Engineered viral particles for targeted delivery

Abstract

Provided herein, among other things, are novel and improved glycoproteins, including modified glycoproteins; engineered viral particles comprising such novel and modified glycoproteins; and methods for targeted delivery, for example, to immune cells such as T cells. In some aspects, the engineered viral particles allow targeted delivery of the cargo for various applications, including treating various conditions such as autoimmune diseases and cancer. In some aspects, the engineered viral particles comprise a viral fusogen that is a glycoprotein of Chandipura, Perinet, Piry, Jurona viruses, or variants thereof. In other aspects, the engineered viral particles comprise a modified vesiculovirus glycoprotein mutant, for example, with mutations at amino acids corresponding to positions I347 or R354 of Vesicular Stomatitis Virus G (VSV-G) that reduce binding to Low Density Lipoprotein Receptor (LDL-R).

Description

ATTORNEY DOCKET NO. ORB-015WO1ENGINEERED VIRAL PARTICLES FOR TARGETED DELIVERYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to, and the benefit of U.S. Provisional Application Number 63 / 720,349, filed on November 14, 2024, the contents of which are incorporated herein by reference in their entireties.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed with an electronically filed Sequence Listing in XML format. The sequence listing file entitled ORB-015WOl_seqlist.XML was created on October 20, 2025, and is 114,688 bytes in size; the information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.BACKGROUND

[0003] Virus tropism, which is the ability of a virus to infect different cell types, is altered or expanded by pseudotyping. Pseudotyped viral particles can be engineered to alter the tropism of a virus by using glycoproteins from other enveloped viruses, e.g., vectors pseudotyped with rabies virus-derived glycoproteins target the central nervous system.

[0004] Lentiviruses and other viruses have been pseudotyped using Vesicular Stomatitis Virus (VSV), an oncolytic rhabdovirus, such as VS V-gly coprotein (VSV-G), which confers a broad tropism and stability to the pseudotyped virus. VSV is known to be used for pseudotyped viruses. Broad tropism of wild-type VSV due to ubiquitous expression of major receptor, low-density lipoprotein receptor (LDL-R), however, can transduce non-specific cells and cause side-effects.

[0005] Further, efficient gene transfer into target cells having a very low expression level of LDL-R, e.g., unstimulated T cells, B cells, and hematopoietic stem cells, remains challenging.

[0006] There is a need for improved viral glycoproteins that can be produced at a scale suitable for manufacturing and can be safely used to effectively transduce immune cells for application such as targeted immunotherapy.SUMMARY OF THE INVENTION

[0007] The present disclosure provides, in part, improved engineered viral particles for targeted delivery of a cargo (for example, and without limitation, a nucleic acid cargoATTORNEY DOCKET NO. ORB-015WO1encoding a therapeutic protein) to cells to treat, for example, cancer, infectious disease, or immune diseases, including autoimmune diseases. The disclosure is based, in part, on the discovery of viral glycoproteins from newly-identified viruses (e.g., Chandipura, Perinet, Piry, and Jurona viruses) that naturally do not bind human cell receptors (e.g., LDL-receptor) but retain the ability to initiate membrane fusion, / .< ., function as fusogens. The present disclosure provides, among other things, engineered viral particles comprising Chandipura, Perinet, Piry, or Jurona vesiculoviruses, or variants thereof, or engineered viral particles comprising viral glycoproteins from said viruses. The discovery of viral glycoproteins from the viruses that naturally do not bind human cell receptors provides safe delivery of the cargo without off-target effects, and also has advantages over known mutations of VSV-G. These advantages include being able to stably produce a high-titer, providing for improved manufacturability, being less sensitive to complement inactivation, and providing for specific delivery to target cells, for example, by incorporating a targeting moiety, thereby increasing the efficacy and safety of the cargo with fewer off-target effects. Delivery of the engineered viral particles of the present disclosure to desired target cells (e.g., immune cells such as T cells, among others) is enhanced by incorporating a targeting moiety, which avoids off-target delivery caused by non-specific glycoprotein binding.

[0008] The present disclosure also provides, in part, a complementary approach to generating improved engineered viral particles comprising novel mutations or mutations at novel residues for LDL-receptor binding in the vesiculovirus glycoprotein (as a non-limiting example, Vesicular Stomatitis Virus glycoprotein (VSV-G)). Wild-type VSV-G is not optimal for delivery to all cell types (e.g., VSV-G is not delivered effectively to immune cells that do not express LDL-R to high levels), and, further, wild-type VSV-G is sensitive to serum complement inactivation. Although VSV-G K47 and R354 are known receptorbinding sites that have been mutated to make modified fusogens, the present disclosure, among other things, provides novel mutations and / or mutations at novel residues, for example, I347N, I347Q, R354G, and R354N of VSV-G. In some embodiments, these novel mutations and / or mutations at novel residues yield a sufficiently high titer of vesiculovirus for commercial scale manufacture and administration, and also yield stable engineered viral particles. Such engineered vesiculoviruses (e.g., Maraba virus variants) are also less susceptible to complement inactivation.

[0009] The discovery of novel residues also expands the repertoire of modified vesiculovirus G proteins for targeted delivery, for example, including in combinatorial multidosing regimens with concomitant or sequential administration of different vesiculovirus GATTORNEY DOCKET NO. ORB-015WO1proteins to optimize delivery in a therapeutic regimen, for example, in treating various diseases and conditions, including but not limited to immune diseases, such as autoimmune diseases, and cancer.

[0010] In some aspects, provided herein is an engineered viral particle comprising a viral fusogen, a targeting moiety, and at least one nucleic acid, wherein the viral fusogen is a glycoprotein of Chandipura virus, Perinet virus, Piry virus, Jurona virus, or a variant thereof. In other aspects, provided herein is an engineered viral particle comprising a viral fusogen, a targeting moiety and at least one nucleic acid, wherein the viral fusogen comprises a modified vesiculovirus glycoprotein (VSV-G), comprising one or more amino acid mutations at residues selected from the group consisting of 1347 and R354.

[0011] In some embodiments, the one or more amino acid mutations decreases binding of the engineered viral particle to Low Density Lipoprotein Receptor (LDL-R).

[0012] In some embodiments, the viral fusogen is a glycoprotein of Chandipura virus.

[0013] In some embodiments, the viral fusogen is a glycoprotein of Perinet virus.

[0014] In some embodiments, the viral fusogen is a glycoprotein of Piry virus.

[0015] In some embodiments, the viral fusogen is a glycoprotein of Jurona virus.

[0016] In some embodiments, the modified vesiculovirus glycoprotein is a modified Vesicular Stomatitis Virus glycoprotein (VSV-G).

[0017] In some embodiments, the modified vesiculovirus glycoprotein is a modified Morreton virus glycoprotein, Cocal virus glycoprotein, Alagoas virus glycoprotein, Carajas virus glycoprotein, New Jersey virus glycoprotein or Maraba virus glycoprotein.

[0018] In some embodiments, the modified vesiculovirus glycoprotein is a modified Maraba virus glycoprotein.

[0019] In some embodiments, the one or more amino acid mutations are selected from the group consisting of I347N, I347Q, R354G, and R354N.

[0020] In some embodiments, the amino acid mutation is I347N or I347Q.

[0021] In some embodiments, the amino acid mutation is R354G or R354N.

[0022] In some embodiments, the viral glycoprotein fusogen lacks human low density lipoprotein (LDL)-receptor binding.

[0023] In some embodiments, the viral glycoprotein comprises a sequence of any one of SEQ ID NOs: 1-10 or 13-71.

[0024] In some embodiments, the engineered viral particle is a pseudotyped viral particle.ATTORNEY DOCKET NO. ORB-015WO1

[0025] In some embodiments, the engineered viral particle is a pseudotyped vesiculovirus, retrovirus, alphavirus, herpesvirus, arenavirus, paramyxovirus, lentivirus, arenavirus, or oncolytic virus.

[0026] In some embodiments, the targeting moiety is an antibody or an antigen binding fragment thereof, or a ligand.

[0027] In some embodiments, the targeting moiety is an antibody or an antigen binding fragment thereof that binds to a cell surface marker.

[0028] In some embodiments, the targeting moiety is a ligand that binds to a receptor on a cell surface.

[0029] In some embodiments, the targeting moiety is immune cell specific.

[0030] In some embodiments, the immune cell is a T cell, a B cell or a natural killer (NK) cell.

[0031] In some embodiments, the immune cell is a T cell.

[0032] In some embodiments, the immune cell is a B cell.

[0033] In some embodiments, the nucleic acid encodes a polypeptide.

[0034] In some embodiments, the polypeptide is a therapeutic or immunogenic polypeptide. In some embodiments, the polypeptide is a therapeutic polypeptide.

[0035] In some embodiments, the immunogenic polypeptide is derived from an infectious pathogen.

[0036] In some embodiments, the immunogenic polypeptide is a cancer antigen. In some embodiments, the immunogenic polypeptide is an oncoantigen.

[0037] In some embodiments, provided herein is a composition comprising the engineered viral particle of the present disclosure.

[0038] In some embodiments, provided herein is a cell comprising the engineered viral particle of the present disclosure.

[0039] In some embodiments, provided herein is a targeted delivery vehicle comprising the engineered viral particle of the present disclosure.

[0040] In some embodiments, provided herein is a modified vesiculovirus envelope glycoprotein, comprising one or more amino acid mutations corresponding to positions 1347 or R354 of wild-type VSV-G of SEQ ID NO: 12. In some embodiments, provided herein is a modified vesiculovirus envelope glycoprotein, comprising an amino acid mutation at 1347. In some embodiments, provided herein is a modified vesiculovirus envelope glycoprotein, comprising an amino acid mutation at R354. In some embodiments, provided herein is a modified vesiculovirus envelope glycoprotein, comprising an amino acid mutation atATTORNEY DOCKET NO. ORB-015WO1positions 1347 and R354. In some embodiments, provided herein is a modified vesiculovirus envelope glycoprotein, comprising an amino acid mutation at K47 and 1347. In some embodiments, provided herein is a modified vesiculovirus envelope glycoprotein, comprising an amino acid mutation at K47 and R354. In some embodiments, provided herein is a modified vesiculovirus envelope glycoprotein, comprising an amino acid mutation at K47, 1347, and R354.

[0041] In some embodiments, provided herein is a modified vesiculovirus glycoprotein, wherein the one or more amino acid mutations are selected from the group consisting of I347N, I347Q, R354G, and R354N.

[0042] In some aspects, provided herein is a modified Vesicular Stomatitis Virus envelope glycoprotein (VSV-G), comprising one or more amino acid mutations at residues selected from the group consisting of 1347 and R354. In some aspects, provided herein is a modified Vesicular Stomatitis Virus envelope glycoprotein (VSV-G), comprising an amino acid mutation at 1347. In some aspects, provided herein is a modified Vesicular Stomatitis Virus envelope glycoprotein (VSV-G), comprising an amino acid mutation at R354. In some aspects, provided herein is a modified Vesicular Stomatitis Virus envelope glycoprotein (VSV-G), comprising an amino acid mutation at 1347 and R354. In some aspects, provided herein is a modified Vesicular Stomatitis Virus envelope glycoprotein (VSV-G), comprising an amino acid mutation at K47 and 1347. In some aspects, provided herein is a modified Vesicular Stomatitis Virus envelope glycoprotein (VSV-G), comprising an amino acid mutation at K47 and R354. In some aspects, provided herein is a modified Vesicular Stomatitis Virus envelope glycoprotein (VSV-G), comprising an amino acid mutation at K47, 1347 and R354.

[0043] In some embodiments, provided herein is a modified VSV glycoprotein, wherein the one or more amino acid mutations are selected from the group consisting of I347N, I347Q, R354G, and R354N.

[0044] In some embodiments, the amino acid mutation is I347N or I347Q.

[0045] In some embodiments, the amino acid mutation is R354G or R354N.

[0046] In some embodiments, the modified viral glycoprotein fusogen lacks human low density lipoprotein (LDL)-receptor binding.

[0047] In some embodiments, wherein the glycoprotein comprises a sequence of any one of SEQ ID NOs: 13-71.

[0048] In some embodiments, provided herein is a Vesicular Stomatitis Virus comprising the modified VSV glycoprotein of the present disclosure.ATTORNEY DOCKET NO. ORB-015WO1

[0049] In some aspects, provided herein is an engineered viral particle comprising a vesiculovirus, a targeting moiety, and at least one nucleic acid, wherein the targeting moiety is on the surface of the engineered viral particle, and wherein the vesiculovirus is a Chandipura virus, a Perinet virus, a Piry virus, or a Jurona virus, or a variant thereof.

[0050] In some embodiments, the vesiculovirus is a Chandipura virus, or a variant thereof.

[0051] In some embodiments, the vesiculovirus is a Perinet virus, or a variant thereof.

[0052] In some embodiments, the vesiculovirus is a Piry virus, or a variant thereof.

[0053] In some embodiments, the vesiculovirus is a Jurona virus, or a variant thereof.

[0054] In some aspects, provided herein is an engineered virus particle, comprising a vesiculovirus, a targeting moiety, and at least one nucleic acid, wherein the targeting moiety is on the surface of the engineered viral particle, or a variant thereof.

[0055] In some embodiments, the vesiculovirus is a Vesicular Stomatitis Virus (VSV) variant.

[0056] In some embodiments, the vesiculovirus is a Morreton virus, a Cocal virus, an Alagoas virus, a Carajas virus, a New Jersey virus or a Maraba virus variant.

[0057] In some embodiments, the vesiculovirus is a Maraba virus variant.

[0058] In some embodiments, provided herein is a method of delivering a nucleic acid into a cell comprising contacting the cell with the engineered viral particle of the present disclosure.

[0059] In some embodiments, the cell is an immune cell.

