[0076] In aspects, the FMG 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 moreconservative amino acid substitutions that do not interfere with the fusion function of the rhabdoviral G glycoprotein or functional fragment or derivative thereof.

[0077] In aspects, the rhabdoviral G glycoprotein 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.

[0078] 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 the 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 fusion protein comprising a rhabdoviral G glycoprotein.

[0079] In aspects, the FMG 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, 3amino 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, 40 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.

[0080] In aspects the recombinant rhabdoviral G glycoprotein is engineered to have a mutation 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.

[0081] Vesicular stomatitis virus (VSV) is a rhabdovirus, having a natural receptor of the low-density lipoprotein receptor (LDL-R) or very low density lipoprotein receptor (VLDL- R), 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 LDL-R or VLDL-R, 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: 14). 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: 14). In aspects, the mutation comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 18, 42, 43, 44, 120, 122, 124, 128, 131, or 135. 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: 14), 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 comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 19, 20, 121, 123, 125, 126, 129, 132, 133, or 136.

[0082] In aspects, preferred full length rhabdoviral G glycoproteins, ectodomains, signal peptides, and engineered mutations of the ectodomains to reduce or abolish its naturalreceptor binding affinity are shown in Table 1. VSIV indicates Vesiculovirus Indiana G glycoprotein, VSNJV indicates Vesiculovirus newjersey G glycoprotein, VSCV indicates Vesiculovirus carajas G glycoprotein, VS AV indicates Vesiculovirus alagoas G glycoprotein, VSCOV indicates Vesiculovirus cocal G glycoprotein, WT indicates wild type, 41 J indicates residue 41 can be an I or L, KIR indicates a R substitution at residue 1 corresponding to SEQ ID NO: 14, G115A indicates an A substitution at residue 115 corresponding to SEQ ID NO: 14, Delta K47 indicates a deletion at residue 47 corresponding to SEQ ID NO: 14, and K47Q+Y209Q+R354Q indicates Q substitutions at residues 47, 209, and 354 corresponding to SEQ ID NO: 14.Table 1

[0083] 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 while unexpectedly 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 nonreplicating virus.

[0084] 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 LDL-R background binding.

[0085] 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(Wl): W276-W279 (2022), which is incorporated herein by reference in its entirety) comparing the base rhabdoviral G glycoprotein to another rhabdoviral G glycoprotein.

[0086] Any suitable affinity tag can be used. In aspects, the affinity tag is a FLAG-tag (SEQ ID NO: 140), strep-tag, GFP, or others. In aspects, the affinity tag is a strep-tag. In aspects, the strep-tag comprises the amino acid sequence WSHPQFEK (SEQ ID NO: 141).

[0087] As used herein, the phrase “displayed at the outer surface of the envelope membrane” refers to the location of a molecule that is at least partially located on the outer surface of an envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle. In aspects, the affinity tag displayed at the outer surface is displayed near the outer surface of the membrane, e.g. in contact with the outer surface of the envelop membrane. In aspects, the affinity tag displayed at the outer surface is displayed far from the outer surface of the membrane, e.g. not directly in contact with the outer surface of the envelop membrane.

[0088] In aspects, the affinity tag is flanked by one or more linkers. In aspects, at least one VMG is within a fusion protein comprising at least one affinity tag, wherein the fusion protein comprises at least one protease cleavable linker between VMG and at least one affinity tag. Any suitable protease cleavable linker can be used. In aspects, at least one protease cleavable linker comprises the amino acid sequence of ENLYFQGG (SEQ ID NO: 142) or ENLYFQGS (SEQ ID NO: 143) and the protease is tobacco etch virus (TEV) protease.

[0089] In aspects, at least one VMG is within a fusion protein comprising at least one affinity tag, wherein the fusion protein comprises at least one protease cleavable linker and one or more linkers different from the protease cleavable linker between VMG and at least one affinity tag. In aspects, at least one protease cleavable linker is between two or more linkers different from the protease cleavable linker. In aspects, the two or more linkers different from the protease cleavable linker are the same linker. In aspects, the two or more linkers different from the protease cleavable linker are not the same linker. Any suitable linker different from the protease cleavable linker can be used, such as those described herein and disclosed in Chen et al., Adv. Drug. Deliv. Rev., 65(1): 1357-1369 (2013).

[0090] In aspects, at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one targeting polypeptide, and wherein at least one targeting polypeptide is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle, and wherein at least one targeting polypeptide comprises Spy-tag.

[0091] In aspects, the membraned vesicle comprises (1) VMGs within a fusion protein comprising exactly one affinity tag; and (2) VMGs that do not comprise an affinity tag and do not comprise a targeting polypeptide; wherein the VMGs are incorporated into the envelope membrane, wherein the VMGs are FMGs, and wherein the VMGs are engineered to reduce or eliminate binding to its natural receptor.

[0092] In aspects, the membraned vesicle comprises (1) VMGs within a fusion protein comprising exactly one affinity tag; (2) FMGs within a fusion protein comprising exactly one targeting polypeptide; and (3) VMGs that do not comprise an affinity tag and do not comprise a targeting polypeptide; wherein the VMGs are incorporated into the envelope membrane, wherein the fusion proteins of (1) and (2) are separate fusion proteins, wherein the VMGs are FMGs, wherein the amino acid sequences of the VMGs are the same and that of vesicular stomatitis virus G glycoprotein (VSV-G), wherein the VMGs are engineered to reduce or eliminate binding to its natural receptor, and wherein the targeting polypeptide is Spy -tag.

[0093] In aspects, the membraned vesicle is an enveloped delivery vehicle (EDV). In aspects, the membraned vesicle is a virus-like particle. In aspects, the membraned vesicle is an exosome.

[0094] In aspects, the present disclosure provides a membraned vesicle comprising: (a) an envelope membrane; and (b) viral membrane glycoproteins (VMGs), each VMG optionally within a fusion protein; wherein at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one affinity tag, wherein at least one VMG is incorporated into the envelope membrane and comprises a fusogenic membrane glycoprotein (FMG), wherein at least one affinity tag is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle; wherein at least one FMG is a rhabdoviral G glycoprotein engineered to have a mutation to reduce or abolish its natural receptor binding specificity, wherein the rhabdoviral G glycoprotein comprises a mutation at one or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO: 14), and wherein the mutation is a substitution at two or more positions or the mutation is a deletion.

[0095] The disclosure further provides a method of generating the membraned vesicles described herein. In aspects, the method comprises: (a) transfecting a host cell with: (i) at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one affinity tag; (ii) optionally, at least one packaging plasmid; and (b) collecting membraned vesicles comprising at least one affinity tag displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle. Any suitable host cell, plasmid, and packaging plasmid can be used.

[0096] In aspects, the generated membraned vesicles are VLPs. Suitable host cells for generating VLPs can include, but are not limited to prokaryotic or eukaryotic cells, e.g., bacteria cells, yeast cells, insect cells, plant cells, or mammalian cells (such as HEK293 cells). In aspects, the generated membraned vesicles are exosomes. Suitable host cells for generating exosomes can include, but are not limited to any eukaryotic cell, e.g., macrophages (such as RAW246.7 cells), dendritic cells, mesenchymal stem cells, tumor cells (such as Panc-1, EL-4, MDA-MB23, and 4T-1 cells).

[0097] In aspects, the method comprises transfecting the host cell with at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one targeting polypeptide. In aspects, the at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one affinity tag and at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one targeting polypeptide are separate plasmids. In aspects, the at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one affinity tag and at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one targeting polypeptide are on the same plasmid.

[0098] In aspects, collecting membraned vesicles comprises purifying the collected membraned vesicles using affinity purification using at least one affinity tag displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle. In aspects, affinity purification comprises affinity chromatography. In aspects, the method of generating the membraned vesicles occurs in a supernatant. In aspects, at least one affinity tag displayed at the outer surface of the membrane, wherein the outer surface is opposite the lumen of the membraned vesicle, is bound to an affinity column comprised of a resin coated with a high affinity binding partner of the affinity tag. In aspects, the high affinity binding partner of the affinity tag is Strep-Tactin.

[0099] In aspects, an affinity tag can be used to capture and purify membraned vesicles that are desired. In aspects, an affinity tag can be used to capture and purify away membraned vesicles that are not desired. For example, in a sample having membraned vesicles of one or another affinity tag, one affinity tag can be used for capturing undesired membraned vesicles while desired membraned vesicles with the other affinity tag are allowed to flow through a column; the other affinity tag can be used for capturing desired membraned vesicles in a separate column.

[0100] In aspects, the fusion protein comprising an affinity tag is eluted using an elution buffer. Any suitable elution buffer can be used. In aspects, the elution buffer comprises a high pH, low pH, or high salt elution buffer. In aspects, the elution buffer disrupts the bond between the affinity tag and high affinity binding partner of the affinity tag that is displayed at the outer surface of the membrane, wherein the outer surface is opposite the lumen of the membraned vesicle, such that purified membraned vesicles are collected.

[0101] In aspects, at least one VMG is within a fusion protein comprising at least one affinity tag, wherein the fusion protein comprises at least one protease cleavable linker between VMG and at least one affinity tag, and wherein the method comprises using at least one protease to cleave at least one cleavable linker to elute at least one cleaved fusion protein. In aspects, the fusion protein comprising an affinity tag is eluted using a protease to cleave a cleavable linker between affinity tag and VMG within a fusion protein that is incorporated into the membrane of the membraned vesicle, such that purified membraned vesicles are collected. In aspects, at least one protease cleavable linker comprises the amino acid sequence of ENLYFQGG (SEQ ID NO: 142) or ENLYFQGS (SEQ ID NO 143) and the protease is tobacco etch virus (TEV) protease.

[0102] The below are certain aspects of the disclosure.

[0103] 1. A membraned vesicle comprising:(a) an envelope membrane; and(b) viral membrane glycoproteins (VMGs), each VMG optionally within a fusion protein; wherein at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one affinity tag, wherein at least one VMG is incorporated into the envelope membrane and comprises a fusogenic membrane glycoprotein (FMG),wherein at least one affinity tag is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle; and wherein the ratio of VMG without an affinity tag to VMG with an affinity tag is from 0: 1 to 5: 1.

[0104] 2 The membraned vesicle of aspect 1, wherein the ratio of VMG with an affinity tag to VMG without an affinity tag is from 1 : 1 to 3 : 1.

[0105] 3. The membraned vesicle of aspect 1 or 2, wherein the ratio of VMG with an affinity tag to VMG without an affinity tag is 3 : 1.

[0106] 4. The membraned vesicle of any one of aspects 1-3, wherein at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one targeting polypeptide, and wherein at least one targeting polypeptide is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

[0107] 5. The membraned vesicle of aspect 4, wherein the ratio of VMG with a targeting polypeptide to VMG without an affinity tag to VMG with an affinity tag is from 1 :0: 1 to 1 :5: 1

[0108] 6. The membraned vesicle of aspect 4 or 5, wherein the ratio of VMG with a targeting polypeptide to VMG without an affinity tag to VMG with an affinity tag is from 1 : 1 : 1 to 1 :3: 1.

[0109] 7 The membraned vesicle of any one of aspects 4-6, wherein the ratio of VMG with a targeting polypeptide to VMG without an affinity tag to VMG with an affinity tag is 1 :3: 1.

[0110] 8. The membraned vesicle of any one of aspects 1-7, wherein at least one FMG is a rhabdoviral G glycoprotein or functional fragment or derivative thereof that is of a Flanders virus glycoprotein (FLAV-G) (SEQ ID NO: 6), a Chandipura virus glycoprotein (CHPV-G) (SEQ ID NO: 7), a Perinet virus glycoprotein (PERV-G) (SEQ ID NO: 8), a Piry virus glycoprotein (PIRYV-G) (SEQ ID NO: 9), a Fukuoka virus glycoprotein (FUKV-G) (SEQ ID NO: 10), a Joinjakaka virus glycoprotein (JOIV-G) (SEQ ID NO: 11), a Kumasi virus glycoprotein (KRV-G) (SEQ ID NO: 12), a Keuraliba virus glycoprotein (KEUV-G) (SEQ ID NO: 13), 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), aKanyawara 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).

[0111] 9. The membraned vesicle of any one of aspects 1-7, wherein at least one FMG is a rhabdoviral G glycoprotein or functional fragment or derivative thereof that is of a Vesiculovirus glycoprotein or a functional fragment or derivative thereof.

[0112] 10. The membraned vesicle of aspect 9, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus Indiana, Vesiculovirus new jersey, Vesiculovirus carajas, or Vesiculovirus alagoas.

[0113] 11. The membraned vesicle of aspect 10, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus indiana (SEQ ID NO: 14).

[0114] 12. The membraned vesicle of aspect 10, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus newjersey (SEQ ID NO: 15).

[0115] 13. The membraned vesicle of any one of aspects 8-12, wherein the rhabdoviral G glycoprotein is substantially intact.

[0116] 14. The membraned vesicle of any one of aspects 8-12, wherein the rhabdoviral G glycoprotein is a functional fragment or derivative thereof.

[0117] 15. The membraned vesicle of aspect 14, wherein the cytoplasmic tail of the glycoprotein is truncated, deleted, or replaced with another sequence.

[0118] 16. The membraned vesicle of any one of aspects 8-15, wherein the rhabdoviral G glycoprotein is engineered to reduce or abolish its natural receptor binding specificity.

[0119] 17. The membraned vesicle of aspect 16, wherein the rhabdoviral G glycoprotein is engineered to have a mutation to reduce or abolish its natural receptor binding specificity.

[0120] 18. The membraned vesicle of aspect 17, wherein the rhabdoviral G glycoprotein comprises a mutation at one or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus indiana glycoprotein (SEQ ID NO: 14).

[0121] 19. The membraned vesicle of aspect 17 or 18, wherein the mutation is a substitution.

[0122] 20. The membraned vesicle of aspect 19, wherein the mutation is a substitution with a Q.

[0123] 21. The membraned vesicle of aspect 19 or 20, wherein the mutation is a substitution at two or more positions.

[0124] 22. The membraned vesicle of aspect 17 or 18, wherein the mutation is a deletion.

[0125] 23. The membraned vesicle of aspect 22, wherein the mutation is a single deletion at the position corresponding to K47 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO: 14).

[0126] 24. The membraned vesicle of any one of aspects 1-23, wherein at least one VMG is within a fusion protein comprising at least one affinity tag, wherein the fusion protein does not comprise a targeting polypeptide.

[0127] 25. The membraned vesicle of aspect 24, wherein two or more VMGs heterooligomerize to form at least one protomer-complex comprising: (i) at least one VMG within a fusion protein comprising at least one affinity tag, wherein the fusion protein does not comprise a targeting polypeptide; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide.

[0128] 26. The membraned vesicle of any one of aspects 1-25, wherein at least one VMG is within a fusion protein comprising at least one targeting polypeptide, wherein the fusion protein does not comprise an affinity tag.

[0129] 27. The membraned vesicle of aspect 26, wherein two or more VMGs heterooligomerize to form at least one protomer-complex comprising: (i) at least one VMG within a fusion protein comprising at least one targeting polypeptide, wherein the fusion protein does not comprise an affinity tag; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide.

[0130] 28. The membraned vesicle of any one of aspects 1-27, wherein at least one VMG is within a fusion protein comprising at least one affinity tag and at least one targeting polypeptide.

[0131] 29. The membraned vesicle of aspect 28, wherein two or more VMGs heterooligomerize to form at least one protomer-complex comprising: (i) at least one VMG within a fusion protein comprising at least one affinity tag and at least one targeting polypeptide; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide.

[0132] 30. The membraned vesicle of any one of aspects 1-29, wherein at least one VMG is a Type 1 membrane glycoprotein.

[0133] 31. The membraned vesicle of aspect 30, wherein the Type 1 membrane glycoprotein is within a fusion protein comprising at least one affinity tag at the N-terminus of the fusion protein, wherein the affinity tag of the fusion protein comprising the Type 1 membrane glycoprotein is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

[0134] 32. The membraned vesicle of aspect 30, wherein the Type 1 membrane glycoprotein is within a fusion protein comprising at least one retroviral gag protein at the C- terminus of the fusion protein wherein the retroviral gag protein is inside the lumen of the membraned vesicle.

[0135] 33. The membraned vesicle of aspect 31, wherein the fusion protein comprising the Type 1 membrane glycoprotein comprises at least one retroviral gag protein at the C- terminus of the fusion protein wherein the retroviral gag protein is inside the lumen of the membraned vesicle.

[0136] 34. The membraned vesicle of any one of aspects 1-33, wherein at least one VMG is a Type 2 membrane glycoprotein.

[0137] 35. The membraned vesicle of aspect 34, wherein the Type 2 membrane glycoprotein is within a fusion protein comprising at least one affinity tag at the C-terminus of the fusion protein, wherein the affinity tag of the fusion protein comprising the Type 2 membrane glycoprotein is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

[0138] 36. The membraned vesicle of aspect 34 or 35, wherein the Type 2 membrane glycoprotein is within a fusion protein comprising at least one retroviral gag protein at the N- terminus of the fusion protein wherein the retroviral gag protein is inside the lumen of the membraned vesicle.

[0139] 37. The membraned vesicle of any one of aspects 1-36, wherein at least one VMG is a Type 1 membrane glycoprotein and at least one VMG is a Type 2 membrane glycoprotein.

[0140] 38. The membraned vesicle of any one of aspects 34-37, wherein at least one Type2 membrane glycoprotein is within a bipartite protomer-complex comprising at least one Type 1 membrane glycoprotein comprising a fusion protein comprising at least one retroviral gag protein at the C-terminus of the fusion protein, wherein the retroviral gag protein is inside the lumen of the membraned vesicle, and wherein the Type 1 membrane glycoprotein is an FMG.

[0141] 39. The membraned vesicle of aspect 32, 33, 36, or 38 wherein at least one retroviral gag protein is tethered to the inner surface of the envelope membrane.

[0142] 40. The membraned vesicle of aspect 32, 33, 36, 38, or 39 wherein at least one retroviral gag protein is truncated at its C-terminus.

[0143] 41. The membraned vesicle of aspect 39 or 40, wherein more than one retroviral gag protein form a polyhedral core structure within the membraned vesicle.

[0144] 42. The membraned vesicle of aspect 41, wherein the gag proteins comprising the polyhedral core structure comprise both nonchimeric gag proteins and gag proteins fused to the C-terminus of a membrane anchored Type 1 membrane glycoprotein which is optionally an FMG.

[0145] 43. The membraned vesicle of any one of aspects 1-42, wherein at least one VMG is a Type 2 membrane glycoprotein comprising Paramyxoviridae canine distemper virus H protein and at least one VMG is a Type 1 membrane glycoprotein comprising Paramyxoviridae canine distemper virus F protein.