[0060] In some aspects, provided herein is a method of delivering a nucleic acid into a target cell or tissue in a subject in need thereof comprising administering to the subject a nucleic acid encapsulated within an engineered viral particle, wherein the engineered viral particle comprises: a viral glycoprotein fusogen of any one of glycoproteins of Chandipura virus, Perinet virus, Piry virus, Jurona virus, or a variant thereof.

[0061] In some aspects, provided herein is a method of delivering a nucleic acid into a target cell or tissue in a subject in need thereof comprising administering to the subject a nucleic acid encapsulated within an engineered viral particle, wherein the engineered viral particle comprises: a viral fusogen comprising a modified vesiculovirus glycoprotein, comprising one or more amino acid mutations corresponding to positions 1347 or R354 of wild-type Vesicular Stomatitis Virus glycoprotein (VSV-G) of SEQ ID NO: 12.

[0062] In some embodiments, the nucleic acid encodes an immunogenic polypeptide.

[0063] In some embodiments, the administering is for therapeutic or prophylactic treatment.ATTORNEY DOCKET NO. ORB-015WO1

[0064] In some embodiments, the nucleic acid encodes a therapeutic molecule.

[0065] In some embodiments, the therapeutic molecule is an enzyme, an antibody or antigen binding fragment thereof, a chimeric antigen receptor, a T cell receptor, or a recombinant polypeptide.

[0066] In some embodiments, the subject is a human subject.

[0067] In some aspects, provided herein is use of an engineered viral particle for delivering a nucleic acid therapeutic to a cell in vivo comprising contacting the cell with a nucleic acid encapsulated within an engineered viral particle which comprises a viral glycoprotein fusogen of any one of Chandipura virus, Perinet virus, Piry virus, Jurona virus, or a variant thereof.

[0068] In some aspects, provided herein is the use of an engineered viral particle for delivering a nucleic acid therapeutic to a cell in vivo comprising: contacting the cell with a nucleic acid encapsulated within an engineered viral particle which comprises a viral fusogen comprising a modified vesiculovirus glycoprotein, comprising one or more amino acid mutations corresponding to positions 1347 or R354 of wild-type Vesicular Stomatitis Virus glycoprotein (VSV-G) of SEQ ID NO: 12.

[0069] In some embodiments, provided herein is an engineered viral particle of the present disclosure for use as a medicament.

[0070] In some embodiments, provided herein is the engineered viral particle of the present disclosure for use in oncotherapy, vaccination, immune therapy and / or gene therapy. In some embodiments, immune therapy comprises immunomodulation. In some embodiments, immune therapy comprises treating autoimmune diseases.

[0071] In some embodiments, provided herein is a vector system for producing the engineered viral particle(s) of the present disclosure, comprising one or more vectors encoding the viral glycoprotein fusogen and the targeting moiety, wherein the fusogen is a viral glycoprotein of any one of Chandipura virus, Perinet virus, Piry virus, Jurona virus, or a variant thereof.

[0072] In some embodiments, provided herein is a vector system for producing the engineered viral particle of the present disclosure, comprising one or more vectors encoding the viral glycoprotein fusogen and the targeting moiety, wherein the fusogen is a modified viral glycoprotein of a vesiculovirus comprising one or more amino acid mutations selected from the group consisting of I347N, I347Q, R354G, and R354N.

[0073] In some embodiments, the vector system comprises one vector expressing the viral glycoprotein fusogen and the targeting moiety.ATTORNEY DOCKET NO. ORB-015WO1

[0074] In some embodiments, provided herein is a host cell for producing engineered viral particle(s) comprising the vector system of the present disclosure.

[0075] In some embodiments, provided herein is a kit comprising the vector system of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The drawings are for illustration purposes only, and are not meant to be limiting.

[0077] FIG. 1A is a schematic diagram showing a traditional entry mechanism of VSV-G wild-type viral particle by binding an LDL-R on the cell surface. VSV-G fuses with the cellular membrane, and this facilitates entry of the cargo into the cell.

[0078] FIG. IB is a schematic diagram showing entry of engineered viral particles of the present disclosure comprising a targeting moiety and an engineered viral fusogen. In the first step of targeted delivery, the engineered viral particle comprising a VSV-G mutant, which lacks LDL-R binding ability, binds to the target / ligand’s receptor on the cell through a ligand-receptor interaction without binding to LDL-R. In the second step of targeted delivery, a mutant VSV-G (that does not bind LDL-R) fuses with the cellular membrane, and the payload is delivered to the cell.

[0079] FIG. 2 is a schematic showing exemplary components of a wild-type viral particle (“WT VLP”), a de-targeted viral particle, and a re-targeted viral particle (“Targeted VLP”). The wild-type viral particle exhibits pan-tropism due to binding LDL-R on diverse cell types. Mutant VSV-G lacking human cell receptor binding does not promote uptake into cells in a de-targeted viral particle. In the present disclosure, engineered viral particles that incorporate a targeting moiety (e.g., scFv, nanobody, endogenous protein) enable specific re-targeting to cells expressing the ligand’s receptor.

[0080] FIG. 3 is a graph showing the transduction efficiency of exemplary engineered viral particles comprising wild-type vesiculovirus G proteins from different viruses, and comparing it with engineered viral particles comprising the same vesiculovirus G proteins but with the addition of anti-CD5 single-chain variable fragment (scFv) as a targeting moiety and a nucleic acid encoding GFP as the cargo (“aCD5 scFv”). As a control, exemplary engineered viral particles without the anti-CD5 scFv targeting moiety (“No targeter” as shown in FIG. 3) were also prepared. Transduction efficiency was measured by the number of cells expressing Green Fluorescent Protein (GFP). Engineered viral particles comprising Chandipura, Perinet, Piry, Jurona or Isfahan viruses showed no transduction by wild-type viral particles, as theirATTORNEY DOCKET NO. ORB-015WO1glycoproteins were natural LDL-R binding mutants, but showed a high transduction efficiency when administered in a viral particle with a targeting moiety, as shown by the high percent expression of GFP+ cells.

[0081] FIG. 4 is a graph displaying the transduction efficiency of exemplary engineered viral particles comprising mutant vesiculovirus G proteins from different virus species in Jurkat cells. Engineered viral particles comprising exemplary mutant vesiculovirus G proteins showed reduced off-target receptor binding and were effectively re-targeted to T cells by the targeting moiety.

[0082] FIG. 5A shows a graph that demonstrates specific T cell targeting in the spleen by engineered viral particles comprising exemplary VSV-G mutants by the anti-CD3 targeting moiety after intravenous administration of the engineered viral particles in mice.

[0083] FIG. 5B shows a graph that demonstrates specific T-cell targeting of engineered viral particles comprising exemplary Maraba virus VSV-G mutants by the anti-CD3 targeting moiety after intravenous administration of the engineered viral particles in mice relative to B cells or other cells, for example, including macrophages, dendritic cells, and other immune cells that are not B cells or T cells.

[0084] FIG. 6 is a molecular model of a VSV-G monomer depicting the K47 and R354 residues in the LDL-R binding pocket as well as depicting other amino acid residues such as H08, 1182, Y209, and 1347.

[0085] FIG. 7 is a graph that demonstrates the change in binding free energy for various exemplary virus mutants and the basis for selection of the mutants from related viruses to the VSV Indiana strain, for example, Maraba virus, Alagoas virus, Morreton virus, and Cocal virus that showed disrupted LDL-R binding and de-targeting, as identified by a change in binding free energy.

[0086] FIG. 8A and FIG. 8B represent flow cytometry results, and showed the number of GFP+ cells, which indicated cell surface expression of exemplary engineered viral particles comprising novel VSV-G protein mutants (VSV-Gmut) after transfection of the VSV-Gmut in an exemplary producer cell line with a GFP transfer plasmid.

[0087] FIG. 9A, FIG. 9B, and FIG. 9C represent flow cytometry results showing the number of GFP+ cells, and demonstrated de-targeting using engineered viral particles comprising novel VSV-G mutants, e.g., I347N, I347Q, R354G, and R354N, in Jurkat and Raji cells relative to targeted, de-targeted and wild-type controls.

[0088] FIG. 10A shows flow cytometry results, and demonstrated that engineered viral particles comprising de-targeted VSV-G I347N and I347Q mutants were successfully re-ATTORNEY DOCKET NO. ORB-015WO1targeted in Jurkat cells by transducing with a targeting moiety. FIG. 10B shows flow cytometry results, and demonstrated that de-targeted VSV-G R354G and R354N mutants were successfully re-targeted in Jurkat cells by transducing with a targeting moiety.DEFINITIONS

[0089] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0090] Engineered viral particle: As used herein, the term “engineered viral particle” or “engineered virus particle” refers to a modified virus particle designed to deliver genetic material to cells. Engineered virus particles include particles derived from viruses, including, as non-limiting examples, adenovirus, adeno-associated virus, alphavirus, herpesvirus, arenavirus, paramyxovirus, retrovirus, e.g., lentivirus, oncolytic virus particles, or pseudotyped vesiculovirus particles. Engineered viral particles are, for example, virus-like particles that have been modified or designed for delivery of a payload, for example, a therapeutic or prophylactic protein, or a heterologous nucleic acid that encodes such a protein.

[0091] Engineered viral vector: As used herein, the term “engineered viral vector” refers to a modified virus vector, e.g., a virus-derived nucleic acid designed to deliver genetic material to cells. Adenovirus, adeno-associated virus, alphavirus, herpesvirus, arenavirus, paramyxovirus, retrovirus, e.g., lentivirus, or oncolytic virus vectors are non-limiting examples of engineered viral vectors used to deliver heterologous nucleic acids to a cell. Engineered virus vectors include a pseudotyped vesiculovirus vector. Engineered viral vectors include nucleic acid sequences engineered, for example, to produce engineered viral particles.

[0092] Fusogen: As used herein, the term “fusogen” refers to an agent or molecule (e.g. a protein) that creates an interaction between two membrane-enclosed lumens, e.g. a fusogen promotes membrane fusion, or one that creates a connection, e.g., a pore, between two lumens (e.g., the lumen of a viral particle and the cytoplasm of a target cell).

[0093] De-targeted: As used herein, the term “de-targeted” refers to a fusogen that does not bind to a human cell (e.g., does not bind to a receptor on a human cell) or lacks receptorbinding residues. For example, a naturally-occurring or wild-type fusogen that naturally does not recognize a human cell receptor, or a mutant counterpart (e.g., a VSV G-protein mutantATTORNEY DOCKET NO. ORB-015WO1comprising mutations in LDL-R binding sites) are de-targeted fusogens, which can be incorporated in a engineered viral particle.

[0094] Re-targeted: As used herein, the term “re-targeted” or “retargeted” refers to a fusogen that comprises a targeting moiety having a sequence that is not part of the naturally-occurring or wild-type fusogen. For example, a retargeted fusogen comprises a different targeting moiety relative to the targeting moiety in the naturally-occurring form of the fusogen. In some embodiments, the naturally-occurring form of the fusogen lacks a targeting domain, and the re-targeted fusogen comprises a targeting moiety that is absent from the naturally-occurring form of the fusogen. In some embodiments, the fusogen is modified to comprise a targeting moiety. In some embodiments, the fusogen comprises one or more sequence alterations outside of the targeting moiety relative to the naturally-occurring form of the fusogen, e.g., in a transmembrane domain, fusogenically active domain, or cytoplasmic domain.

[0095] Virus-like Particle: Virus-like particles (VLPs) are molecules that closely resemble viruses, but are non-infectious because they contain no viral genetic material. As used herein, the term “virus-like particle” is used interchangeably with “viral like particles.” VLPs can be naturally occurring or synthesized through the individual expression of viral structural proteins, which can then self-assemble into the virus-like particle. As used herein, VLPs are used interchangeably with “engineered viral particles.”

[0096] Selective Endogenous Encapsidation for Cellular Delivery System (SEND): As used herein, “Selective Endogenous Encapsidation for Cellular Delivery System” (SEND) is a delivery system that comprises non-naturally occurring self-assembling polypeptides for transferring nucleic acids and / or proteins to a cell, as described in Segel, M., et al., Science, 373.6557 (2021): 882-889, and US20200347100A1 which are incorporated herein in its entirety.

[0097] Nanoblade: As used herein, a “nanoblade” is a delivery system that comprises a virus-derived particle as described in Mangeot, PE., et al., Nature Communications, 10.1 (2019): 1-15, and US2021 / 0284697A1, which are incorporated herein in their entireties.DETAILED DESCRIPTION

[0098] Provided herein, among other things, are engineered viral particles and methods for targeted therapy, e.g., oncotherapy, autoimmune or infectious disease therapy, wherein the viral fusogen is a glycoprotein of any one of glycoproteins of Chandipura, Perinet, Piry,ATTORNEY DOCKET NO. ORB-015WO1Jurona viruses, or variants thereof. Also provided herein, among other things, are engineered viral particles and methods for targeted therapy for various applications, e.g., cancer therapy, immune therapy including, for example, immunomodulation and autoimmune therapy, or infectious disease therapy, wherein the viral fusogen is a modified vesiculovirus glycoprotein mutant comprising one or more amino acid mutations corresponding to positions 1347 or R354 of wild-type Vesicular Stomatitis Virus glycoprotein (VSV-G), which reduces or abolishes non-specific human cell entry.

[0099] The present invention provides, among other things, engineered viral particles comprising novel and improved glycoproteins from vesiculoviruses and methods of targeted delivery of the cargo (also known as “payload”), e.g., nucleic acids, including nucleic acids encoding therapeutic proteins, to cells. The disclosure is based, in part, on the discovery of novel and improved viral glycoproteins from novel viruses (e.g., Chandipura, Perinet, Piry, Jurona viruses) that do not bind human cell receptors but retain the ability to initiate cell membrane fusion, z.e., function as fusogens. Delivery of the engineered viral particles of the present disclosure to desired target cells (e.g., immune cells) is controlled by a targeting moiety, avoiding off-target delivery caused by non-specific glycoprotein binding. Provided herein, among other things, is an engineered viral particle comprising a viral fusogen, a targeting moiety, and at least one nucleic acid, wherein the viral fusogen is an envelope glycoprotein of any one of the glycoproteins of Chandipura virus, Perinet virus, Piry virus, and Jurona virus, or a variant thereof. Also provided herein are methods of delivering a nucleic acid into a cell comprising contacting the cell with the engineered viral particle of the present disclosure.