[0146] 44. The membraned vesicle of aspect 43, wherein at least one VMG is within a fusion protein comprising a Type 2 membrane glycoprotein comprising Paramyxoviridae canine distemper virus H protein and at least one affinity tag at the C-terminus of the fusion protein, and wherein at least one VMG is within a fusion protein comprising a Type 1 membrane glycoprotein comprising Paramyxoviridae canine distemper virus F protein and at least one retroviral gag protein at the C-terminus of the fusion protein.

[0147] 45. The membraned vesicle of any one of aspects 1-44, wherein at least one VMG is within a fusion protein comprising at least one affinity tag, wherein the fusion protein comprises at least one protease cleavable linker between VMG and at least one affinity tag.

[0148] 46. The membraned vesicle of aspect 45, wherein at least one protease cleavable linker comprises the amino acid sequence of ENLYFQGG (SEQ ID NO: 142) or ENLYFQGS (SEQ ID NO: 143) and the protease is tobacco etch virus (TEV) protease.

[0149] 47. The membraned vesicle of any one of aspects 1-46, wherein at least one affinity tag is a strep-tag.

[0150] 48. The membraned vesicle of any one of aspects 1-47, wherein at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one targeting polypeptide, and wherein at least one targeting polypeptide is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle, and wherein at least one targeting polypeptide comprises Spy-tag.

[0151] 49. The membraned vesicle of any one of aspects 1-48, comprising:(1) VMGs within a fusion protein comprising exactly one affinity tag; and(2) VMGs that do not comprise an affinity tag and do not comprise a targeting polypeptide; wherein the VMGs are incorporated into the envelope membrane, wherein the VMGs are FMGs, and wherein the VMGs are engineered to reduce or eliminate binding to its natural receptor.

[0152] 50. The membraned vesicle of aspect 1, comprising:(1) VMGs within a fusion protein comprising exactly one affinity tag;(2) FMGs within a fusion protein comprising exactly one targeting polypeptide; and(3) VMGs that do not comprise an affinity tag and do not comprise a targeting polypeptide; wherein the VMGs are incorporated into the envelope membrane, wherein the fusion proteins of (1) and (2) are separate fusion proteins, wherein the VMGs are FMGs, wherein the amino acid sequences of the VMGs are the same and that of vesicular stomatitis virus G glycoprotein (VSV-G), wherein the VMGs are engineered to reduce or eliminate binding to its natural receptor, and wherein the targeting polypeptide is Spy-tag.

[0153] 51. The membraned vesicle of any one of aspects 1-50, wherein the membraned vesicle is an enveloped delivery vehicle.

[0154] 52. The membraned vesicle of any one of aspects 1-51, wherein the membraned vesicle is a virus-like particle.

[0155] 53. The membraned vesicle of any one of aspects 1-51, wherein the membraned vesicle is an exosome.

[0156] 54. A membraned vesicle comprising:(a) an envelope membrane; and(b) viral membrane glycoproteins (VMGs), each VMG optionally within a fusion protein;wherein at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one affinity tag, wherein at least one VMG is incorporated into the envelope membrane and comprises a fusogenic membrane glycoprotein (FMG), wherein at least one affinity tag is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle; wherein at least one FMG is a rhabdoviral G glycoprotein engineered to have a mutation to reduce or abolish its natural receptor binding specificity, wherein the rhabdoviral G glycoprotein comprises a mutation at one or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO: 14), and wherein the mutation is a substitution at two or more positions or the mutation is a deletion.

[0157] 55. The membraned vesicle of aspect 54, wherein at least one FMG is a rhabdoviral G glycoprotein or functional fragment or derivative thereof that is of a Flanders virus glycoprotein (FLAV-G) (SEQ ID NO: 6), a Chandipura virus glycoprotein (CHPV-G) (SEQ ID NO: 7), a Perinet virus glycoprotein (PERV-G) (SEQ ID NO: 8), a Piry virus glycoprotein (PIRYV-G) (SEQ ID NO: 9), a Fukuoka virus glycoprotein (FUKV-G) (SEQ ID NO: 10), a Joinjakaka virus glycoprotein (JOIV-G) (SEQ ID NO: 11), a Kumasi virus glycoprotein (KRV-G) (SEQ ID NO: 12), a Keuraliba virus glycoprotein (KEUV-G) (SEQ ID NO: 13), 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).

[0158] 56. The membraned vesicle of aspect 54, wherein at least one FMG is a rhabdoviral G glycoprotein or functional fragment or derivative thereof that is of a Vesiculovirus glycoprotein or a functional fragment or derivative thereof.

[0159] 57. The membraned vesicle of aspect 56, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus Indiana, Vesiculovirus new jersey, Vesiculovirus carajas, or Vesiculovirus alagoas.

[0160] 58. The membraned vesicle of aspect 57, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus indiana (SEQ ID NO: 14).

[0161] 59. The membraned vesicle of aspect 57, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus newjersey (SEQ ID NO: 15).

[0162] 60. The membraned vesicle of any one of aspects 54-59, wherein the rhabdoviralG glycoprotein is substantially intact.

[0163] 61. The membraned vesicle of any one of aspects 54-59, wherein the rhabdoviralG glycoprotein is a functional fragment or derivative thereof.

[0164] 62. The membraned vesicle of aspect 61, wherein the cytoplasmic tail of the glycoprotein is truncated, deleted, or replaced with another sequence.

[0165] 63. The membraned vesicle of any one of aspects 54-62, wherein the mutation is a substitution at two or more positions.

[0166] 64. The membraned vesicle of aspect 63, wherein the substitution is with Q.

[0167] 65. The membraned vesicle of any one of aspects 54-62, wherein the mutation is a deletion.

[0168] 66. The membraned vesicle of aspect 65, wherein the mutation is a single deletion at the position corresponding to K47 on the Vesiculovirus indiana glycoprotein (SEQ ID NO: 14).

[0169] 67. The membraned vesicle of any one of aspects 54-66, wherein at least oneVMG is within a fusion protein comprising at least one affinity tag, wherein the fusion protein does not comprise a targeting polypeptide.

[0170] 68. The membraned vesicle of aspect 67, wherein two or more VMGs heterooligomerize to form at least one protomer-complex comprising: (i) at least one VMG within a fusion protein comprising at least one affinity tag, wherein the fusion protein does not comprise a targeting polypeptide; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide.

[0171] 69. The membraned vesicle of any one of aspects 54-68, wherein at least oneVMG is within a fusion protein comprising at least one targeting polypeptide, wherein the fusion protein does not comprise an affinity tag.

[0172] 70. The membraned vesicle of aspect 69, wherein two or more VMGs hetero-oligomerize to form at least one protomer-complex comprising: (i) at least one VMG within a fusion protein comprising at least one targeting polypeptide, wherein the fusion protein does not comprise an affinity tag; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide.

[0173] 71. The membraned vesicle of any one of aspects 54-70, wherein at least oneVMG is within a fusion protein comprising at least one affinity tag and at least one targeting polypeptide.

[0174] 72. The membraned vesicle of aspect 71, wherein two or more VMGs hetero-oligomerize to form at least one protomer-complex comprising: (i) at least one VMG within a fusion protein comprising at least one affinity tag and at least one targeting polypeptide; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide.

[0175] 73. The membraned vesicle of any one of aspects 54-72, wherein at least oneVMG is a Type 1 membrane glycoprotein.

[0176] 74. The membraned vesicle of aspect 73, wherein the Type 1 membrane glycoprotein is within a fusion protein comprising at least one affinity tag at the N-terminus of the fusion protein, wherein the affinity tag of the fusion protein comprising the Type 1 membrane glycoprotein is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

[0177] 75. The membraned vesicle of aspect 73, wherein the Type 1 membrane glycoprotein is within a fusion protein comprising at least one retroviral gag protein at the C- terminus of the fusion protein wherein the retroviral gag protein is inside the lumen of the membraned vesicle.

[0178] 76. The membraned vesicle of aspect 74, wherein the fusion protein comprising the Type 1 membrane glycoprotein comprises at least one retroviral gag protein at the C- terminus of the fusion protein wherein the retroviral gag protein is inside the lumen of the membraned vesicle.

[0179] 77. The membraned vesicle of any one of aspects 54-76, wherein at least oneVMG is a Type 2 membrane glycoprotein.

[0180] 78. The membraned vesicle of aspect 77, wherein the Type 2 membrane glycoprotein is within a fusion protein comprising at least one affinity tag at the C-terminus of the fusion protein, wherein the affinity tag of the fusion protein comprising the Type 2membrane glycoprotein is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

[0181] 79. The membraned vesicle of aspect 77 or 78, wherein the Type 2 membrane glycoprotein is within a fusion protein comprising at least one retroviral gag protein at the N- terminus of the fusion protein wherein the retroviral gag protein is inside the lumen of the membraned vesicle.

[0182] 80. The membraned vesicle of any one of aspects 54-79, wherein at least oneVMG is a Type 1 membrane glycoprotein and at least one VMG is a Type 2 membrane glycoprotein.

[0183] 81. The membraned vesicle of any one of aspects 77-80, wherein at least one Type2 membrane glycoprotein is within a bipartite protomer-complex comprising at least one Type 1 membrane glycoprotein comprising a fusion protein comprising at least one retroviral gag protein at the C-terminus of the fusion protein, wherein the retroviral gag protein is inside the lumen of the membraned vesicle, and wherein the Type 1 membrane glycoprotein is an FMG.

[0184] 82. The membraned vesicle of aspect 75, 76, 79, or 81 wherein at least one retroviral gag protein is tethered to the inner surface of the envelope membrane.