[0100] The present disclosure provides, in part, engineered viral particles for targeted delivery of the cargo, for example, and without limitation, a nucleic acid or therapeutic protein to cells for various applications, including oncolytic therapy, autoimmune disease therapy, or infectious disease therapy. The disclosure is based, in part, on the discovery of viral glycoproteins from viruses (e.g., Chandipura, Perinet, Piry, Jurona viruses) that do not bind human cell receptors (e.g., LDL-receptor) but retain the ability to initiate membrane fusion, z.e., function as fusogens. The disclosure provides, among other things, engineered viral particles comprising Chandipura, Perinet, Piry, or Jurona vesiculoviruses, or variants thereof, or comprising viral glycoproteins from said viruses. The discovery of viral glycoproteins from other viruses that naturally do not bind human cell receptors provides safe delivery of the cargo without off-target effects, and also has advantages over known mutations of VSV-G, such as being able to be produced to a high-titer, being less sensitive toATTORNEY DOCKET NO. ORB-015WO1complement inactivation and, thus, more stable as well as safe and showing improved efficacy with fewer off-target effects.

[0101] The present disclosure also provides, in part, engineered viral particles comprising novel mutations or mutations at novel residues of VSV Indiana and related viruses for LDL-receptor binding in the Vesicular Stomatitis Virus glycoprotein (VSV-G), for example, I347N, I347Q, R354G, and / or R354N, and combinations thereof, that yield a sufficiently high titer of vesiculovirus for commercial-scale manufacture and administration, and that also yield stable engineered viral particles that are less susceptible to complement inactivation.

[0102] In another aspect, the present disclosure provides use of an engineered viral particle for delivering a nucleic acid therapeutic to a cell in vivo comprising contacting the cell with a nucleic acid encapsulated within an engineered viral particle which comprises a viral glycoprotein fusogen of any one of Chandipura virus, Perinet virus, Piry virus, Jurona virus, or a variant thereof.

[0103] In another aspect, the present disclosure provides use of an engineered viral particle for delivering a nucleic acid therapeutic to a cell in vivo comprising contacting the cell with a nucleic acid encapsulated within an engineered viral particle which comprises a viral glycoprotein fusogen, comprising a modified glycoprotein comprising one or more amino acid mutations selected from the group consisting of I347N, I347Q, R354G, and R354N.Engineered Viral ParticlesVesiculoviruses

[0104] Vesicular stomatitis virus (VSV), Chandipura virus, Perinet virus, Piry virus, Jurona virus, are all non-limiting examples of vesiculoviruses. Vesiculoviruses are ubiquitous viruses that infect both animal and human cells. They belong to the rhabdovirus family and mononegavirales order, which are enveloped and contain a non- segmented, single (mono) strand of negative sense genomic RNA. Vesiculoviruses have bullet-shaped virions encapsulating a simple and efficient 11 kb to 11.3 kb genome encoding five viral proteins, nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large protein (L), in order, from the 3' genomic end. There is a small 47-50 nucleotide (nt) leader (1) sequence transcribed from the 3' end, and a 57-60 nucleotide trailer sequence at the 5' end involved in viral replication. Besides the 60-nucleotide trailer, there are two non-transcrib ed nucleotides at each of the four gene junctions and three non-coding nucleotides at the 1-NATTORNEY DOCKET NO. ORB-015WO1junction, adding up to about 70 non-transcribed nucleotides in the vesiculovirus genome. The four non-coding gene junctions separating the five mRNAs contain a 23 nucleotide conserved sequence: 3'-AUACUUUUUUUNNUUGUCNNUAG-5' (SEQ ID NO: 72) that functions as polyadenylation signal terminating each cistron, signals the start of the next mRNA species, and templates the capped mRNA 5' end (5'-m7Gppp(m)Am-A(m)CAGNNAUC-3' (SEQ ID NO: 73)). Within the P protein, two small highly basic proteins, C and C9, are encoded in a second open reading frame (ORF) of most vesiculoviruses.

[0105] Transcription begins at the 3' end of the genome. The first transcript is the 3' leader, which is neither capped nor polyadenylated but transported to the nucleus where it inhibits host cell transcription. The leader transcript is followed by the N mRNA, which is capped during synthesis by the virion polymerase complex (composed of N, P and L).Proteins necessary in larger amounts (such as N) are located near the 3 '-end and are transcribed in larger amounts, and those needed in smaller amounts are located towards the 5 '-end and are transcribed less frequently, providing an efficient way of regulating gene expression. An intergenic sequence (5'-AGUUUUUUUCAUA-3' (SEQ ID NO: 74)) signals polyadenylation, termination, and re-initiation of transcription in decreasing amounts as the polymerase complex moves away from the transcription initiation site.

[0106] Following translation of mRNAs to yield viral proteins, genome replication is initiated by the L protein at the 3 '-end of the genome and a full-length complementary antigenome is synthesized, serving as a template for the synthesis of a 45-nt minus sense leader RNA and full-length progeny genomes, which can further serve as templates for transcription or be assembled into viral particles. Following replication, further rounds of transcription (secondary transcription), translation and replication continue.

[0107] In some aspects, provided herein is an engineered viral particle comprising a viral fusogen, a targeting moiety and at least one nucleic acid, wherein the viral fusogen is a glycoprotein of any one of glycoproteins of Chandipura virus, Perinet virus, Piry virus, Jurona virus, or a variant thereof.

[0108] In some embodiments, the one or more amino acid mutations decreases the binding of the engineered viral particle to Low Density Lipoprotein Receptor (LDL-R).

[0109] In some embodiments, the viral fusogen is a glycoprotein of Chandipura virus.

[0110] In some embodiments, the viral fusogen is a glycoprotein of Perinet virus.[oni] In some embodiments, the viral fusogen is a glycoprotein of Piry virus.

[0112] In some embodiments, the viral fusogen is a glycoprotein of Jurona virus.ATTORNEY DOCKET NO. ORB-015WO1

[0113] In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 1-10. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 6. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10.

[0114] In some embodiments, the glycoprotein comprises a sequence of any one of SEQ ID NO: 1-10. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 1. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 2. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 3. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 4. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 5. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 6. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 7. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 8. In someATTORNEY DOCKET NO. ORB-015WO1embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 9. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 10.

[0115] In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 11.

[0116] In some embodiments, the glycoproteins in this disclosure are Vesiculovirus glycoproteins lacking LDL-R binding, as shown in Table 1, or variants thereof.Table 1. Vesiculovirus glycoproteins lacking LDL-R bindingATTORNEY DOCKET NO. ORB-015WO1ATTORNEY DOCKET NO. ORB-015WO1LDL Receptor

[0117] Low-density lipoprotein receptor (LDL-R) and other members of the receptor family are ubiquitous VSV receptors (Finkelshtein et al., 2013). The LDL-R gene family consists of transmembrane receptors that are involved in endocytic uptake of lipoproteins, and require Ca2+for ligand binding. All these receptors have in common several CR repeats (up to several tens), EGF precursor-like repeats, a membrane-spanning region, and an intracellular domain containing at least one internalization signal sequence.

[0118] The LDL-R is a type I transmembrane protein that regulates cholesterol homeostasis in mammalian cells (Brown and Goldstein, 1986) by removing cholesterol carrying lipoproteins from serum. At neutral pH, ligands bind LDL-R, which are subsequently internalized and then released in the acidic environment of the endosomes leading to lysosomal degradation. The receptor then recycles back to the cell surface. LDL-R ectodomain is composed of a ligand-binding domain, an epidermal growth factor (EGF) precursor homology domain, and a C-terminal domain enriched in O-linked oligosaccharides. The ligand binding domain is made of 7 cysteine-rich repeats (CR1 to CR7), and each repeat consists of approximately 40 amino acids and contains 6 cysteine residues, engaged in 3 disulfide bridges, and an acidic residues cluster that coordinates a Ca2+ion. The intracellular release of the cargo is driven by a low-pH-induced conformational change of LDL-R from an open to a closed conformation.

[0119] Viral glycoproteins, for example, vesiculovirus glycoproteins, including Vesicular Stomatitis Virus glycoprotein, are one example of a ligand that binds LDL-R family receptors for entry into mammalian cells.Cell-specific Surface Glycoproteins / Fusogens

[0120] In some embodiments, the targeting moiety on the surface of the vesiculovirus is a glycoprotein. Glycoproteins (G proteins) of vesicular stomatitis virus (VSV) and related rhabdoviruses (e.g., rabies virus) carry out cell attachment and membrane fusion. Vesicular stomatitis virus (VSV) enters cells via endocytosis. At low pH, glycoprotein G catalyzes fusion between viral and endosomal membranes undergoing a conformational change from a pre-fusion trimeric conformation to a post-fusion trimeric conformation. The fusogenic conformation is reversible, in contrast to many other low pH-induced viral fusion proteins.ATTORNEY DOCKET NO. ORB-015WO1For example, VSV glycoprotein mutants bearing mutations in the low density lipoprotein receptor (LDL-R) binding sites, K47 and R354, do not bind human LDL-R and are fusogens used for retargeting engineered viral particles. VSV-G also has a low seroprevalence in humans and facilitates high-titer engineered viral particle.

[0121] In some embodiments of the present disclosure, the glycoprotein is a Chandipura virus glycoprotein G (CNV-G). In some embodiments, the glycoprotein is a Perinet virus glycoprotein. In some embodiments, the glycoprotein is a Piry virus glycoprotein. In some embodiments, the glycoprotein is a Jurona virus glycoprotein.

[0122] The glycoproteins of Chandipura, Perinet, Piry, Jurona virus, and variants thereof are highly selective towards specific cell types, e.g., primary cells and tumor cells. Notably, cellular entry is independent of the LDL-R.Modified Vesiculovirus Glycoproteins

[0123] In some aspects, provided herein is an engineered viral particle comprising a viral fusogen, a targeting moiety, and at least one nucleic acid, wherein the viral fusogen comprises a modified vesiculovirus glycoprotein, comprising one or more amino acid mutations corresponding to positions 1347 or R354 of wild-type Vesicular Stomatitis Virus glycoprotein (VSV-G) of SEQ ID NO: 12.

[0124] In some aspects, provided herein is a modified vesiculovirus G protein, comprising one or more amino acid mutations at residues selected from the group consisting of 1347 and R354. In some aspects, provided herein is a modified vesiculovirus envelope glycoprotein (VSV-G), comprising an amino acid mutation at 1347. In some aspects, provided herein is a modified vesiculovirus envelope glycoprotein (VSV-G), comprising an amino acid mutation at R354. In some aspects, provided herein is a modified vesiculovirus envelope glycoprotein (VSV-G), comprising an amino acid mutation at 1347 and R354. In some aspects, provided herein is a modified vesiculovirus envelope glycoprotein (VSV-G), comprising an amino acid mutation at K47 and 1347. In some aspects, provided herein is a modified vesiculovirus envelope glycoprotein (VSV-G), comprising an amino acid mutation at K47 and R354. In some aspects, provided herein is a modified vesiculovirus envelope glycoprotein (VSV-G), comprising an amino acid mutation at K47, 1347 and R354.

[0125] In some embodiments, the modified vesiculovirus glycoprotein is a modified Vesicular Stomatitis Virus glycoprotein (VSV-G). In some aspects, provided herein is a modified Vesicular Stomatitis Virus envelope glycoprotein (VSV-G), comprising one or more amino acid mutations at residues selected from the group consisting of 1347 and R354.ATTORNEY DOCKET NO. ORB-015WO1In some aspects, provided herein is a modified Vesicular Stomatitis Virus envelope glycoprotein (VSV-G), comprising an amino acid mutation at 1347. In some aspects, provided herein is a modified Vesicular Stomatitis Virus envelope glycoprotein (VSV-G), comprising an amino acid mutation at R354. In some aspects, provided herein is a modified Vesicular Stomatitis Virus envelope glycoprotein (VSV-G), comprising an amino acid mutation at 1347 and R354. In some aspects, provided herein is a modified Vesicular Stomatitis Virus envelope glycoprotein (VSV-G), comprising an amino acid mutation at K47 and 1347. In some aspects, provided herein is a modified Vesicular Stomatitis Virus envelope glycoprotein (VSV-G), comprising an amino acid mutation at K47 and R354. In some aspects, provided herein is a modified Vesicular Stomatitis Virus envelope glycoprotein (VSV-G), comprising an amino acid mutation at K47, 1347 and R354.

[0126] In some embodiments, the mutation is at a position corresponding to 1347 or R354 of VSV-G in another vesiculovirus. Some non-limiting examples are provided in Table 3 herein.

[0127] In some embodiments, the modified vesiculovirus glycoprotein is a modified Morreton, Cocal, Alagoas, Carajas, New Jersey, or Maraba virus glycoprotein. In some embodiments, the modified vesiculovirus glycoprotein is a modified Morreton virus glycoprotein. In some embodiments, the modified vesiculovirus glycoprotein is a modified Cocal virus glycoprotein. In some embodiments, the modified vesiculovirus glycoprotein is a modified Alagoas virus glycoprotein. In some embodiments, the modified vesiculovirus glycoprotein is a modified Carajas virus glycoprotein. In some embodiments, the modified vesiculovirus glycoprotein is a modified New Jersey virus glycoprotein. In some embodiments, the modified vesiculovirus glycoprotein is a modified Maraba virus glycoprotein.