[0185] 83. The membraned vesicle of aspect 75, 76, 79, 81, or 82 wherein at least one retroviral gag protein is truncated at its C-terminus.

[0186] 84. The membraned vesicle of aspect 82 or 83, wherein more than one retroviral gag protein form a polyhedral core structure within the membraned vesicle.

[0187] 85. The membraned vesicle of aspect 84, wherein the gag proteins comprising the polyhedral core structure comprise both nonchimeric gag proteins and gag proteins fused to the C-terminus of a membrane anchored Type 1 membrane glycoprotein which is optionally an FMG.

[0188] 86. The membraned vesicle of any one of aspects 54-85, wherein at least oneVMG is a Type 2 membrane glycoprotein comprising Paramyxoviridae canine distemper virus H protein and at least one VMG is a Type 1 membrane glycoprotein comprising Paramyxoviridae canine distemper virus F protein.

[0189] 87. The membraned vesicle of aspect 86, wherein at least one VMG is within a fusion protein comprising a Type 2 membrane glycoprotein comprising Paramyxoviridae canine distemper virus H protein and at least one affinity tag at the C-terminus of the fusion protein, and wherein at least one VMG is within a fusion protein comprising a Type 1membrane glycoprotein comprising Paramyxoviridae canine distemper virus F protein and at least one retroviral gag protein at the C-terminus of the fusion protein.

[0190] 88. The membraned vesicle of any one of aspects 54-87, wherein at least oneVMG is within a fusion protein comprising at least one affinity tag, wherein the fusion protein comprises at least one protease cleavable linker between VMG and at least one affinity tag.

[0191] 89. The membraned vesicle of aspect 88, wherein at least one protease cleavable linker comprises the amino acid sequence of ENLYFQGG (SEQ ID NO: 142) or ENLYFQGS (SEQ ID NO: 143) and the protease is tobacco etch virus (TEV) protease.

[0192] 90. The membraned vesicle of any one of aspects 54-89, wherein at least one affinity tag is a strep-tag.

[0193] 91. The membraned vesicle of any one of aspects 54-90, wherein at least oneVMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one targeting polypeptide, and wherein at least one targeting polypeptide is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle, and wherein at least one targeting polypeptide comprises Spy-tag.

[0194] 92. The membraned vesicle of any one of aspects 54-91, comprising:(1) VMGs within a fusion protein comprising exactly one affinity tag; and(2) VMGs that do not comprise an affinity tag and do not comprise a targeting polypeptide; wherein the VMGs are incorporated into the envelope membrane, wherein the VMGs are FMGs, and wherein the VMGs are engineered to reduce or eliminate binding to its natural receptor.

[0195] 93. The membraned vesicle of aspect 54, comprising:(1) VMGs within a fusion protein comprising exactly one affinity tag;(2) FMGs within a fusion protein comprising exactly one targeting polypeptide; and(3) VMGs that do not comprise an affinity tag and do not comprise a targeting polypeptide; wherein the VMGs are incorporated into the envelope membrane, wherein the fusion proteins of (1) and (2) are separate fusion proteins,wherein the VMGs are FMGs, wherein the amino acid sequences of the VMGs are the same and that of vesicular stomatitis virus G glycoprotein (VSV-G), wherein the VMGs are engineered to reduce or eliminate binding to its natural receptor, and wherein the targeting polypeptide is Spy-tag.

[0196] 94. The membraned vesicle of any one of aspects 54-93, wherein the membraned vesicle is an enveloped delivery vehicle.

[0197] 95. The membraned vesicle of any one of aspects 54-94, wherein the membraned vesicle is a virus-like particle.

[0198] 96. The membraned vesicle of any one of aspects 54-94, wherein the membraned vesicle is an exosome.

[0199] 97. A method of generating the membraned vesicle of any one of aspects 1-96, the method comprising:(a) transfecting a host cell with:(i) at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one affinity tag;(ii) optionally, at least one packaging plasmid; and(b) collecting membraned vesicles comprising at least one affinity tag displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

[0200] 98. The method of aspect 97, comprising transfecting the host cell with at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one targeting polypeptide.

[0201] 99. The method of aspect 98, wherein at least one plasmid encoding at least oneVMG that is within a fusion protein comprising at least one affinity tag and at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one targeting polypeptide are separate plasmids.

[0202] 100. The method of aspect 98, wherein at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one affinity tag and at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one targeting polypeptide are on the same plasmid.

[0203] 101. The method of any one of aspects 97-100, wherein collecting membraned vesicles comprises purifying the collected membraned vesicles using affinity purification using at least one affinity tag displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

[0204] 102. The method of aspect 101, wherein at least one VMG is within a fusion protein comprising at least one affinity tag, wherein the fusion protein comprises at least one protease cleavable linker between VMG and at least one affinity tag, and wherein the method comprises using at least one protease to cleave at least one cleavable linker to elute at least one cleaved fusion protein.Example 1

[0205] This Example demonstrates lentiviral purification using the Strep-Tactin® Sepharose affinity column. Strep-Tactin® is a streptavidin variant. The Strep-Tactin® Sepharose affinity column contains 4% agarose coupled with Strep-Tactin®.

[0206] Three lentiviruses were constructed to have the following structures: VSV-G- QQQ; UCHT1-VSV-G-QQQ; and Strep-(G4S)3-VSV-G-QQQ. Strep refers to Strep-tag®II. UCHT1 is a targeting molecule. VSV-G-QQQ refers to a VSV-G protein with K47Q+R354Q+Y209Q substitution. 1.5 mL of (1) VSV-G-QQQ alone and (2) a combination of UCHT1- VSV-G-QQQ and Strep- VSV-G-QQQ in a 1 :3 ratio were each added in OPTI-MEM to individual Strep-Tactin® Sepharose affinity columns. The flow through was collected. The affinity columns were washed three times with 1 mL of buffer comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, and ImM EDTA. The flow through resulting from the three washes was collected. 3 mL of an elution buffer comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA and 2.5 mM desthiobiotin was then added to the columns, allowing the Strep-tagged lentiviruses that bound to the affinity column to be eluted. The eluate was collected. Figure 1 A shows the experimental protocol. Western blot analysis was then performed on the lentiviruses and their supernatant, the flow through after adding the lentiviruses and their supernatant to the affinity column, the flow through after washing the column three times, and the eluate (FIG. IB). FIG. IB shows that strep-tagged lentiviruses did bind to the affinity column and were eluted, but not all of the strep-tagged lentiviruses bound to the affinity column (see column Fl of FIG. IB).Example 2

[0207] This Example demonstrates purification of strep-tagged lentiviruses using Strep- Tactin® Magnetic Microbeads.

[0208] 1.5 mL of a combination of VSV-G-QQQ and Strep- VS V-G-QQQ in a 1 :3 ratio was resuspended with 200 pL of beads in a reaction tube and incubated on ice for one hour. The tube was removed from the ice and placed in the magnetic separator. The reaction tube was removed from the magnetic separator and 25 pL of buffer BXT per 1 pL of beads was added to the reaction tube. The reaction tube was incubated for 10 minutes and vortexed two to three times to resuspend the beads. The reaction tube was places back into the magnetic separator and the supernatant containing the target protein was transferred into a new reaction tube. The experimental protocol is shown in FIG. 2A. Western blot analysis was performed on the lentiviruses, the supernatant removed after, and the eluate (FIG. 2B). The strep-tagged lentiviruses did not completely bind to the beads as shown in the SI column of FIG. 2B.Example 3

[0209] This Example demonstrates purification of strep-tagged lentiviruses using Strep- Tactin®XT 4flow affinity column.

[0210] Three lentiviruses were constructed to have the following structures: VSV-G-WT; UCHT1-VSV-G-QQQ; and Strep- VS V-G-QQQ. 1.5 mL of (1) a combination of VSV-G-WT and Strep- VS V-G-QQQ in a 1 :3 ratio and (2) a combination of UCHT1-VSV-G- QQQ and Strep- VS V-G-QQQ in a 1 :3 ratio were each added in OPTI-MEM to individual Strep-Tactin®XT 4Flow® affinity columns and Strep-Tactin®XT 4Flow® high capacity affinity columns. The flow through was collected. The affinity columns were washed three times with 1 mL of a solution comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA. The flow through resulting from the three washes was collected. 3 mL of an elution buffer comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 50 mM biotin was then added to the columns, allowing the Strep-tagged lentiviruses that bound to the affinity columns to be eluted. The eluate was collected. Figure 3 A shows the experimental protocol. Western blot analysis was then performed on the lentiviruses and their supernatant, the flow through after adding the lentivirus to the affinity columns, the flow through after washing the columns three times, and the eluate (FIG. 3B). FIG. 3B shows that strep-tagged lentiviruses bind to the affinity column well and can be eluted.Example 4

[0211] This Example demonstrates use of a linker between Strep-Tag®II and VSV-G- QQQ

[0212] Three lentiviruses were constructed to have the following structures: UCHT1- VSV-G-QQQ; Strep-VSV-G-QQQ and Strep-(G4S)3-VSV-G-QQQ. (G4S)3 is a linker. 1.5 mL of (1) a combination of UCHT1-VSV-G-QQQ and Strep-VSV-G-QQQ in a 1 :3 ratio and (2) a combination of UCHT1-VSV-G-QQQ and Strep-(G4S)3-VSV-G-QQQ in a 1 :3 ratio were each added in OPTI-MEM to individual Strep-Tactin®XT 4Flow® high capacity affinity columns. The flow through was collected. The affinity columns were washed three times with 1 mL of a solution comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA. The flow through resulting from the three washes was collected. 3 mL of an elution buffer comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 50 mM biotin was then added to the columns, allowing the Strep-tagged lentiviruses that bound to the affinity columns to be eluted. The eluate was collected. Figure 4A shows the experimental protocol. Western blot analysis was then performed on the lentiviruses and their supernatant, the flow through after adding the lentiviruses and their supernatant to the affinity columns, the flow through after washing the affinity columns three times, and the eluate (FIG. 4B). FIG. 4B shows that strep-tagged lentiviruses with a linker between Strep- Tag®II and VSV-G-QQQ bind to the affinity column better than the strep-tagged lentiviruses without a linker between Strep-Tag®II.Example 5

[0213] This Example quantifies the amount of recovered Strep-tagged lentiviruses using the disclosed purification method.