[0128] In some embodiments, the one or more amino acid mutations are selected from the group consisting of I347N, I347Q, R354G, and R354N.

[0129] In some embodiments, the amino acid mutation is I347N or I347Q.

[0130] In some embodiments, the amino acid mutation is R354G or R354N.

[0131] In some embodiments, the viral glycoprotein comprises a sequence of any one of SEQ IDNOs: 13-71.

[0132] In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are selected from the group consisting of I347N, I347Q, R354G, and R354N. In some embodiments, the modified VSV glycoprotein contains one or more amino acid substitutions selected from the group consisting of I347N, I347Q,ATTORNEY DOCKET NO. ORB-015WO1R354G, and R354N. In some embodiments, the 1347 mutation comprises any amino acid equivalent to asparagine or glutamine or modified forms of asparagine or glutamine. In some embodiments, the R354 mutation comprises any amino acid equivalent to asparagine or glycine or modified forms of asparagine or glycine. In some embodiments, the R354 mutation comprises an alanine.

[0133] In some embodiments, the amino acid mutation is I347N or I347Q. In some embodiments, the amino acid mutation is I347N. In some embodiments, the amino acid mutation is I347Q.

[0134] In some embodiments, the amino acid mutation is R354G or R354N. In some embodiments, the amino acid mutation is R354G. In some embodiments, the amino acid mutation is R354N.

[0135] In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are I347N and R354G. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are I347N and R354N. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are I347Q and R354G. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are I347Q and R354N.

[0136] In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47A and R354G. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47Q and R354G. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47A and R354N. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47A and R354G.

[0137] In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47A and I347N. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47Q and I347N. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47A and I347N. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47A and I347Q.

[0138] In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47Q, I347N and R354G. In some embodiments,ATTORNEY DOCKET NO. ORB-015WO1provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47Q, I347N and R354N. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47Q, I347Q and R354G. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47Q, I347Q and R354N.

[0139] In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47A, I347N and R354G. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47A, I347N and R354N. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47A, I347Q and R354G. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47A, I347Q and R354N.

[0140] In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47Q, I347N and R354Q. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47A, I347N and R354Q. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47Q, I347Q and R354Q. In some embodiments, provided herein is a modified VSV glycoprotein wherein the one or more amino acid mutations are K47A, I347Q and R354Q.

[0141] In some embodiments, the viral glycoprotein fusogen lacks human low density lipoprotein (LDL)-receptor binding.

[0142] In some embodiments, the glycoprotein comprises a sequence having at least 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 13-71. In some embodiments, the glycoprotein comprises a sequence having at least 95% identity to SEQ ID NOs: 13-71. In some embodiments, the glycoprotein comprises a sequence having at least 96% identity to SEQ ID NOs: 13-71. In some embodiments, the glycoprotein comprises a sequence having at least 97% identity to SEQ ID NOs: 13-71. In some embodiments, the glycoprotein comprises a sequence having at least 98% identity to SEQ ID NOs: 13-71. In some embodiments, the glycoprotein comprises a sequence having at least 99% identity to SEQ ID NOs: 13-71.

[0143] In some embodiments, the glycoprotein comprises a sequence of any one of SEQ ID NOs: 13-71. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 13. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 14. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 15. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 16. In someATTORNEY DOCKET NO. ORB-015WO1embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 17. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 18. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 19. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 20. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 21. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 22. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 23. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 24. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 25. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 26. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 27. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 28. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 29. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 30. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 31. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 32. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 33. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 34. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 35. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 36. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 37. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 38. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 39.In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 40.

[0144] In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 41. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 42. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 43. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 44. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 45. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 46. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 47. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 48. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 49. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 50. In someATTORNEY DOCKET NO. ORB-015WO1embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 51. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 52. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 53. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 54. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 55. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 56. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 57. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 58. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 59. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 60. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 61. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 62. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 63. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 64. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 65. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 66. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 67. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 68. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 69. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 70. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 71.

[0145] In some embodiments, provided herein are modified VSV-G proteins as shown in Table 2 herein, or variants thereof.

[0146] Table 2. Modified VSV-G glycoprotein sequencesATTORNEY DOCKET NO. ORB-015WO1ATTORNEY DOCKET NO. ORB-015WO1ATTORNEY DOCKET NO. ORB-015WO1ATTORNEY DOCKET NO. ORB-015WO1ATTORNEY DOCKET NO. ORB-015WO1

[0147] In some embodiments, provided herein is a Vesicular Stomatitis Virus variant comprising the modified VSV glycoprotein of the present disclosure. In some embodiments, provided herein are vesiculovirus variants, for example, among others, Alagoas virus, Carajas virus, Cocal virus, Indiana virus, Maraba virus, Morreton virus, and New Jersey virus variants, comprising modified vesiculovirus glycoproteins. Some non-limiting examples of such variants are provided in Table 3.

[0148] Table 3. Exemplary Vesiculovirus G protein mutantsATTORNEY DOCKET NO. ORB-015WO1ATTORNEY DOCKET NO. ORB-015WO1ATTORNEY DOCKET NO. ORB-015WO1ATTORNEY DOCKET NO. ORB-015WO1Virus-like Particles (VLPs)ATTORNEY DOCKET NO. ORB-015WO1Virus-like particles (VLPs) are self-assembled viral proteins that have a virion-like structure, but are non-infectious, as they lack an intact viral genome so are unable to replicate or infect the host. Engineered Viral Particles are modified VLPs that are very useful in delivering nucleic acids or proteins, for example, therapeutic nucleic acids or proteins. Using VSV G-protein is a known method for synthesizing VLPs. The present disclosure provides improved viral glycoproteins from newly-identified viruses (e.g., Chandipura, Perinet, Piry, Jurona viruses, or a variant thereof) or novel VSV-G mutants or sites (e.g., I347N, I347Q, R354G, and / or R354N, and combinations thereof) that do not bind human cell receptors but retain fusogenic ability to provide engineered viral particles with improved specificity and targeting efficacy as well as fewer off-target effects, providing a safe and efficacious delivery system.

[0149] In some embodiments of the engineered viral particle, the nucleic acid encodes a polypeptide.

[0150] In some embodiments, the polypeptide is a therapeutic or immunogenic polypeptide. In some embodiments, the polypeptide is a therapeutic polypeptide. In some embodiments, the polypeptide is an immunogenic polypeptide.

[0151] In some embodiments, the nucleic acid encodes a therapeutic molecule. In some embodiments, the therapeutic molecule is an enzyme, an antibody or antigen binding fragment thereof, a chimeric antigen receptor, a T cell receptor, or a recombinant polypeptide. In some embodiments, the therapeutic molecule is an enzyme. In some embodiments, the therapeutic molecule is an antibody or antigen binding fragment thereof. In some embodiments, the therapeutic molecule is an antibody. In some embodiments, the therapeutic molecule is an antigen binding fragment thereof. In some embodiments, the therapeutic molecule is a chimeric antigen receptor (CAR). In some embodiments, the therapeutic molecule is a T cell receptor. In some embodiments, the therapeutic molecule is a recombinant polypeptide.

[0152] In some embodiments, the immunogenic polypeptide is derived from an infectious pathogen. In some embodiments, the immunogenic polypeptide comprises a cancer antigen.

[0153] In some embodiments, the nucleic acid is a non-coding polynucleotide.

[0154] In some aspects, provided herein is an engineered viral particle comprising a viral fusogen, a targeting moiety, and at least one nucleic acid, wherein the viral fusogen is an envelope glycoprotein of any one of glycoproteins of Chandipura virus, Perinet virus, Piry virus, Jurona virus, or a variant thereof. In some embodiments, the viral fusogen is an envelope glycoprotein of Chandipura virus, or a variant thereof. In some embodiments, the viral fusogen is an envelope glycoprotein of Perinet virus, or a variant thereof. In someATTORNEY DOCKET NO. ORB-015WO1embodiments, the viral fusogen is an envelope glycoprotein of Piry virus, or a variant thereof. In some embodiments, the viral fusogen is an envelope glycoprotein of Jurona virus, or a variant thereof.

[0155] In some embodiments, the viral glycoprotein fusogen does not bind human LDL-receptor.

[0156] In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 1-10 and 13-71. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 1-10. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 6. In some embodiments, the glycoprotein comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9. In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10.

[0157] In some embodiments, the glycoprotein comprises a sequence of any one of SEQ ID NO: 1-10. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 1. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 2. In someATTORNEY DOCKET NO. ORB-015WO1embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 3. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 4. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 5. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 6. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 7. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 8. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 9. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 10.

[0158] In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 11.

[0159] In some embodiments, the glycoprotein comprises a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12. In some embodiments, the glycoprotein comprises a sequence of SEQ ID NO: 12. In some aspects, provided herein is an engineered virus particle comprising a vesiculovirus, a targeting moiety, and at least one nucleic acid, wherein the targeting moiety is on the surface of the engineered viral particle, and wherein the vesiculovirus is a Chandipura virus, a Perinet virus, a Piry virus, or a Jurona virus, or a variant thereof.

[0160] In some embodiments, the vesiculovirus is Chandipura virus, or a variant thereof.

[0161] In some embodiments, the vesiculovirus is Perinet virus, or a variant thereof.

[0162] In some embodiments, the vesiculovirus is Piry virus, or a variant thereof.

[0163] In some embodiments, the vesiculovirus is Jurona virus, or a variant thereof.

[0164] In some embodiments, the engineered viral particle is a pseudotyped viral particle.

[0165] In some embodiments, the engineered viral particle is a pseudotyped vesiculovirus, retrovirus, alphavirus, herpesvirus, arenavirus, paramyxovirus, lentivirus or oncolytic virus. In some embodiments, the engineered viral particle is a pseudotyped viral particle. In some embodiments, the pseudotyped viral particle is a pseudotyped vesiculovirus, retrovirus, alphavirus, herpesvirus, arenavirus, paramyxovirus, lentivirus or oncolytic virus. In some embodiments, the pseudotyped viral particle is a pseudotyped vesiculovirus. In some embodiments, the pseudotyped viral particle is a pseudotyped retrovirus. In some embodiments, the pseudotyped viral particle is a pseudotyped alphavirus. In some embodiments, the pseudotyped viral particle is a pseudotyped herpesvirus. In some embodiments, the pseudotyped viral particle is a pseudotyped arenavirus. In someATTORNEY DOCKET NO. ORB-015WO1embodiments, the pseudotyped viral particle is a pseudotyped paramyxovirus. In some embodiments, the pseudotyped viral particle is a pseudotyped lentivirus. In some embodiments, the engineered viral particle is a pseudotyped oncolytic virus.

[0166] In some aspects, provided herein is an engineered viral particle comprising a vesiculovirus, a targeting moiety, and at least one nucleic acid, wherein the targeting moiety is on the surface of the engineered viral particle, and wherein the vesiculovirus is a vesiculovirus variant of the present disclosure comprising a modified vesiculovirus glycoprotein mutated in an LDL-R binding site.

[0167] In some embodiments, the vesiculovirus is a Vesicular Stomatitis Virus (VSV) variant.

[0168] In some embodiments, the vesiculovirus is an Alagoas virus variant, a Carajas virus variant, a Cocal virus variant, an Indiana virus variant, a Maraba virus variant, a Morreton virus variant, or a New Jersey virus variant.

[0169] In some embodiments, the vesiculovirus is an Alagoas virus variant. In some embodiments, the Alagoas virus variant comprises a modified glycoprotein comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 45-47. In some embodiments, the Alagoas virus variant comprises a modified glycoprotein comprising the sequence of any one of SEQ ID NO: 45-47.

[0170] In some embodiments, the vesiculovirus is a Carajas virus variant. In some embodiments, the Carajas virus variant comprises a modified glycoprotein comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 48-51. In some embodiments, the Carajas virus variant comprises a modified glycoprotein comprising the sequence of any one of SEQ ID NO: 48-51.

[0171] In some embodiments, the vesiculovirus is a Cocal virus variant. In some embodiments, the Cocal virus variant comprises a modified glycoprotein comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 52-55. In some embodiments, the Cocal virus variant comprises a modified glycoprotein comprising the sequence of any one of SEQ ID NO: 52-55.

[0172] In some embodiments, the vesiculovirus is an Indiana virus variant. In some embodiments, the Indiana virus variant comprises a modified glycoprotein comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,ATTORNEY DOCKET NO. ORB-015WO196%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 56-59. In some embodiments, the Indiana virus variant comprises a modified glycoprotein comprising the sequence of any one of SEQ ID NO: 56-59.

[0173] In some embodiments, the vesiculovirus is a Maraba virus variant. In some embodiments, the Maraba virus variant comprises a modified glycoprotein comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 60-63. In some embodiments, the Maraba virus variant comprises a modified glycoprotein comprising the sequence of any one of SEQ ID NO: 60-63.

[0174] In some embodiments, the vesiculovirus is a Morreton virus variant. In some embodiments, the Morreton virus variant comprises a modified glycoprotein comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 64-67. In some embodiments, the Morreton virus variant comprises a modified glycoprotein comprising the sequence of any one of SEQ ID NO: 64-67.

[0175] In some embodiments, the vesiculovirus is a New Jersey virus variant. In some embodiments, the New Jersey virus variant comprises a modified glycoprotein comprising a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO: 68-71. In some embodiments, the New Jersey virus variant comprises a modified glycoprotein comprising the sequence of any one of SEQ ID NO: 68-71.

[0176] In some embodiments, the present invention provides a composition comprising an engineered viral particle described herein. In some embodiments, the present invention provides a cell comprising an engineered viral particle described herein. In some embodiments, the present invention provides a targeted delivery vehicle comprising an engineered viral particle described herein.Targeted Delivery VehicleTargeting moiety

[0177] In some embodiments, the engineered viral particle comprises a targeting moiety specific for a target cell of interest. In some embodiments, the targeting moiety is an antibody or antigen binding fragment thereof. In some embodiments, the targeting moiety is an antibody or an antigen binding fragment thereof that binds to a cell surface marker.ATTORNEY DOCKET NO. ORB-015WO1

[0178] In some embodiments, the targeting moiety is a ligand. In some embodiments, the targeting moiety is a ligand that binds a receptor on a cell surface.