[0214] Two lentiviruses were constructed to have the following structures: UCHT1-VSV- G-QQQ; and Strep-(G4S)3 -VSV-G-QQQ. 15 mL of a combination of UCHT1- VSV-G-QQQ and Strep-(G4S)3 -VSV-G-QQQ in a 1 :3 ratio was added in OPTI-MEM to a Strep- Tactin®XT 4Flow® high-capacity affinity column. The flow through was collected. The affinity column was washed three times with 5 mL of a solution comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA. The flow through resulting from the three washes was collected. 15 mL of an elution buffer comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 50 mM biotin was then added to the column allowingthe Strep-tagged lentiviruses that bound to the affinity column to be eluted. The eluate was collected. Figure 5A shows the experimental protocol. Western blot analysis was then performed on the lentiviruses and their supernatant, the flow through after adding the lentiviruses to the column, the flow through after washing the column three times, and the eluate (FIG. 5B). The amount of p24, measured as viral particles per mL (VP / mL), in each sample was quantified by p24 ELISA. p24 is a capsid protein encoded by the gag gene of the Human Immunodeficiency Virus (HIV) that can be measured in an ELISA assay to calculate the amount of lentivirus in a sample. Flow titer was determined by the amount of GFP+ cells of cells transduced with serial dilution of lentivirus supernatants. An ELISA assay against HEK293 host cell protein (HCP) was used to quantify total host cell proteins. The p24, flow titer, and total host cell proteins of EX50 were all normalized to those of F0X50. Table 2 indicates that 6.5% as determined by ELISA and 4.1% as determined by Flow Titer of the original amount of Strep-tagged lentivirus were recovered.Table 2

[0215] Jurkat cell were then transduced with either the lentiviruses supernatants (F0), 50X concentrated lentiviruses supernatants (F0 (50X)), 50x concentrated eluate (Eluate (50X)), or a no virus control. 50X concentration was performed using VIVASPIN concentrator according to the manufacturer’s instructions. The 50X concentrated lentiviruses supernatants (F0 (50X) were concentrated to 2076 viral particles per mL (VP / mL) and the 50x concentrated eluate (Eluate (50X)) was concentrated to 1344 VP / mL. Jurkat cells were plated in a 96-well plate at 50,000 cells / well using 50 pL of 20% RPMI. Then 2e6 of each kind of cells were taken, spun down, and resuspended in 2 mL of 20% RPMI and seeded at 50 pL cells / well. 20 pL of each virus treatment was combined with 30 pL of OPTLMEM and 50 pL of the virus and OPTI-MEM mix was added to each well. Next the 96-well plates were wrapped with parafilm and spun at 2000 rpm for 30 minutes at room temperature. Thenthe cells incubated at 37°C in an incubator. FIG. 5C shows micrographs of the Jurkat cells 72 hours post transduction taken using a Nikon microscope.Example 6

[0216] This Example demonstrates that using more elution buffer elutes more Strep- tagged lentivirus.

[0217] Three lentiviruses were constructed to have the following structures: UCHT1- VSV-G-QQQ VSV-G-QQQ, and Strep-(G4S)3-VSV-G-QQQ. 1.5 mL of a combination of UCHT1-VSV-G-QQQ, VSV-G-QQQ, and Strep-(G4S)3-VSV-G-QQQ in a 1 :1 :2 ratio was added in OPTI-MEM to a Strep-Tactin®XT 4Flow® high-capacity affinity column. This experiment was the second use of the affinity column. The flow through was collected. The affinity column was washed three times with 1 mL of a solution comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA. The flow through resulting from the three washes was collected. 1.5 mL of an elution buffer comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 50 mM biotin was then added to the columns allowing the Strep-tagged lentiviruses that bound to the affinity column to be eluted. This step was repeated nine times. The eluate was collected after each step. Figure 6A shows the experimental protocol. Western blot analysis was then performed on the lentiviruses and their supernatant, the flow through after adding the lentiviruses to the column, the flow through after washing the column three times, and the eluate after each elution step (FIG.6B). FIG. 6B indicates that performing more elution steps elutes more Strep-tagged lentivirus from the affinity column.Example 7

[0218] This Example demonstrates that a decreased ratio of strep-tagged lentivirus to non-strep-tagged lentivirus can still be purified using the disclosed method.

[0219] Three lentiviruses were constructed to have the following structures: UCHT1- VSV-G-QQQ VSV-G-QQQ, and Strep-(G4S)3-VSV-G-QQQ. 1.5 mL of a combination of UCHT1 -VSV-G-QQQ, VSV-G-QQQ, and Strep-(G4S)3 -VSV-G-QQQ in the ratios described in Table 3 were each added in OPTI-MEM to a Strep-Tactin®XT 4Flow® high- capacity affinity column. The flow through was collected. The affinity columns were washed three times with 1 mL of a solution comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA. 4.5 mL of an elution buffer comprising 100 mM Tris / HCl (pH 8.0),150 mM NaCl, 1 mM EDTA, and 50 mM biotin was then added to each of the columns allowing the Strep-tagged lentiviruses that bound to the affinity column to be eluted. The eluate was collected. Figure 7A shows the experimental protocol. Western blot analysis was then performed on the lentiviruses and their supernatant, the flow through after adding the lentiviruses to the columns, the flow through after washing the columns three times, and the eluate (FIG. 7B). FIG. 7B indicates that almost all of the tested combinations of lentivirus bound to the affinity column. Some lentivirus produced with a VSV-G-QQQ & Strep- (G4S)3-VSV-G-QQQ ratio of 9: 1 (1 :2.7:0.3) were eluted from the affinity column during the washes (see UCHT1-VSV-G and VSV-G labeled bands in Fl column).Table 3Example 8

[0220] This Example demonstrates that lentivirus elutes faster from Strep-Tactin®XT 4Flow® column than Strep-Tactin®XT 4Flow® high-capacity column, including with 30 minutes of incubation.

[0221] Three lentiviruses were constructed to have the following structures: UCHT1- VSV-G-QQQ VSV-G-QQQ, and Strep-(G4S)3-VSV-G-QQQ. 1.5 mL of a combination of UCHT1 -VSV-G-QQQ, VSV-G-QQQ, and Strep-(G4S)3 -VSV-G-QQQ in a 1 : 1.5: 1.5 ratio was added in OPTI-MEM to a Strep-Tactin®XT 4Flow® high capacity affinity column, Strep-Tactin®XT 4Flow® and Strep-Tactin®XT4Flow® high capacity affinity column that was incubated for 30 minutes. The flow through was collected. The affinity columns were washed three times with 1 mL of a solution comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA. 1.5 mL of an elution buffer comprising 100 mM Tris / HCl (pH 8.0),150 mM NaCl, 1 mM EDTA, and 50 mM biotin was then added to the columns allowing the Strep-tagged lentiviruses that bound to the affinity column to be eluted. This step was repeated ten times. The eluate was collected after each step. Figure 8 A shows the experimental protocol. Western blot analysis was then performed on the lentiviruses and their supernatant, the flow through after adding the lentiviruses to the columns, the flow through after washing the columns three times, and the eluate after each elution step (FIG. 8B). FIG. 8B indicates that the Strep-tagged lentivirus elutes faster from the Strep- Tactin®XT 4Flow® column.Example 9

[0222] This Example demonstrates that reducing the ratio of Strep-tagged lentivirus and increasing the amount of elution buffer improves elution of Strep-tagged lentivirus.

[0223] Three lentiviruses were constructed to have the following structures: UCHT1- VSV-G-QQQ; VSV-G-QQQ; and Strep-(G4S)3-VSV-G-QQQ. 12.5 mL of a combination of UCHT1-VSV-G-QQQ, VSV-G-QQQ and Strep-(G4S)3 -VSV-G-QQQ in a 1 : 1 :2 ratio was added in OPTI-MEM to a Strep-Tactin®XT 4Flow® affinity column. The flow through was collected. The affinity column was washed three times with 10 mL of a solution comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA. The flow through resulting from the three washes was collected. 7.5 mL of an elution buffer comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 50 mM biotin was then added to the columns five times allowing the Strep-tagged lentiviruses that bound to the affinity column to be eluted. The eluate was collected. Figure 9A shows the experimental protocol. Western blot analysis was then performed on lentiviruses and their supernatants, the flow through after adding the lentiviruses to the column, the flow through after washing the column three times, and the eluate (FIG. 9B). The amount of p24, measured as viral particles per mL (VP / mL), in each sample was quantified by p24 ELISA. Transduction ration was determined by transducing Jurkat cells with each lentivirus and quantifying GFP+ cell ratio via flow analysis. Total protein was calculated by BCA assay. dsDNA amount, measured as ng / ML, was determined by PicoGreen dsDNA kit. The p24, transduction ratio, total protein, and dsDNA of EX40 were all normalized to those of F0X40. Table 4 indicates that 8.7% as determined by ELISA of the original amount of Strep-tagged lentivirus was recovered. Table 4 also indicates that the transduction ratio in Jurkat cells was 67.0%, indicating a 67% recovery ratio.Table 4

[0224] Jurkat cells and Nalm6 cells were transduced as described in Example 5. FIG. 9C shows the transduction ratio, as measured by the percentage of GFP+ cells, of 40 pL of each lentivirus treatment in Jurkat cells and FIG. 9E shows the same transduction ration normalized to F0X40. 40X concentration of samples was performed using VIVASPIN concentrator according to the manufacturer’s instructions. FIG. 9D shows the transduction ratio, as measured by the percentage of GFP+ cells, of 40 pL of each lentivirus in Nalm6 cells. FIG. 9F shows micrographs of the Jurkat and Nalm6 cells 72 hours post transduction taken using a Nikon microscope.Example 10

[0225] This Example demonstrates that protease cleavage can successfully occur during the purification process.

[0226] Three lentiviruses were constructed to have the following structures: UCHT1- VSV-G-QQQ, Strep-(G4S)5-Tev site-(G4S)5-VSV-G-QQQ, and Strep-(G4S)3-Tev site- (G4S)3-VSV-G-QQQ. Tev site refers to TEV protease, which is the common name for the 27 kDa catalytic domain of the Nuclear Inclusion a (NIa) protein encoded by the tobacco etch virus (TEV). (G4S)5 is a linker. 1.5 mL of a combination of (1) UCHT1-VSV-G-QQQ and Strep-(G4S)5-Tev site-(G4S)5-VSV-G-QQQ in a 1 :3 ratio and (2) UCHT1-VSV-G-QQQ and Strep-(G4S)3-Tev site-(G4S)3-VSV-G-QQQ in a 1 :3 ratio were added in OPTI-MEM to individual Strep-Tactin®XT 4Flow® high capacity affinity columns. The flow through was collected. Each affinity column was washed two times with 1 mL of a solution comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA. The affinity columns were then filled with 2 mL of TEV protease containing buffer, containing 50 units per mL, and stored overnight at 4 °C. The flow through was collected. 4.5 mL of an elution buffer comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 50 mM biotin wasthen added to the columns allowing the Strep-tagged lentiviruses that bound to the affinity column to be eluted. The eluate was collected. Figure 10A shows the experimental protocol. Western blot analysis was then performed on the lentiviruses and their supernatant, the flow through after adding the lentivirus to the columns, the flow through after storing the columns with TEV protease containing buffer overnight, and the eluate (FIG. 10B). FIG. 10B indicates that TEV protease cleavage occurred in the affinity column prior to elution (see F2 columns in FIG. 10B).Example 11

[0227] This Example demonstrates that TEV protease cleavage can occur in 90 minutes when stored at 30 °C.

[0228] Two lentiviruses were constructed to have the following structures: UCHT1-VSV- G-QQQ and Strep-(G4S)5-Tev site-(G4S)5-VSV-G-QQQ. 1.5 mL of a combination of UCHT1-VSV-G-QQQ and Strep-(G4S)5-Tev site-(G4S)5-VSV-G-QQQ in a 1 :3 ratio was added in OPTI-MEM to a Strep-Tactin®XT 4Flow® high-capacity affinity column. This Experiment was the second use of the affinity column. The flow through was collected. The affinity column was washed two times with 1 mL of a solution comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA. The affinity column was then filled with 2 mL of TEV protease containing buffer, containing 50 units per mL, and stored for 90 minutes at 30 °C. The flow through was collected. 4.5 mL of an elution buffer comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 50 mM biotin was then added to the column allowing the Strep-tagged lentiviruses that bound to the affinity column to be eluted. The eluate was collected. Figure 11 A shows the experimental protocol. Western blot analysis was then performed the lentiviruses and their supernatant, the flow through after adding the lentivirus to the column, the flow through after storing the column with TEV protease containing buffer for 90 minutes, and the eluate (FIG. 1 IB). FIG. 1 IB indicates that TEV protease cleavage occurred for most lentiviruses in the affinity column prior to elution (column F2 of FIG. 1 IB).Example 12

[0229] This Example demonstrates that the addition of washing steps improves recovery of cleaved lentivirus.

[0230] Two lentiviruses were constructed to have the following structures: UCHT1-VSV- G-QQQ and Strep-(G4S)5-Tev site-(G4S)5-VSV-G-QQQ. 1.5 mL of a combination of UCHT1-VSV-G-QQQ and Strep-(G4S)5-Tev site-(G4S)5-VSV-G-QQQ in a 1 :3 ratio was added in OPTI-MEM to a Strep-Tactin®XT 4Flow® affinity column. The flow through was collected. The affinity column was washed two times with 1 mL of a solution comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA. The flow through was collected. The affinity column was then filled with 2 mL of TEV protease containing buffer, containing 50 units per mL, and stored for 90 minutes at 30 °C. The affinity column was washed three times. The flow through was collected. 2 mL of an elution buffer comprising 100 mM Tris / HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, and 50 mM biotin was then added three times to the column allowing the Strep-tagged lentiviruses that bound to the affinity column to be eluted. The eluate was collected. Figure 12A shows the experimental protocol. Western blot analysis was then performed on the lentiviruses and their supernatant, the flow through after adding the lentivirus to the column, the flow through after the first set of two washes, the flow through after storing the column with TEV protease containing buffer for 90 minutes and the second set of washes, and the eluate (FIG. 12B). FIG. 12B indicates that TEV protease cleavage occurred for most lentiviruses in the affinity column prior to elution and most of the cleaved viruses were washed out before elution.

[0231] 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.

[0232] 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,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as ashorthand 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.

[0233] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects 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.SEQUENCES

Claims

CLAIMS:

1. A membraned vesicle comprising:(a) an envelope membrane; and(b) viral membrane glycoproteins (VMGs), each VMG optionally within a fusion protein; wherein at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one affinity tag, wherein at least one VMG is incorporated into the envelope membrane and comprises a fusogenic membrane glycoprotein (FMG), wherein at least one affinity tag is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle; and wherein the ratio of VMG without an affinity tag to VMG with an affinity tag is from 0: 1 to 5: 1.

2. The membraned vesicle of claim 1, wherein the ratio of VMG with an affinity tag to VMG without an affinity tag is from 1 : 1 to 3 : 1.

3. The membraned vesicle of claim 1 or 2, wherein the ratio of VMG with an affinity tag to VMG without an affinity tag is 3 : 1.

4. The membraned vesicle of any one of claims 1-3, wherein at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one targeting polypeptide, and wherein at least one targeting polypeptide is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

5. The membraned vesicle of claim 4, wherein the ratio of VMG with a targeting polypeptide to VMG without an affinity tag to VMG with an affinity tag is from 1:0:1 to 1:5:

16. The membraned vesicle of claim 4 or 5, wherein the ratio of VMG with a targeting polypeptide to VMG without an affinity tag to VMG with an affinity tag is from 1:1:1 to 1:3:1.

7. The membraned vesicle of any one of claims 4-6, wherein the ratio of VMG with a targeting polypeptide to VMG without an affinity tag to VMG with an affinity tag is 1:3:1.

8. The membraned vesicle of any one of claims 1-7, wherein at least one FMG is a rhabdoviral G glycoprotein or functional fragment or derivative thereof that is of a Flanders virus glycoprotein (FLAV-G) (SEQ ID NO: 6), a Chandipura virus glycoprotein (CHPV-G) (SEQ ID NO: 7), a Perinet virus glycoprotein (PERV-G) (SEQ ID NO: 8), a Piry virus glycoprotein (PIRYV-G) (SEQ ID NO: 9), a Fukuoka virus glycoprotein (FUKV-G) (SEQ ID NO: 10), a Joinjakaka virus glycoprotein (JOIV-G) (SEQ ID NO: 11), a Kumasi virus glycoprotein (KRV-G) (SEQ ID NO: 12), a Keuraliba virus glycoprotein (KEUV-G) (SEQ ID NO: 13), 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 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).

9. The membraned vesicle of any one of claims 1-7, wherein at least one FMG is a rhabdoviral G glycoprotein or functional fragment or derivative thereof that is of a Vesiculovirus glycoprotein or a functional fragment or derivative thereof.

10. The membraned vesicle of claim 9, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus Indiana, Vesiculovirus new jersey, Vesiculovirus carajas, or Vesiculovirus alagoas.

11. The membraned vesicle of claim 10, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus indiana (SEQ ID NO:14).

12. The membraned vesicle of claim 10, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus newjersey (SEQ ID NO:15).

13. The membraned vesicle of any one of claims 8-12, wherein the rhabdoviral G glycoprotein is substantially intact.

14. The membraned vesicle of any one of claims 8-12, wherein the rhabdoviral G glycoprotein is a functional fragment or derivative thereof.

15. The membraned vesicle of claim 14, wherein the cytoplasmic tail of the glycoprotein is truncated, deleted, or replaced with another sequence.

16. The membraned vesicle of any one of claims 8-15, wherein the rhabdoviral G glycoprotein is engineered to reduce or abolish its natural receptor binding specificity.

17. The membraned vesicle of claim 16, wherein the rhabdoviral G glycoprotein is engineered to have a mutation to reduce or abolish its natural receptor binding specificity.