[0179] In some embodiments, the targeting moiety is on the surface of the engineered viral particle. In some embodiments, the targeting moiety is on the surface of the vesiculovirus. In some embodiments, the targeting moiety is on the surface of the pseudotyped viral particle.

[0180] In some embodiments, the targeting moiety is immune cell specific. In some embodiments, the immune cell is a T cell, a B cell or a natural killer (NK) cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a natural killer (NK) cell.

[0181] In some embodiments, the targeting moiety binds to a cell surface marker on the target cell of interest. In some embodiments, the targeting moiety binds to a receptor on the cell surface of the target cell of interest.

[0182] In some embodiments, the targeting moiety binds to a cell surface marker on the immune cell. In some embodiments, the targeting moiety binds to a receptor on the cell surface of the immune cell. In some embodiments, the targeting moiety binds to any immune cell surface marker or receptor commonly known in the art.

[0183] In some embodiments, the targeting moiety binds to a cell surface marker on a T cell. Exemplary markers on T cells include, but are not limited to, CD3, TCRa, TCRP, TCRy, TCRi ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD40, TIM1, SLAM, CD2, CD5 and CD266. In some embodiments, the targeting moiety binds to any T cell surface marker or receptor commonly known in the art.

[0184] In some embodiments, the targeting moiety binds to a cell surface marker on a B cell. Exemplary markers on B cells include, but are not limited to, CD2, CD5, CD7, CD8, CD 19, CD20, CD21, CD22, CD23, CD37, CD40, CD40L, CD52, CD80, and HLA-DR. In some embodiments, the targeting moiety binds to any B cell surface marker or receptor commonly known in the art.

[0185] In some embodiments, the targeting moiety binds to a cell surface marker on an NK cell. Exemplary markers on NK cells include, but are not limited to, NKp30, NKp40, NKp44, NKp46, NKG2D, DNAM1, DAP10, CD16, CRTAM, CD27, PSGL1, CD96, CD 100, NKp80, CD244, SLAMF6, SLAMF7, KIR2DS2, KIR3DS1, KIR2DS5, KIR2DS1, CD94, NKG2C, NKG2E, and CD 160. In some embodiments, the targeting moiety binds to any NK cell surface marker or receptor commonly known in the art.Targeting AntibodyATTORNEY DOCKET NO. ORB-015WO1

[0186] In some embodiments, the engineered viral particle comprises a targeting antibody or antigen binding fragment thereof. In some embodiments, the targeting antibody or antigen binding fragment thereof is on the surface of the engineered viral particle. In some embodiments, the targeting antibody or antigen binding fragment thereof is on the surface of the vesiculovirus. In some embodiments, the targeting antibody or antigen binding fragment thereof is on the surface of the pseudotyped viral particle.

[0187] In some embodiments, the targeting antibody or antigen binding fragment thereof comprises a Fab, a Fab’, a F(ab’)2, an Fd, an Fc, single chain antibody (e.g., a scFv), a single domain antibody (e.g., a VHH), a complementary determining region, a maxibody, a minibody, an intrabody, a diabody (e.g., bivalent and / or bispecific), a triabody, a tetrabody, or a chimeric e.g., humanized) antibody. In some embodiments, the targeting antibody or antigen binding fragment thereof comprises a Fab. In some embodiments, the targeting antibody or antigen binding fragment thereof comprises a Fab.’ In some embodiments, the targeting antibody or antigen binding fragment thereof comprises a F(ab’)2. In some embodiments, the targeting antibody or antigen binding fragment thereof comprises an Fd. In some embodiments, the targeting antibody or antigen binding fragment thereof comprises an Fc. In some embodiments, the targeting antibody or antigen binding fragment thereof comprises single chain antibody (e.g., a scFv). In some embodiments, the targeting antibody or antigen binding fragment thereof comprises a single domain antibody (e.g., a VHH). In some embodiments, the targeting antibody or antigen binding fragment thereof comprises a complementary determining region. In some embodiments, the targeting antibody or antigen binding fragment thereof comprises a maxibody. In some embodiments, the targeting antibody or antigen binding fragment thereof comprises a minibody. In some embodiments, the targeting antibody or antigen binding fragment thereof comprises an intrabody. In some embodiments, the targeting antibody or antigen binding fragment thereof comprises a diabody (e.g., bivalent and / or bispecific). In some embodiments, the targeting antibody or antigen binding fragment thereof comprises a triabody. In some embodiments, the targeting antibody or antigen binding fragment thereof comprises a tetrabody. In some embodiments, the targeting antibody or antigen binding fragment thereof comprises a chimeric (e.g., humanized) antibody.

[0188] In some embodiments, the engineered viral particle comprises a targeting moiety that binds a receptor on the surface of a targeted cell and enters the targeted cell.

[0189] Engineered viral particle targeting moieties provide specific cellular targeting, reduce immune responses and facilitate extravasation. Most ligands are incorporated intoATTORNEY DOCKET NO. ORB-015WO1engineered viral particles through covalent attachments to native or non-natural amino acids, although P22 can display ligands through non-covalent bonds. Various exemplary ligands include, but are not limited to, Green Fluorescent Protein (GFP), flagellin, transferrin, DNA aptamers, granulocyte macrophage colony stimulating factor, PEG, HIV-Tat cell-penetrating peptide, glycans, foreign epitopes, peptide tags, human epidermal growth factor, ganglioside GM2 tumor-associated carbohydrate antigen, metalloporphyrin derivative, CD47 selfpeptide, CD40L, MIANS (fluorescent probe), S9 peptide, alkynes, biotin, RGD peptide (integrin-binding), pan-bombesin analogue (with fluorescent probes and PEG), folic acid-PEG, among others.Cargo

[0190] In some embodiments, the present invention provides engineered viral particles for targeted delivery of a cargo to a target cell.

[0191] In some embodiments, the cargo comprises at least one nucleic acid. In some embodiments, the cargo comprises at least one polypeptide. In some embodiments, the cargo comprises at least one nucleic acid and at least one polypeptide. In some embodiments, the cargo is a nucleic acid, for example, linear RNA, such as mRNA, or circular RNA, for example, encoding a chimeric antigen receptor (CAR) or an antigen. Incorporated herein by reference in entirety is the disclosure in patent applications PCT / US24 / 48786, PCT / US25 / 33446, PCT / US25 / 34866, 63 / 763,317 and 63 / 846,699 by Applicants including, among other things, disclosure related to circular RNA, including but not limited to design elements and methods of circularization. In some embodiments, the cargo is linear RNA. In some embodiments, the cargo is messenger RNA. In some embodiments, the cargo is circular RNA. In some embodiments, the VLP is used in a SEND system. SEND is ‘Selective Endogenous Encapsidation for cellular delivery’ which uses humanized VLPs to deliver exogenous mRNA cargo, for example, Cre and Cas9. Integrated long terminal repeat (LTR) retroelements, in particular, gag homologs prevalent in the human genome, e.g., Arc or PEG10 form capsid-like particles and bind mRNA. In some embodiments, SEND-VLPs are comprised of optimized mouse or human PEG10, VSV-G, and the cargo mRNA containing the relevant parts of the PEG10 UTRs, for example, in a modular set of three plasmids.Alternatively, a non-viral fusogen, for example, mouse fusogenic syncytin gene SYNA (evolved from a retroviral envelope gene) is used. In some embodiments, a viral fusogen of the present disclosure that does not bind LDL-R is used, e.g., Chandipura, Perinet, Piry, Jurona, or a variant thereof, or a modified vesiculovirus glycoprotein comprising one or moreATTORNEY DOCKET NO. ORB-015WO1amino acid mutations corresponding to I347N, I347Q, R354G, and R354N of Vesicular Stomatitis Virus (VSV) glycoprotein.

[0192] In some embodiments, the nucleic acid encodes a chimeric antigen receptor (CAR). In some embodiments, the nucleic acid encodes an antigen.

[0193] In some embodiments, the nucleic acid encodes a polypeptide. In some embodiments, the cargo is a polypeptide.

[0194] In some embodiments, the nucleic acid targets a repressor of a signaling pathway, gene, or nucleic acid, or represses a signaling pathway, gene, or nucleic acid. In some embodiments, the nucleic acid encodes a transcription factor that upregulates or downregulates a signaling pathway, gene, or nucleic acid. In some embodiments, the activator or repressor is a nuclease-inactive cas9 (dCas9) linked to a transcriptional activator or repressor that is targeted to the signaling pathway, gene, or nucleic acid by a guide RNA. In some embodiments, a genetic modification epigenetically modifies expression of an endogenous signaling pathway, gene, or nucleic acid. In some embodiments, the epigenetic activator is a nuclease-inactive cas9 (dCas9) linked to a epigenetic modifier that is targeted to the signaling pathway, gene, or nucleic acid by a guide RNA. In some embodiments, a cell’s DNA is edited prior to generating the fusosome to alter (e.g., upregulate or downregulate) the expression of signaling pathways (e.g., the Wnt / Beta-catenin pathway), gene, or nucleic acid. In some embodiments, the DNA is edited using a guide RNA and CRISPR-Cas9 / Cpfl or other gene editing technology.

[0195] In some embodiments, a cargo is chemically conjugated to, loaded onto the exterior or interior of, or encapsulated within Virus Like Particles (VLPs). VLPs with tolerance to domain insertions, whether at the N-terminus, C-terminus, or both termini or within the polypeptide, are useful for inserting chemically reactive handles, adjuvants, or binding moieties into the VLP.

[0196] In some embodiments, engineered protein-protein interactions are used, often in tandem with a domain insertion, to associate cargo with a VLP. For example, the SpyCatcher / SpyTag system creates isopeptide bonds between appended for cargo-attachment to the interior and exterior of VLPs.

[0197] VLPs are used in drug delivery (e.g., paclitaxel, doxorubicin), delivery of proteins (e.g., epitopes), nucleic acids (e.g., siRNA, mRNA, circular RNA), enzymes (e.g., AADC), and as vaccines (e.g., against malaria). Besides therapeutics, they are also used in delivery of cargo for diagnostics (e.g., markers, tracers, etc.).ATTORNEY DOCKET NO. ORB-015WO1Methods of Use in Targeted Delivery

[0198] Specific cell targeting is used to direct effective uptake of a therapeutic and / or diagnostic agent by a specific cell type, for example, a tumor in oncotherapy. Targeting is achieved through proteins, for example, antibodies, peptides, nucleic acids, for example, aptamers, small molecules, vitamins and carbohydrates. By attaching targeting moieties to de-targeted VLPs which lack the ability to bind to human cell receptors, for example, LDL-R, specificity for targeting is achieved by receptor mediated endocytosis, providing a safe and efficacious therapeutic method while minimizing off-target effects.

[0199] In some aspects, provided herein is a method of delivering a nucleic acid into a cell comprising contacting the cell with the engineered viral particle of the present disclosure.

[0200] In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a natural killer (NK) cell.

[0201] In some aspects, provided herein is a method of delivering a nucleic acid into a target cell or tissue in a subject in need thereof comprising administering to the subject an engineered viral particle, wherein the engineered viral particle comprises a viral glycoprotein fusogen of any one of glycoproteins of Chandipura virus, Perinet virus, Piry virus, Jurona virus, or variant thereof. In some embodiments, the viral glycoprotein fusogen is of Chandipura virus, or variant thereof. In some embodiments, the viral glycoprotein fusogen is of Perinet virus, or variant thereof. In some embodiments, the viral glycoprotein fusogen is of Piry virus, or variant thereof. In some embodiments, the viral glycoprotein fusogen is of Jurona virus, or variant thereof.

[0202] In some aspects, provided herein is a method of delivering a nucleic acid into a target cell or tissue in a subject in need thereof comprising administering to the subject a nucleic acid encapsulated within an engineered viral particle, wherein the engineered viral particle comprises a viral fusogen comprising a modified vesiculovirus glycoprotein, comprising one or more amino acid mutations corresponding to positions 1347 or R354 of wild-type Vesicular Stomatitis Virus glycoprotein (VSV-G) of SEQ ID NO: 12.

[0203] In some embodiments, the one or more amino acid mutations are selected from the group consisting of I347N, I347Q, R354G and R354N. In some embodiments, the one or more amino acid mutations is I347N. In some embodiments, the one or more amino acid mutations is I347Q. In some embodiments, the one or more amino acid mutations is R354G. In some embodiments, the one or more amino acid mutations is R354N. In some embodiments, the one or more amino acid mutations is I347N and R354G. In someATTORNEY DOCKET NO. ORB-015WO1embodiments, the one or more amino acid mutations is I347N and R354N. In some embodiments, the one or more amino acid mutations is I347Q and R354G. In some embodiments, the one or more amino acid mutations is I347Q and R354N.

[0204] In some embodiments, the nucleic acid encodes an immunogenic polypeptide.

[0205] In some embodiments, the administering is for therapeutic treatment or prophylaxis. In some embodiments, the administering is for therapeutic treatment. In some embodiments, the administering is for prophylaxis.

[0206] In some embodiments, the nucleic acid encodes a therapeutic molecule.

[0207] In some embodiments, the therapeutic molecule is an enzyme, an antibody or antigen binding fragment thereof, a chimeric antigen receptor, a T cell receptor, or a recombinant polypeptide. In some embodiments, the therapeutic molecule is an enzyme. In some embodiments, the therapeutic molecule is an antibody or antigen binding fragment thereof. In some embodiments, the therapeutic molecule is a chimeric antigen receptor. In some embodiments, the therapeutic molecule is a T cell receptor. In some embodiments, the therapeutic molecule is a recombinant polypeptide.