18. The membraned vesicle of claim 17, wherein the rhabdoviral G glycoprotein comprises a mutation at one or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO: 14).

19. The membraned vesicle of claim 17 or 18, wherein the mutation is a substitution.

20. The membraned vesicle of claim 19, wherein the mutation is a substitution with a Q.

21. The membraned vesicle of claim 19 or 20, wherein the mutation is a substitution at two or more positions.

22. The membraned vesicle of claim 17 or 18, wherein the mutation is a deletion.

23. The membraned vesicle of claim 22, wherein the mutation is a single deletion at the position corresponding to K47 on the Vesiculovirus indiana glycoprotein (SEQ ID NO: 14).

24. The membraned vesicle of any one of claims 1-23, wherein at least one VMG is within a fusion protein comprising at least one affinity tag, wherein the fusion protein does not comprise a targeting polypeptide.

25. The membraned vesicle of claim 24, wherein two or more VMGs hetero-oligomerize to form at least one protomer-complex comprising: (i) at least one VMG within a fusion protein comprising at least one affinity tag, wherein the fusion protein does notcomprise a targeting polypeptide; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide.

26. The membraned vesicle of any one of claims 1-25, wherein at least one VMG is within a fusion protein comprising at least one targeting polypeptide, wherein the fusion protein does not comprise an affinity tag.

27. The membraned vesicle of claim 26, wherein two or more VMGs hetero-oligomerize to form at least one protomer-complex comprising: (i) at least one VMG within a fusion protein comprising at least one targeting polypeptide, wherein the fusion protein does not comprise an affinity tag; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide.

28. The membraned vesicle of any one of claims 1-27, wherein at least one VMG is within a fusion protein comprising at least one affinity tag and at least one targeting polypeptide.

29. The membraned vesicle of claim 28, wherein two or more VMGs hetero-oligomerize to form at least one protomer-complex comprising: (i) at least one VMG within a fusion protein comprising at least one affinity tag and at least one targeting polypeptide; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide.

30. The membraned vesicle of any one of claims 1-29, wherein at least one VMG is a Type 1 membrane glycoprotein.

31. The membraned vesicle of claim 30, wherein the Type 1 membrane glycoprotein is within a fusion protein comprising at least one affinity tag at the N-terminus of the fusion protein, wherein the affinity tag of the fusion protein comprising the Type 1membrane glycoprotein is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

32. The membraned vesicle of claim 30, wherein the Type 1 membrane glycoprotein is within a fusion protein comprising at least one retroviral gag protein at the C-terminus of the fusion protein wherein the retroviral gag protein is inside the lumen of the membraned vesicle.

33. The membraned vesicle of claim 31, wherein the fusion protein comprising the Type 1 membrane glycoprotein comprises at least one retroviral gag protein at the C- terminus of the fusion protein wherein the retroviral gag protein is inside the lumen of the membraned vesicle.

34. The membraned vesicle of any one of claims 1-33, wherein at least one VMG is a Type 2 membrane glycoprotein.

35. The membraned vesicle of claim 34, wherein the Type 2 membrane glycoprotein is within a fusion protein comprising at least one affinity tag at the C-terminus of the fusion protein, wherein the affinity tag of the fusion protein comprising the Type 2 membrane glycoprotein is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

36. The membraned vesicle of claim 34 or 35, wherein the Type 2 membrane glycoprotein is within a fusion protein comprising at least one retroviral gag protein at the N-terminus of the fusion protein wherein the retroviral gag protein is inside the lumen of the membraned vesicle.

37. The membraned vesicle of any one of claims 1-36, wherein at least one VMG is a Type 1 membrane glycoprotein and at least one VMG is a Type 2 membrane glycoprotein.

38. The membraned vesicle of any one of claims 34-37, wherein at least one Type 2 membrane glycoprotein is within a bipartite protomer-complex comprising at least one Type 1 membrane glycoprotein comprising a fusion protein comprising at least one retroviral gag protein at the C-terminus of the fusion protein, wherein the retroviral gag protein is inside the lumen of the membraned vesicle, and wherein the Type 1 membrane glycoprotein is an FMG.

39. The membraned vesicle of claim 32, 33, 36, or 38 wherein at least one retroviral gag protein is tethered to the inner surface of the envelope membrane.

40. The membraned vesicle of claim 32, 33, 36, 38, or 39 wherein at least one retroviral gag protein is truncated at its C-terminus.

41. The membraned vesicle of claim 39 or 40, wherein more than one retroviral gag protein form a polyhedral core structure within the membraned vesicle.

42. The membraned vesicle of claim 41, wherein the gag proteins comprising the polyhedral core structure comprise both nonchimeric gag proteins and gag proteins fused to the C-terminus of a membrane anchored Type 1 membrane glycoprotein which is optionally an FMG.

43. The membraned vesicle of any one of claims 1-42, wherein at least one VMG is a Type 2 membrane glycoprotein comprising Paramyxoviridae canine distemper virus H protein and at least one VMG is a Type 1 membrane glycoprotein comprising Paramyxoviridae canine distemper virus F protein.

44. The membraned vesicle of claim 43, wherein at least one VMG is within a fusion protein comprising a Type 2 membrane glycoprotein comprising Paramyxoviridae canine distemper virus H protein and at least one affinity tag at the C-terminus of the fusion protein, and wherein at least one VMG is within a fusion protein comprising a Type 1 membrane glycoprotein comprising Paramyxoviridae canine distemper virus F protein and at least one retroviral gag protein at the C-terminus of the fusion protein.

45. The membraned vesicle of any one of claims 1-44, wherein at least one VMG is within a fusion protein comprising at least one affinity tag, wherein the fusion protein comprises at least one protease cleavable linker between VMG and at least one affinity tag.

46. The membraned vesicle of claim 45, wherein at least one protease cleavable linker comprises the amino acid sequence of ENLYFQGG (SEQ ID NO: 142) or ENLYFQGS (SEQ ID NO: 143) and the protease is tobacco etch virus (TEV) protease.

47. The membraned vesicle of any one of claims 1-46, wherein at least one affinity tag is a strep-tag.

48. The membraned vesicle of any one of claims 1-47, wherein at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one targeting polypeptide, and wherein at least one targeting polypeptide is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle, and wherein at least one targeting polypeptide comprises Spy-tag.

49. The membraned vesicle of any one of claims 1-48, comprising:(1) VMGs within a fusion protein comprising exactly one affinity tag; and(2) VMGs that do not comprise an affinity tag and do not comprise a targeting polypeptide; wherein the VMGs are incorporated into the envelope membrane, wherein the VMGs are FMGs, and wherein the VMGs are engineered to reduce or eliminate binding to its natural receptor.

50. The membraned vesicle of claim 1, comprising:(1) VMGs within a fusion protein comprising exactly one affinity tag;(2) FMGs within a fusion protein comprising exactly one targeting polypeptide; and(3) VMGs that do not comprise an affinity tag and do not comprise a targeting polypeptide; wherein the VMGs are incorporated into the envelope membrane, wherein the fusion proteins of (1) and (2) are separate fusion proteins, wherein the VMGs are FMGs, wherein the amino acid sequences of the VMGs are the same and that of vesicular stomatitis virus G glycoprotein (VSV-G), wherein the VMGs are engineered to reduce or eliminate binding to its natural receptor, and wherein the targeting polypeptide is Spy-tag.

51. The membraned vesicle of any one of claims 1-50, wherein the membraned vesicle is an enveloped delivery vehicle.

52. The membraned vesicle of any one of claims 1-51, wherein the membraned vesicle is a virus-like particle.

53. The membraned vesicle of any one of claims 1-51, wherein the membraned vesicle is an exosome.

54. A membraned vesicle comprising:(a) an envelope membrane; and(b) viral membrane glycoproteins (VMGs), each VMG optionally within a fusion protein; wherein at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one affinity tag, wherein at least one VMG is incorporated into the envelope membrane and comprises a fusogenic membrane glycoprotein (FMG), wherein at least one affinity tag is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle; wherein at least one FMG is a rhabdoviral G glycoprotein engineered to have a mutation to reduce or abolish its natural receptor binding specificity, wherein the rhabdoviral G glycoprotein comprises a mutation at one or more positions corresponding to H8, K47, Y209, and K354 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO: 14), and wherein the mutation is a substitution at two or more positions or the mutation is a deletion.

55. The membraned vesicle of claim 54, wherein at least one FMG is a rhabdoviral G glycoprotein or functional fragment or derivative thereof that is of a Flanders virus glycoprotein (FLAV-G) (SEQ ID NO: 6), a Chandipura virus glycoprotein (CHPV-G) (SEQ ID NO: 7), a Perinet virus glycoprotein (PERV-G) (SEQ ID NO: 8), a Piryvirus glycoprotein (PIRYV-G) (SEQ ID NO: 9), a Fukuoka virus glycoprotein (FUKV-G) (SEQ ID NO: 10), a Joinjakaka virus glycoprotein (JOIV-G) (SEQ ID NO: 11), a Kumasi virus glycoprotein (KRV-G) (SEQ ID NO: 12), a Keuraliba virus glycoprotein (KEUV-G) (SEQ ID NO: 13), 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).

56. The membraned vesicle of claim 54, wherein at least one FMG is a rhabdoviral G glycoprotein or functional fragment or derivative thereof that is of a Vesiculovirus glycoprotein or a functional fragment or derivative thereof.

57. The membraned vesicle of claim 56, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus Indiana, Vesiculovirus new jersey, Vesiculovirus carajas, or Vesiculovirus alagoas.