[0208] In some embodiments, the subject is a mammalian subject. In some embodiments, the subject is a human subject.

[0209] In some aspects, provided herein is use of an engineered viral particle for delivering a nucleic acid therapeutic to a cell in vivo comprising contacting the cell with the engineered viral particle which comprises a viral glycoprotein fusogen of any one of Chandipura virus, Perinet virus, Piry virus, Jurona virus, or a variant thereof. In some embodiments, the viral glycoprotein fusogen is of Chandipura virus, or a variant thereof. In some embodiments, the viral glycoprotein fusogen is of Perinet virus, or a variant thereof. In some embodiments, the viral glycoprotein fusogen is of Piry virus, or a variant thereof. In some embodiments, the viral glycoprotein fusogen is of Jurona virus, or a variant thereof.

[0210] In some aspects, provided herein is use of an engineered viral particle for delivering a nucleic acid therapeutic to a cell in vivo comprising contacting the cell with the engineered viral particle comprising a modified vesiculovirus glycoprotein, comprising one or more amino acid mutations corresponding to positions 1347 or R354 of wild-type Vesicular Stomatitis Virus glycoprotein (VSV-G) of SEQ ID NO: 12.

[0211] In some embodiments, the one or more amino acid mutations are selected from the group consisting of I347N, I347Q, R354G and R354N. In some embodiments, the one or more amino acid mutations is I347N. In some embodiments, the one or more amino acid mutations is I347Q. In some embodiments, the one or more amino acid mutations is R354G.ATTORNEY DOCKET NO. ORB-015WO1In some embodiments, the one or more amino acid mutations is R354N. In some embodiments, the one or more amino acid mutations is I347N and R354G. In some embodiments, the one or more amino acid mutations is I347N and R354N. In some embodiments, the one or more amino acid mutations is I347Q and R354G. In some embodiments, the one or more amino acid mutations is I347Q and R354N.

[0212] In some embodiments, the engineered viral particle of the present disclosure is for use as a medicament.

[0213] In some embodiments, the engineered viral particle is for use in oncotherapy, vaccination, immune therapy and / or gene therapy. In some embodiments, the engineered viral particle is for use in oncotherapy. In some embodiments, the engineered viral particle is for use in vaccination. In some embodiments, the engineered viral particle is for use in immune therapy. In some embodiments, the engineered viral particle is for use in immunomodulation. In some embodiments, the engineered viral particle is for use in autoimmune therapy. In some embodiments, the engineered viral particle is for use in gene therapy.Immunotherapy

[0214] In some embodiments, the engineered viral particle is used for immunotherapy. In some embodiments, the engineered viral particle used in immunotherapy comprises a polypeptide or a particle encoding the polypeptide. In some embodiments, the engineered viral particle comprises at least one antigen of interest and / or molecules involved in immune response recognition, for example, cytokines, adjuvants, immune checkpoint modulators, antibodies targeting cytokines, among others. In some embodiments, the engineered viral particle is used for immunomodulation. In some embodiments, the engineered viral particle is used in autoimmune therapy.

[0215] In some embodiments, the immunotherapy is oncotherapy targeting cancer, for example, using engineered viral particles comprising oncolytic virus particles.Oncotherapy

[0216] In some embodiments, engineered viral particles are targeted or retargeted to cancer cells for cancer immunotherapy. Cancer immunotherapy requires specific targeting of prophylactic or therapeutic agents to specific tumor cell types, without risk of off-target effects. Re-targeted engineered viral particles are useful for targeted drug delivery in providing safe and efficacious cancer immunotherapy.

[0217] Engineered viral particles and VLPs stimulate an immune response by interacting with various components of the immune system. For example, VLPs are first internalized byATTORNEY DOCKET NO. ORB-015WO1dendritic cells, and subsequently processed for antigen presentation by MHC class I and MHC class II inducing both cytotoxic CD8+ T cells (MHC class I) and helper CD4+ cells (MHC class II). Short epitopes binding the MHC class I are generated from proteins derived from intracellular pathogens, while those binding the MHC-class II are generated from extracellular proteins upon internalization.

[0218] T helper cells differentiate into either Thl or Th2 subtypes which stimulate T cells that sustain the adaptive immune response, important for anticancer immunity in both preventive and therapeutic cancer vaccines. Thl cells release proinflammatory cytokines IFN-y and TNF-a, and Th2 cells, which produce IL-4 and IL-10, together damage the tumor microenvironment. CD8+ T cells differentiate into effector cytotoxic T lymphocytes lyse cancer cells.

[0219] Prophylactic Oncotherapy: For example, VLP based prophylactic vaccines against oncogenic viruses, e.g., Hepatitis B Virus (HBV) and Human Papillomavirus (HPV) are approved and in use, e.g., Engerix-B (GSK), Recombivax (Merck), PreHeybrio (SCIgen), and Heplisav-B (Dynavax) against HBV consisting ofHbsAg; Cervarix (GlaxoSmithKline) quadrivalent vaccine Gardasil and Gardasil 9 to prevent infection by nine HPV genotypes (Merck Sharp & Dohme Corp.). For example, Heplisav-B was also effective in inducing antibody responses in patients also receiving immunosuppressive drug treatment. VLP vaccines can also be used to target other oncogenic viruses, such as human herpesvirus type 8 (HHV-8), also known as Kaposi sarcoma-associated herpes virus (KSHV), and the Epstein-Barr virus (EBV, HHV-4), which is associated with Burkitt’s lymphoma.

[0220] Therapeutic Oncotherapy: An antigen suitable for targeting in cancer immunotherapy should ideally be exclusively expressed in cancer cells, not found in healthy cells, be vital for the survival of the cells, and stimulate a strong immune response. Various antigens, including oncoantigens, neoantigens, overexpressed antigens, cancer-testicular antigens (MAGE and NY-ESO-1), and foreign “non-self’ antigens are used.

[0221] In some embodiments, the targeted antigen is a neoantigen. Neoantigens, e.g., KRAS, BRAF, and PIK3CA driver genes, among others, are novel proteins unique to tumor cells, which are produced when somatic genomic alterations, for example, single-nucleotide variants (SNVs), base insertions and deletions (INDELs), and gene fusions, arise within the DNA of the tumor. Although the resultant tumor mutational burden poses the challenge of acquired resistance in treatment with immune checkpoint inhibitors, it leads to specific targeting in immunotherapy, minimizing off-target effects. Neoantigen-specific T cells are less likely to be eliminated during tumor immune evasion.ATTORNEY DOCKET NO. ORB-015WO1

[0222] In some embodiments, the targeted antigen is an oncoantigen. Oncoantigens, e.g., EGFR, HER-2, the mucin MUC1, CD20, and the idiotypes of neoplastic clones of B and T cells, IGF1R involved in epithelial and mesenchymal tumors, among others, are persistent tumor antigens that are typically expressed at low levels in normal cells but overexpressed in tumor cells and play a role in tumor progression and do not escape from immune recognition.

[0223] In some embodiments, the targeted antigen is a tissue lineage and differentiation antigen, e.g., Prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), glycoprotein 100 (gplOO), and melanoma antigen recognized by T cells 1 (MART-1), among others, are found in both normal and tumor cells originating from the same tissue.

[0224] In some embodiments, the targeted antigen is a cancer germline antigen (CGA), which is only expressed in germ cells of immune-privileged organs, epigenetically silenced in somatic tissues, and re-expressed in high levels in several carcinomas and sarcomas (such as MAGE and NY-ESO-1).

[0225] In some embodiments, the targeted antigen is a molecule involved in epithelial-to-mesenchymal transition (EMT) and stem-ness, for example, OCT-4, CD44, and CD133, or xCT in breast cancer.Vaccines

[0226] In some embodiments, engineered viral particles are used in infectious disease vaccines. For example, engineered viral particles are used to produce vaccines that result in stronger or more durable immune response to reduce the frequency of boosters and greater protection against related viral strains.

[0227] In some embodiments, engineered viral particles have one target antigen per engineered viral particle and protect against a specific virus. In some embodiments, engineered viral particles have multiple different antigens on a single engineered viral particle and protect against different viruses in combination.

[0228] For example, VLP based vaccines are used in treatment of various infectious diseases, for example, HIV, influenza, hepatitis B, hepatitis E, malaria, Ebola virus, SARS-CoV-2, Zika virus, Dengue, and foot and mouth disease, among others. For example, VLP based vaccines are used to treat or prevent Varicella Zoster Virus (VZV).

[0229] Further, as discussed previously, vaccines against HBV and HPV are approved. Hecolin ® (Xiamen Innovax Biotech Co.) is approved for treatment of HEV and Mosquirix™ is approved against malaria.ATTORNEY DOCKET NO. ORB-015WO1Gene Therapy

[0230] In some embodiments, engineered viral particles are used to deliver nucleic acids or proteins for gene therapy. Aside from virus particles, VLPs are used to deliver nucleic acid or protein to treat genetic diseases. For example, some VLPs exhibit natural tropism to central nervous system cells which are otherwise challenging to target. John Cunningham polyomavirus (JCPyV) response for oligodendrocyte infections and CNS disease is used for drug delivery to the brain. Similarly, vesiculoviruses of the present invention are used in gene therapy for delivery to various target tissues.Gene Editing

[0231] In some embodiments, engineered viral particles or VLPs deliver nucleic acids and enzymes for efficient in vivo base editing in multiple organs.

[0232] In some embodiments, DNA is edited using engineered viral particle or VLP -based delivery of guide RNA and CRISPR-Cas9 / Cpfl or other gene editing technology. In some embodiments, VLPs are used to deliver Cas9 or a dead Cas9 (dCas9) linked to a transcriptional activator or repressor or epigenetic modifier that is targeted to a signaling pathway, gene, or nucleic acid by a guide RNA. In some embodiments, a genetic modification epigenetically modifies expression of an endogenous signaling pathway, gene, or nucleic acid.

[0233] Engineered viral particles or VLPs offer transient delivery of base editors, minimizing off-target effects in genome editing.

[0234] In some embodiments, VLPs are used in nanoblades. For example, murine leukemia virus (MLV)-like VLPs or other VLPs of the present disclosure are loaded with Cas9-gRNA ribonucleoproteins which can deliver CRISPR to multiple target cells, e.g., primary cells, embryos, etc. In some embodiments, nanoblades carry cargo for indel formation via Non-Homologous End Joining, precise Homology Directed Repair, CRISPR activation or repression, transgenic mouse creation, in vivo gene editing of mice, and other CRISPR applications.Treatment of Autoimmune Disease

[0235] In some embodiments, engineered viral particles or VLPs of the present invention are used in a method of treating autoimmune disease. In some embodiments, engineered viral particles are used in treating B-cell associated autoimmune diseases. In some embodiments, engineered viral particles are used in treating Systemic Lupus Erythematosus (SLE), as aATTORNEY DOCKET NO. ORB-015WO1non-limiting example of an autoimmune disease. In some embodiments, provided herein is a method of treating one or more symptoms or indications of SLE (e.g., lupus nephritis).Treatment of Allergies

[0236] In some embodiments, engineered viral particles or VLPs are used to deliver allergens to provoke an immune response and treat allergies. The modular nature of VLPs allows for fine-tuning of the immune response. For example, in some embodiments, engineered viral particles or VLPs deliver exemplary antigens, including but not limited to, cat allergen Fel dl, house dust mite Der pl peptide, mugwort pollen allergen Art vl, house dust mite extract, and including in some embodiments, adjuvants, for example, A-type CpG adjuvant, among others. In some embodiments, engineered viral particle or VLP treatment inhibits basophil and mast cell degranulation and abolishes IgE memory responses, ameliorating symptoms of rhinitis and allergic asthma.Screening for Receptor-Ligand Specificity

[0237] In some embodiments, engineered viral particles or VLPs are used in screening for membrane receptors and in interactome studies. Cell surface receptors are important for cell signaling, however, despite their importance and abundance, receptor interaction networks are challenging to decipher because of difficulties in maintaining membrane proteins in their native conformation and their typically weak interactions. In some embodiments, engineered viral particles or VLPs are used for membrane protein display enabling a purification-free high-throughput detection of receptor-ligand interactions in membranes. In some embodiments, engineered viral particles or VLPs provide a broad platform for enhanced detection of extracellular interactions of membrane proteins over a wide range of binding affinities, including weak interactions.Particle Systems and Methods of Engineered Viral Particle Production

[0238] The engineered viral particles described herein can be produced by any method commonly known in the art. In some embodiments, the engineered viral particles are produced by a vector system.

[0239] In some aspects, provided herein is a vector system for producing the engineered viral particle of the present disclosure, comprising one or more vectors encoding the viral glycoprotein fusogen and the targeting moiety, wherein the fusogen is a viral glycoprotein of any one of Chandipura virus, Perinet virus, Piry virus, Jurona virus, or a variant thereof. In some aspects, provided herein is a vector system for producing the engineered viral particleATTORNEY DOCKET NO. ORB-015WO1of the present disclosure, comprising one or more vectors encoding the viral glycoprotein fusogen and the targeting moiety, wherein the fusogen is a modified viral glycoprotein of a vesiculovirus comprising one or more amino acid mutations selected from the group consisting of I347N, I347Q, R354G, and R354N.

[0240] In some embodiments, the vector system comprises one vector expressing the viral glycoprotein fusogen and the targeting moiety.

[0241] In some embodiments, the present invention provides a vector system for producing an engineered viral particle described herein. In some embodiments, the vector system comprises one or more vectors encoding the viral glycoprotein fusogen and the targeting moiety. In some embodiments, the vector system comprises one vector encoding the viral glycoprotein fusogen and the targeting moiety. In some embodiments, the vector system comprises one vector encoding the viral fusogen and another vector encoding the targeting moiety. In some embodiments, the vector system further comprises a vector expressing a cargo.