58. The membraned vesicle of claim 57, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus indiana (SEQ ID NO:14).

59. The membraned vesicle of claim 57, wherein the rhabdoviral G glycoprotein or functional fragment or derivative thereof is of Vesiculovirus newjersey (SEQ ID NO:15).

60. The membraned vesicle of any one of claims 54-59, wherein the rhabdoviral G glycoprotein is substantially intact.

61. The membraned vesicle of any one of claims 54-59, wherein the rhabdoviral G glycoprotein is a functional fragment or derivative thereof.

62. The membraned vesicle of claim 61, wherein the cytoplasmic tail of the glycoprotein is truncated, deleted, or replaced with another sequence.

63. The membraned vesicle of any one of claims 54-62, wherein the mutation is a substitution at two or more positions.

64. The membraned vesicle of claim 63, wherein the substitution is with Q.

65. The membraned vesicle of any one of claims 54-62, wherein the mutation is a deletion.

66. The membraned vesicle of claim 65, wherein the mutation is a single deletion at the position corresponding to K47 on the Vesiculovirus Indiana glycoprotein (SEQ ID NO: 14).

67. The membraned vesicle of any one of claims 54-66, wherein at least one VMG is within a fusion protein comprising at least one affinity tag, wherein the fusion protein does not comprise a targeting polypeptide.

68. The membraned vesicle of claim 67, wherein two or more VMGs hetero-oligomerize to form at least one protomer-complex comprising: (i) at least one VMG within afusion protein comprising at least one affinity tag, wherein the fusion protein does not comprise a targeting polypeptide; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide.

69. The membraned vesicle of any one of claims 54-68, wherein at least one VMG is within a fusion protein comprising at least one targeting polypeptide, wherein the fusion protein does not comprise an affinity tag.

70. The membraned vesicle of claim 69, wherein two or more VMGs hetero-oligomerize to form at least one protomer-complex comprising: (i) at least one VMG within a fusion protein comprising at least one targeting polypeptide, wherein the fusion protein does not comprise an affinity tag; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide.

71. The membraned vesicle of any one of claims 54-70, wherein at least one VMG is within a fusion protein comprising at least one affinity tag and at least one targeting polypeptide.

72. The membraned vesicle of claim 71, wherein two or more VMGs hetero-oligomerize to form at least one protomer-complex comprising: (i) at least one VMG within a fusion protein comprising at least one affinity tag and at least one targeting polypeptide; and (ii) at least one VMG that does not comprise an affinity tag and does not comprise a targeting polypeptide.

73. The membraned vesicle of any one of claims 54-72, wherein at least one VMG is a Type 1 membrane glycoprotein.

74. The membraned vesicle of claim 73, wherein the Type 1 membrane glycoprotein is within a fusion protein comprising at least one affinity tag at the N-terminus of thefusion protein, wherein the affinity tag of the fusion protein comprising the Type 1 membrane glycoprotein is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

75. The membraned vesicle of claim 73, wherein the Type 1 membrane glycoprotein is within a fusion protein comprising at least one retroviral gag protein at the C-terminus of the fusion protein wherein the retroviral gag protein is inside the lumen of the membraned vesicle.

76. The membraned vesicle of claim 74, wherein the fusion protein comprising the Type 1 membrane glycoprotein comprises at least one retroviral gag protein at the C- terminus of the fusion protein wherein the retroviral gag protein is inside the lumen of the membraned vesicle.

77. The membraned vesicle of any one of claims 54-76, wherein at least one VMG is a Type 2 membrane glycoprotein.

78. The membraned vesicle of claim 77, wherein the Type 2 membrane glycoprotein is within a fusion protein comprising at least one affinity tag at the C-terminus of the fusion protein, wherein the affinity tag of the fusion protein comprising the Type 2 membrane glycoprotein is displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

79. The membraned vesicle of claim 77 or 78, wherein the Type 2 membrane glycoprotein is within a fusion protein comprising at least one retroviral gag protein at the N-terminus of the fusion protein wherein the retroviral gag protein is inside the lumen of the membraned vesicle.

80. The membraned vesicle of any one of claims 54-79, wherein at least one VMG is a Type 1 membrane glycoprotein and at least one VMG is a Type 2 membrane glycoprotein.

81. The membraned vesicle of any one of claims 77-80, wherein at least one Type 2 membrane glycoprotein is within a bipartite protomer-complex comprising at least one Type 1 membrane glycoprotein comprising a fusion protein comprising at least one retroviral gag protein at the C-terminus of the fusion protein, wherein the retroviral gag protein is inside the lumen of the membraned vesicle, and wherein the Type 1 membrane glycoprotein is an FMG.

82. The membraned vesicle of claim 75, 76, 79, or 81 wherein at least one retroviral gag protein is tethered to the inner surface of the envelope membrane.

83. The membraned vesicle of claim 75, 76, 79, 81, or 82 wherein at least one retroviral gag protein is truncated at its C-terminus.

84. The membraned vesicle of claim 82 or 83, wherein more than one retroviral gag protein form a polyhedral core structure within the membraned vesicle.

85. The membraned vesicle of claim 84, wherein the gag proteins comprising the polyhedral core structure comprise both nonchimeric gag proteins and gag proteins fused to the C-terminus of a membrane anchored Type 1 membrane glycoprotein which is optionally an FMG.

86. The membraned vesicle of any one of claims 54-85, wherein at least one VMG is a Type 2 membrane glycoprotein comprising Paramyxoviridae canine distemper virusH protein and at least one VMG is a Type 1 membrane glycoprotein comprising Paramyxoviridae canine distemper virus F protein.

87. The membraned vesicle of claim 86, wherein at least one VMG is within a fusion protein comprising a Type 2 membrane glycoprotein comprising Paramyxoviridae canine distemper virus H protein and at least one affinity tag at the C-terminus of the fusion protein, and wherein at least one VMG is within a fusion protein comprising a Type 1 membrane glycoprotein comprising Paramyxoviridae canine distemper virus F protein and at least one retroviral gag protein at the C-terminus of the fusion protein.

88. The membraned vesicle of any one of claims 54-87, wherein at least one VMG is within a fusion protein comprising at least one affinity tag, wherein the fusion protein comprises at least one protease cleavable linker between VMG and at least one affinity tag.

89. The membraned vesicle of claim 88, wherein at least one protease cleavable linker comprises the amino acid sequence of ENLYFQGG (SEQ ID NO: 142) or ENLYFQGS (SEQ ID NO: 143) and the protease is tobacco etch virus (TEV) protease.

90. The membraned vesicle of any one of claims 54-89, wherein at least one affinity tag is a strep-tag.

91. The membraned vesicle of any one of claims 54-90, wherein at least one VMG is incorporated into the envelope membrane and is within a fusion protein comprising at least one targeting polypeptide, and wherein at least one targeting polypeptide is displayed at the outer surface of the envelope membrane, wherein the outer surface isopposite the lumen of the membraned vesicle, and wherein at least one targeting polypeptide comprises Spy-tag.

92. The membraned vesicle of any one of claims 54-91, comprising:(1) VMGs within a fusion protein comprising exactly one affinity tag; and(2) VMGs that do not comprise an affinity tag and do not comprise a targeting polypeptide; wherein the VMGs are incorporated into the envelope membrane, wherein the VMGs are FMGs, and wherein the VMGs are engineered to reduce or eliminate binding to its natural receptor.

93. The membraned vesicle of claim 54, comprising:(1) VMGs within a fusion protein comprising exactly one affinity tag;(2) FMGs within a fusion protein comprising exactly one targeting polypeptide; and(3) VMGs that do not comprise an affinity tag and do not comprise a targeting polypeptide; wherein the VMGs are incorporated into the envelope membrane, wherein the fusion proteins of (1) and (2) are separate fusion proteins, wherein the VMGs are FMGs, wherein the amino acid sequences of the VMGs are the same and that of vesicular stomatitis virus G glycoprotein (VSV-G),wherein the VMGs are engineered to reduce or eliminate binding to its natural receptor, and wherein the targeting polypeptide is Spy-tag.

94. The membraned vesicle of any one of claims 54-93, wherein the membraned vesicle is an enveloped delivery vehicle.

95. The membraned vesicle of any one of claims 54-94, wherein the membraned vesicle is a virus-like particle.

96. The membraned vesicle of any one of claims 54-94, wherein the membraned vesicle is an exosome.

97. A method of generating the membraned vesicle of any one of claims 1-96, the method comprising:(a) transfecting a host cell with:(i) at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one affinity tag;(ii) optionally, at least one packaging plasmid; and(b) collecting membraned vesicles comprising at least one affinity tag displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

98. The method of claim 97, comprising transfecting the host cell with at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one targeting polypeptide.

99. The method of claim 98, wherein at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one affinity tag and at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one targeting polypeptide are separate plasmids.

100. The method of claim 98, wherein at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one affinity tag and at least one plasmid encoding at least one VMG that is within a fusion protein comprising at least one targeting polypeptide are on the same plasmid.

101. The method of any one of claims 97-100, wherein collecting membraned vesicles comprises purifying the collected membraned vesicles using affinity purification using at least one affinity tag displayed at the outer surface of the envelope membrane, wherein the outer surface is opposite the lumen of the membraned vesicle.

102. The method of claim 101, wherein at least one VMG is within a fusion protein comprising at least one affinity tag, wherein the fusion protein comprises at least one protease cleavable linker between VMG and at least one affinity tag, and wherein the method comprises using at least one protease to cleave at least one cleavable linker to elute at least one cleaved fusion protein.

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