[0242] In some embodiments, the vector system comprises at least one vector encoding the viral glycoprotein and the targeting moiety. In some embodiments, the vector system comprises one vector. In some embodiments, the vector system comprises two vectors. In some embodiments, the vector system comprises three vectors. In some embodiments, the vector system comprises four vectors. In some embodiments, the vector system comprises five vectors. In some embodiments, the vector system comprises six vectors.

[0243] In some embodiments, the vector system comprises one vector encoding both the viral glycoprotein and the targeting moiety. In some embodiments, the vector system comprises one vector encoding the viral glycoprotein and one vector encoding the targeting moiety. In some embodiments, the vector system further comprises a vector encoding a cargo.

[0244] In some embodiments, the vector system comprises one vector encoding the viral glycoprotein, the targeting moiety, and the cargo. In some embodiments, the vector system comprises two vectors encoding the viral glycoprotein, the targeting moiety, and the cargo. In some embodiments, the vector system comprises three vectors encoding the viral glycoprotein, the targeting moiety, and the cargo. In some embodiments, the vector system comprises four vectors encoding the viral glycoprotein, the targeting moiety, and the cargo. In some embodiments, the vector system comprises five vectors encoding the viral glycoprotein, the targeting moiety, and the cargo. In some embodiments, the vector system comprises six vectors encoding the viral glycoprotein, the targeting moiety, and the cargo. InATTORNEY DOCKET NO. ORB-015WO1some embodiments, the vector system of the present disclosure is an oncolytic virus vector system. VSV Indiana as well as related viruses, Maraba, Cocal, Morreton, Alagoas, New Jersey, and Carajas are oncolytic and may thus be used. In some embodiments, the vector system is an immunotherapy virus vector system. In some embodiments, the vector system is a gene therapy vector system.

[0245] In some embodiments, the present invention provides a host cell comprising the vector system described herein. In some embodiments, the present invention provides a host cell for producing the engineered viral particle of the present invention. In some embodiments, the engineered viral particles are produced by a host cell comprising the vector system. The host cell may be any host cell commonly used in the art for the production of engineered viral particles and / or other therapeutics. In some embodiments, the host cell is a mammalian cell.

[0246] In some embodiments, engineered viral particles or VLPs are produced by introducing one or more viral particles of the invention into a suitable producer cell, culturing the producer cell under suitable conditions allowing the production of engineered viral particle or VLP, recovering the produced viral particles from the culture and optionally purifying the viral particle or VLP. In some embodiments, the viral glycoproteins of the present disclosure increase yield of engineered viral particle or VLPs from the production process.Compositions and Kits

[0247] In some embodiments, provided herein is a composition comprising a vesiculovirus glycoprotein from any one of Chandipura virus, Perinet virus, Piry virus, Jurona virus or a variant thereof, or a modified vesiculovirus comprising mutations in one or more of I347N, I347Q, R354G, and R354N. In some embodiments, the composition comprises the glycoprotein lacking LDL-R binding. In some embodiments, provided herein is a composition comprising an engineered viral particle comprising the glycoproteins of the present disclosure. In some embodiments, the composition comprises the vector, host cell or any combination thereof.

[0248] In some embodiments, the composition is a pharmaceutical composition comprising a therapeutically effective amount of the active agent (mutant VSV-G glycoprotein, for example, in some embodiments, with a targeting moiety, or any one of natural glycoproteins lacking LDL-R binding described herein, as well as one or more engineered viral particles or host cells comprising the same). A pharmaceutically acceptableATTORNEY DOCKET NO. ORB-015WO1vehicle may include any and all carriers, solvents, diluents, excipients, adjuvants, dispersion media, coatings, antibacterial or antifungal agents, adsorption delaying agents and the like, suitable for human administration. As used herein, the composition is administered in a therapeutically effective amount which is a dose sufficient to achieve a therapeutic effect.

[0249] Provided herein is a kit comprising the vector system of the present disclosure, for example, one or more vectors encoding the viral glycoproteins of the present disclosure and / or a targeting moiety.EXAMPLES

[0250] Various aspects of the invention are described in further detail in the following Examples. The following examples describe some of the exemplary modes of making and practicing the present invention. However, it should be understood that these Examples are for illustrative purposes only and are not meant to limit the scope of the invention.Example 1. Screening for novel viral fusogens that did not bind LDL-R and allowed for targeted delivery via engineered viral particles

[0251] Vesicular stomatitis virus G protein (VSV-G) is a known fusogen in viral particles (as known as “virus-like particles” or “VLP”). In a traditional delivery mechanism, as shown in FIG. 1 A, wild-type VSV-G expressed on VLP binds to human low-density lipoprotein receptors (LDL-R); VSV-G fuses with the cellular membrane; and this facilitates entry of the cargo into the cell, as shown in FIG. 1A.

[0252] In the present disclosure, in some aspects, the engineered viral particles use a different entry system that includes incorporation of a targeting moiety and an engineered viral fusogen, as illustrated in FIG. IB. In FIG. IB, during the first step of targeted delivery, there is binding through a ligand-receptor interaction (e.g., binding of the ligand / targeting moiety to the receptor / target), without binding to LDL-R. In the second step of targeted delivery, a mutant VSV-G (that does not bind LDL-R) fuses with the cellular membrane, and the payload is delivered. Two residues, K47 and R354, in VSV-G have been identified as the main binding sites to LDL-R. Mutations at these sites in VSV-G disrupt LDL-R binding and do not facilitate uptake by human cells; thus, the mutations result in a de-targeted engineered viral particle. In the present disclosure, incorporation of a targeting moiety (also referred to as “ligand,” e.g., scFv, nanobody, or endogenous protein) to the de-targeted engineered viral particle enables retargeting specifically to cells expressing the cognate receptor (also referredATTORNEY DOCKET NO. ORB-015WO1to as “target”) of the targeting moiety, resulting in a targeted engineered viral particle or retargeted engineered viral particle, as further illustrated in FIG.2.

[0253] In this example, novel viral fusogens were identified and screened for targeting capacity using Green Fluorescent Protein (GFP) as a reporter protein. Fourteen exemplary vesiculovirus G proteins were identified as being unlikely to bind to LDL-R due to the lack of the K47 / R354 binding motif.

[0254] Exemplary engineered viral particles comprising the vesiculovirus G proteins were prepared with an anti-CD5 single-chain variable fragment (scFv) as the targeting moiety and a nucleic acid encoding GFP as the cargo (“aCD5 scFv” as shown in FIG.3). As a control, exemplary engineered viral particles without the anti-CD5 scFv targeting moiety (“No targeter” as shown in FIG.3) were also prepared. The engineered viral particles were tested for transduction in Jurkat cells by measuring the percent expression of GFP after four days. As shown in FIG.3, vesiculovirus G proteins from Chandipura virus, Isfahan virus, Perinet virus, Piry virus, and Jurona virus displayed high transduction efficiency, as shown by the high percent expression of GFP+ cells.

[0255] Overall, this study identified novel vesiculoviruses (e.g., Chandipura virus, Isfahan virus, Perinet virus, Piry virus, and Jurona virus) that do not bind LDL-R. These newly-identified viruses were naturally de-targeted because they naturally do not bind LDL-R, and the viruses were subsequently engineered to be effectively re-targeted to T cells.Example 2. Re-targeting using Engineered Viral Particles Comprising Vesiculovirus G proteins

[0256] This example illustrates exemplary vesiculovirus G proteins with reduced LDL-R binding, and without a reduction in cell uptake and targeting efficiency.

[0257] Exemplary vesiculovirus G proteins from VSV Indiana strain virus, and related viruses, for example, Moreton virus, Cocal virus, Alagoas virus, Carajas virus, and New Jersey virus, were prepared with the mutations K47Q and R354A to disrupt LDL-R binding. Exemplary engineered viral particles comprising the mutant vesiculovirus G proteins were prepared with an anti-CD5 scFv as the targeting moiety and a nucleic acid encoding GFP as the exemplary cargo. As controls, exemplary engineered viral particles with the wild-type vesiculovirus G proteins and without the targeting moiety were also prepared. The engineered viral particles were tested for transduction on Jurkat cells by measuring the percent expression of GFP after four days. As shown in FIG. 4, mutants (“Muf ’) in glycoproteins from VSV Indiana strain, Moreton virus, Cocal virus, Alagoas virus, Carajas virus, and New Jersey virus decreased targeting to Jurkat cells in comparison to wild-type (“Wf ’). However,ATTORNEY DOCKET NO. ORB-015WO1exemplary mutant vesiculovirus G proteins, VSV Indiana, and Alagoas virus mutants, for example, were re-targeted to Jurkat cells when transduced with an anti-CD5 targeting moiety (“Mut + aCD5”) in contrast to wild-type virus transduced with an anti-CD5 targeting moiety (“Wt + aCD5”).

[0258] Overall, this study showed that vesiculovirus G proteins from some viruses reduced or eliminated off-target binding (e.g., LDL-R binding), and showed comparable or improved transduction efficiency relative to the wild-type.Example 3. Exemplary engineered viral particles exhibited a targeted phenotype in vivo

[0259] This example demonstrates that exemplary engineered viral particles exhibited a targeted phenotype in vivo. In this example engineered viral particles comprising VSV-G mutants or other glycoprotein variants that were de-targeted to LDL-R, and successfully retargeted to T cells in the spleen by inclusion of a targeting moiety.

[0260] In Study A, exemplary engineered viral particles were prepared with mutant VSV-G (with K47Q / R354A mutations) and an exemplary targeting moiety (an anti-CD3 scFv). As a control, exemplary engineered viral particles comprising wild-type VSV-G and no targeting moiety were also prepared. Each engineered viral particle contained a nucleic acid encoding a GFP reporter. The engineered viral particles were administered intravenously to NCG-hCD34 mice on Day 0, and spleen, blood, and lung samples were collected and analyzed on Day 7. As shown in FIG. 5A, the exemplary targeted engineered viral particle (“Targeted (VSV-Gmut+aCD3)”) displayed significantly higher GFP expression in T cells, which was the targeted cell type, compared to other cells of the spleen.

[0261] In Study B, exemplary engineered viral particles were prepared with Maraba virus G protein (MARAV-G) with K47Q / R354A mutations and a targeting moiety (an anti-CD3 scFv) (“MARAV Targeted” as shown in FIG. 5B). As a control, exemplary engineered viral particles comprising wild-type MARAV-G and no targeting moiety (“MARAV Non-Targeted”) were also prepared. Each engineered viral particle contained a nucleic acid encoding a GFP reporter. The engineered viral particles were administered intravenously to NCG-hCD34 mice on Day 0, and spleen, blood, and lung samples were collected on Day 7. GFP expression was measured and compared in different human cell subsets. As shown in FIG. 5B, the exemplary targeted engineered viral particle (“MARAV Targeted”) displayed significantly higher GFP expression in T cells, which was the targeted cell type, compared to B cells and “other cells.” “Other cells” include macrophages, dendritic cells, and other immune cells that are not B or T cells.ATTORNEY DOCKET NO. ORB-015WO1

[0262] Overall, this example illustrates that the exemplary engineered viral particles were targeted to T cells in vivo with specificity.Example 4. Identification of new VSV-G mutants and novel mutation residues in VSV-G

[0263] Two VSV-G residues, K47 and R354, have been previously identified as the main binding sites to LDL-R, and mutants of these residues have been used to disrupt LDL-R binding while still retaining VSV-G function as a fusogen (FIG. 6). In particular, K47A and R354Q are known VSV-G mutations that disrupt LDL-R binding (that is, have an LDL-R ablating effect). In addition, H08 and Y209 have been previously identified as additional sites that are important for LDL-R binding.

[0264] In this example, by molecular modeling, novel mutations at known sites as well as novel residues were identified and subsequently screened for activity in reducing LDL-R binding. Briefly, a two-pass computational strategy was employed as described below to identify these novel mutations and novel residues of VSV-G.

[0265] The first-pass computation strategy included molecular modeling. Molecular modeling tools known in the art, for example, AlphaFold 2.3 and molecular dynamics simulation, among others, were used to simulate target engagement of VSV G-protein with individual cysteine-rich domains CR2 and CR3 of the LDL-R protein. Stabilizing reactions between the proteins were identified using atom-resolution energy fields, and change in binding free energy (AAG) was screened using molecular dynamics simulations, exhaustively scanning single-amino acid mutants to test binding ablation.

[0266] The second-pass computation strategy included refolding. High scoring mutants were refolded using the complete AlphaFold and molecular dynamics simulation pipeline to model global interactions between VSV-G Mutants with LDL-R CR2 and CR3. Top variants were selected for experimental testing, which minimized interaction of refolded VSV-G and the target domain.

[0267] In this example, some exemplary novel mutations identified include H008G, R354G, R354N, and Y209A of VSV-G. Some exemplary novel sites tested included I182Q, I347N, and I347Q of VSV-G.

[0268] Among related strains to VSV Indiana, computational modeling was carried out comparing energetics as measured by a change in binding free energy (AAG) of refolded G-protein and target CR2 or CR3 domains of LDL-R. Binding disruption potential was simulated. Residues of interest from energy simulations were mutagenized in-silico and then fully refolded into minimum energy complexes, comprising the following steps: (a) a Google AlphaFold multimer was used to model the combined structure of CR2 and CR3 with eachATTORNEY DOCKET NO. ORB-015WO1wild-type protein; (b) molecular dynamics physics-based relaxation was carried out to refine complexes identified in step (a); (c) relaxed models from step (b) were used to compute intramolecular forces from residue-residue interactions and compute contributions of residue to overall binding affinity; (d) substitutions were carried out for residues identified as highly interactive; complexes were relaxed and energy was calculated to predict favorability of any tested mutation for binding; and (e) molecules with selected mutations which were predicted to reduce binding were then refolded using Alphafold to ensure that changes were local and had no effect on the folding of the entire protein.

[0269] Mutations were also explored for VSG-Indiana related organisms. Mutants that showed a change in binding free energy (AAG » 0) were identified as candidates that disrupted LDL-R binding. Mutations leading to AAG >4 were tested for de-targeting.

[0270] Results shown in FIG. 7 demonstrated that other viruses, for example, Maraba virus, Alagoas virus, Morreton virus, and Cocal virus showed similar refolding, which suggested functional conservation of equivalent residues.

[0271] After sequence analysis, it was identified that the VSV-G 1347 mutation is conserved in Morreton and Cocal viruses. Alagoas, Carajas, and New Jersey viruses contained a valine at the corresponding residue, while Maraba virus had a methionine.Exemplary mutations corresponding to VSV-G mutations in other vesiculoviruses are included in Table 3 herein.

[0272] Overall, the results from this study identified new VSV-G candidate mutations and expanded the repertoire of VSV-G mutants for, for example, retargeting and use in engineered viral particles for pseudotyping.Example 5: Cell surface expression of mutant glycoprotein in VSV-G protein mutants

[0273] Cell surface expression was evaluated for exemplary novel VSV-G protein mutants, for example, H008G, I182Q, I347N, I347Q, R354G, R354N, Y209A (“VSV-Gmut), and targeted engineered viral particles with an anti-CD5 scFv targeting moiety (“hCD5 scFv T2A VSV-Gmut”), and untransfected controls. GFP expression was evaluated 48 hours after transfection of VSV-Gmut in an exemplary producer cell line with a GFP transfer plasmid.

[0274] As shown in FIG. 8 A and FIG. 8B, cells transfected with VSV-G protein mutants showed cell surface expression of the mutant glycoprotein.

[0275] Overall, this example showed that VSV-G glycoprotein mutants identified were expressed on the viral cell surface.ATTORNEY DOCKET NO. ORB-015WO1Example 6: De-targeting of engineered viral particles comprising VSV-G mutants in T cells and B cells

[0276] This example showed de-targeting of engineered viral particles comprising VSV-G mutants described herein in T cells and B cells.

[0277] Briefly, Jurkat (T cells; CD3+ CD19-) and Raji (B cells; CD3- CD19+) cells were transduced with anti-CD5 targeting moiety (hCD5), VSV-G mutant control, VSV-G wildtype, and exemplary novel mutations, H008G, I182Q, I347N, I347Q, R345G, R354N, Y209A, hCD5 scFv T2A VSV-G mut, and a gating control for each cell type.

[0278] The results in FIG. 9A-FIG. 9C showed that the VSV-G novel mutants tested (I347N, I347Q, R354G, and R354N) showed de-targeting in Jurkat and Raji cells.Example 7: Re-targeting of VSV-G mutants in T cells

[0279] This example showed re-targeting of de-targeted novel VSV-G mutants on exemplary T cell lines.

[0280] Briefly, Jurkat and Raji cells were transduced with VSV-G mutant alone or VSV-G mutant and targeting moiety, and GFP transduction was measured. As shown in FIG. 10A, de-targeted VSV-G I347N and I347Q mutants were successfully re-targeted in Jurkat cells by transducing with a targeting moiety.

[0281] Similarly, as shown in FIG. 10B, de-targeted VSV-G R354G and R354N mutants were successfully re-targeted in Jurkat cells by transducing with a targeting moiety.

[0282] Overall, this example showed that exemplary mutations in the novel 1347 site of VSV-G glycoprotein, e.g., I347N and I347Q, were successfully retargeted to T cells. Further, this example also showed that novel mutations, R354G and R354N, were successfully retargeted to T cells.ATTORNEY DOCKET NO. ORB-015WO1EQUIVALENTS AND SCOPE

[0283] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

Claims

ATTORNEY DOCKET NO. ORB-015WO1CLAIMS1. An engineered viral particle comprising:a viral fusogen,a targeting moiety, andat least one nucleic acid,wherein the viral fusogen is a glycoprotein of Chandipura virus, Perinet virus, Piry virus, Jurona virus, or a variant thereof.

2. An engineered viral particle comprising:a viral fusogen,a targeting moiety, andat least one nucleic acid,wherein the viral fusogen comprises a modified vesiculovirus glycoprotein, comprising one or more amino acid mutations corresponding to positions 1347 or R354 of wild-type Vesicular Stomatitis Virus glycoprotein (VSV-G) of SEQ ID NO: 12.

3. The engineered viral particle of claim 2, wherein the one or more amino acid mutations decreases binding of the engineered viral particle to Low Density Lipoprotein Receptor (LDL-R).

4. The engineered viral particle of claim 1, wherein the viral fusogen is a glycoprotein of Chandipura virus, or a variant thereof.

5. The engineered viral particle of claim 1, wherein the viral fusogen is a glycoprotein of Perinet virus, or a variant thereof.

6. The engineered viral particle of claim 1, wherein the viral fusogen is a glycoprotein of Piry virus, or a variant thereof.

7. The engineered viral particle of claim 1, wherein the viral fusogen is a glycoprotein of Jurona virus, or a variant thereof.

8. The engineered viral particle of claim 2, wherein the modified vesiculovirus glycoprotein is a modified Vesicular Stomatitis Virus glycoprotein (VSV-G).ATTORNEY DOCKET NO. ORB-015WO19. The engineered viral particle of claim 2, wherein the modified vesiculovirus glycoprotein is a modified Alagoas virus glycoprotein, Carajas virus glycoprotein, Cocal virus glycoprotein, Indiana virus glycoprotein, Morreton virus glycoprotein, New Jersey virus glycoprotein, or Maraba virus glycoprotein.

10. The engineered viral particle of claim 2, wherein the modified vesiculovirus glycoprotein is a modified Maraba virus glycoprotein.

11. The engineered viral particle of claim 2, wherein the one or more amino acid mutations are selected from the group consisting of I347N, I347Q, R354G, and R354N.

12. The engineered viral particle of claim 11, wherein the amino acid mutation is I347N or I347Q.

13. The engineered viral particle of claim 11, wherein the amino acid mutation is R354G or R354N.

14. The engineered viral particle of any one of the preceding claims, wherein the viral glycoprotein comprises a sequence of any one of SEQ ID NOs: 1-10 and 13-71.

15. The engineered viral particle of any one of the preceding claims, wherein the engineered viral particle is a pseudotyped viral particle.

16. The engineered viral particle of claim 15, wherein the engineered viral particle is a pseudotyped vesiculovirus, retrovirus, alphavirus, herpesvirus, arenavirus, paramyxovirus, lentivirus, arenavirus, or oncolytic virus.

17. The engineered viral particle of any one of the preceding claims, wherein the targeting moiety is an antibody or an antigen binding fragment thereof, or a ligand.

18. The engineered viral particle of claim 17, wherein the targeting moiety is an antibody or an antigen binding fragment thereof that binds to a cell surface marker.

19. The engineered viral particle of claim 18, wherein the targeting moiety is a ligand that binds to a receptor on a cell surface.

20. The engineered viral particle of any one of the preceding claims, wherein the targeting moiety is immune cell specific.ATTORNEY DOCKET NO. ORB-015WO121. The engineered viral particle of claim 20, wherein the immune cell is a T cell, a B cell, or a natural killer (NK) cell.

22. The engineered viral particle of claim 21, wherein the immune cell is a T cell.

23. The engineered viral particle of claim 21, wherein the immune cell is a B cell.

24. The engineered viral particle of any one of the preceding claims, wherein the nucleic acid encodes a polypeptide.

25. The engineered viral particle of claim 24, wherein the polypeptide is a therapeutic or immunogenic polypeptide.

26. The engineered viral particle of claim 25, wherein the polypeptide is a therapeutic polypeptide.

27. The engineered viral particle of claim 25, wherein the immunogenic polypeptide is derived from an infectious pathogen.

28. The engineered viral particle of claim 25, wherein the immunogenic polypeptide is a cancer antigen.

29. A composition comprising the engineered viral particle of any one of claims 1-28.

30. A cell comprising the engineered viral particle of any one of claims 1-28.

31. A targeted delivery vehicle comprising the engineered viral particle of any one of claims 1-28.

32. A modified vesiculovirus envelope glycoprotein, comprising one or more amino acid mutations corresponding to position 1347 or R354 of wild-type VSV-G of SEQ ID NO: 12.

33. The modified vesiculovirus glycoprotein of claim 32, wherein the one or more amino acid mutations are selected from the group consisting of I347N, I347Q, R354G, and R354N.

34. The modified vesiculovirus glycoprotein of claim 33, wherein the amino acid mutation is I347N or I347Q.ATTORNEY DOCKET NO. ORB-015WO135. The modified vesiculovirus glycoprotein of claim 33, wherein the amino acid mutation is R354G or R354N.

36. The modified vesiculovirus glycoprotein of any one of claims 30-35, wherein the glycoprotein comprises a sequence of any one of SEQ ID NOs: 13-71.

37. A vesiculovirus comprising the modified vesiculovirus glycoprotein of any one of claims 32-36.

38. An engineered virus particle comprisinga vesiculovirus,a targeting moiety, andat least one nucleic acid,wherein the targeting moiety is on the surface of the engineered viral particle, and wherein the vesiculovirus is a Chandipura virus, a Perinet virus, a Piry virus, a Jurona virus, or a variant thereof.

39. The engineered viral particle of claim 38, wherein the vesiculovirus is a Chandipura virus, or a variant thereof.

40. The engineered viral particle of claim 38, wherein the vesiculovirus is a Perinet virus, or a variant thereof.

41. The engineered viral particle of claim 38, wherein the vesiculovirus is a Piry virus, or a variant thereof.

42. The engineered viral particle of claim 38, wherein the vesiculovirus is a Jurona virus, or a variant thereof.

43. An engineered virus particle, comprisinga vesiculovirus,a targeting moiety, andat least one nucleic acid,wherein the targeting moiety is on the surface of the engineered viral particle, and wherein the vesiculovirus is of claim 37.ATTORNEY DOCKET NO. ORB-015WO144. The engineered viral particle of claim 43, wherein the vesiculovirus is a Vesicular Stomatitis Virus (VSV) variant.

45. The engineered viral particle of claim 43, wherein the vesiculovirus is an Alagoas virus variant, a Carajas virus variant, a Cocal virus variant, an Indiana virus variant, a Morreton virus variant, a New Jersey virus variant, or a Maraba virus variant.

46. The engineered viral particle of claim 43, wherein the vesiculovirus is a Maraba virus variant.

47. A method of delivering a nucleic acid into a cell comprising contacting the cell with the engineered viral particle of any one of claims 1-28 or 38-46.

48. The method of claim 47, wherein the cell is an immune cell.

49. A method of delivering a nucleic acid into a target cell or tissue in a subject in need thereof comprising administering to the subject a nucleic acid encapsulated within an engineered viral particle, wherein the engineered viral particle comprises:a viral glycoprotein fusogen of any one of glycoproteins of Chandipura virus, Perinet virus, Piry virus, Jurona virus, or a variant thereof.

50. A method of delivering a nucleic acid into a target cell or tissue in a subject in need thereof comprising administering to the subject a nucleic acid encapsulated within an engineered viral particle, wherein the engineered viral particle comprises:a viral fusogen comprising a modified vesiculovirus glycoprotein, comprising one or more amino acid mutations corresponding to positions 1347 or R354 of wild-type Vesicular Stomatitis Virus glycoprotein (VSV-G) of SEQ ID NO: 12.

51. The method of claim 49 or 50, wherein the nucleic acid encodes an immunogenic polypeptide.

52. The method of claim 49 or 50, wherein the administering is for therapeutic or prophylactic treatment.

53. The method of claim 49 or 50, wherein the nucleic acid encodes a therapeutic molecule.ATTORNEY DOCKET NO. ORB-015WO154. The method of claim 53, wherein the therapeutic molecule is an enzyme, an antibody or antigen binding fragment thereof, a chimeric antigen receptor, a T cell receptor, or a recombinant polypeptide.

55. The method of any one of claims 49-54, wherein the subject is a human subject.

56. Use of an engineered viral particle for delivering a nucleic acid therapeutic to a cell in vivo comprising:contacting the cell with a nucleic acid encapsulated within an engineered viral particle which comprises a viral glycoprotein fusogen of any one of Chandipura virus, Perinet virus, Piry virus, Jurona virus, or a variant thereof.

57. Use of an engineered viral particle for delivering a nucleic acid therapeutic to a cell in vivo comprising:contacting the cell with a nucleic acid encapsulated within an engineered viral particle which comprises a viral fusogen comprising a modified vesiculovirus glycoprotein, comprising one or more amino acid mutations corresponding to positions 1347 or R354 of wild-type Vesicular Stomatitis Virus glycoprotein (VSV-G) of SEQ ID NO: 12.

58. An engineered viral particle of any one of claims 1-28 or 38-46 for use as a medicament.

59. The engineered viral particle of claim 58 for use in oncotherapy, vaccination, immune therapy, and / or gene therapy.

60. A vector system for producing the engineered viral particle of any one of claims 1-28, comprising one or more vectors encoding the viral glycoprotein fusogen and the targeting moiety, wherein the fusogen is a viral glycoprotein of any one of Chandipura virus, Perinet virus, Piry virus, Jurona virus, or a variant thereof.

61. A vector system for producing the engineered viral particle of any one of claims 1-28, comprising one or more vectors encoding the viral glycoprotein fusogen and the targeting moiety, wherein the fusogen is a modified viral glycoprotein of a vesiculovirus comprising one or more amino acid mutations selected from the group consisting of I347N, I347Q, R354G, and R354N.

62. The vector system of any one of claims 60 or 61, wherein the vector system comprises one vector expressing the viral glycoprotein fusogen and the targeting moiety.ATTORNEY DOCKET NO. ORB-015WO163. A host cell for producing an engineered viral particle system of claim 62.

64. A kit comprising the vector system of any one of claims 60 or 61.

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