Engineered human endogenous virus-like particles and methods of use thereof for delivery to cells - Patent Application 20070122997

Abstract

A human-derived virus-like particle (heVLP) comprising a membrane comprising a phospholipid bilayer on the outside with one or more HERV-derived envelope proteins, one or more HERV-derived GAG proteins in the heVLP core inside the membrane, and a cargo molecule, e.g., a biological molecule and / or chemical cargo molecule, disposed in the core of the heVLP, and not containing gag protein except for gag protein encoded in the human genome or gag protein encoded by a consensus sequence derived from gag protein found in the human genome, and a method of using the same for delivering cargo molecules to cells.

Description

[Technical Field]

[0001] Priority claim This application claims the benefit of U.S. Patent Application No. 62 / 861,186, filed June 13, 2019, the entire contents of which are incorporated herein by reference.

[0002] Federally funded research or development This invention was made with government support under Grant No. GM118158 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.

[0003] Technical Field Described herein are engineered human endogenous virus-like particles (heVLPs) that contain an outer phospholipid bilayer membrane and a cargo, e.g., a biological molecule and / or chemical cargo, located in the core of the heVLP inside the membrane, and that do not contain non-human gag- or pol-derived proteins, and methods for their use to deliver cargo to cells. [Background technology]

[0004] The delivery of cargo, such as proteins, nucleic acids, and / or chemicals, into the cytosol of living cells represents a significant challenge in the development of biological therapeutics. Summary of the Invention

[0005] Described herein are heVLPs capable of packaging and delivering DNA, RNA, proteins, chemical compounds and / or molecules, and any combination of these four entities to eukaryotic cells. The non-viral heVLP system described herein may be simpler, more efficient, and safer than conventional artificially derived lipid / metal nanoparticle and viral particle-based delivery systems because heVLPs are composed of human-derived components. While the internal cargo may or may not be human-derived, heVLPs are composed entirely of human and synthetic, non-immunogenic components. The "synthetic" components include surface scFv / nanobody / darpin peptides that have been shown to be non-immunostimulatory and can be used to enhance the targeting and cellular uptake of heVLPs. This means that the particle's exterior surface lacks significant immunostimulatory components, minimizing the immunogenicity and antibody neutralization of these particles. Aside from the cargo, heVLPs do not contain the exogenous viral components inherent in other VLPs, representing a significant and novel advancement in technology.Additionally, heVLPs can (but do not require) utilize chemical-based dimerizers, and are capable of packaging and delivering cargo molecules, including therapeutic or diagnostic agents, such as biomolecules and chemicals, such as specialized single- and / or double-stranded DNA molecules (e.g., plasmids, minicircles, closed circular linear DNA, AAV DNA, episomes, bacteriophage DNA, homologous recombination templates, etc.), single- and / or double-stranded RNA molecules (e.g., single-stranded guide RNA, prime-edited guide RNA, messenger RNA, transfer RNA, long non-coding RNA, circular RNA, RNA replicon, circular or linear splicing RNA, microRNA, small interfering RNA, small hairpin RNA, piwi-interacting RNA, toehold switch RNA, RNA to which an RNA-binding protein can bind, bacteriophage RNA, internal ribosome entry site-containing RNA, etc.), proteins, chemical compounds and / or molecules (e.g., small molecules), and combinations of the cargoes listed above (e.g., AAV particles).

[0006] The heVLPs described herein differ from conventional retroviral particles, virus-like particles (VLPs), exosomes, and other previously reported extracellular vesicles capable of delivering cargo because, at least, heVLPs can be produced by strategic overexpression of human-derived components in human cells, heVLPs have a wide range of possible cargoes and delivery strategies, heVLPs lack limited DNA / RNA length constraints, heVLPs lack pol and exogenous gag-derived proteins, and heVLPs have a unique cell entry mechanism.

[0007] Described herein are compositions and methods for cargo delivery that can be used with a variety of protein and nucleic acid molecules, such as genome editing, epigenome modulating, transcriptome editing, and proteome modulating reagents, applicable to many disease therapies.

[0008] Thus, provided herein are engineered heVLPs comprising a membrane comprising a phospholipid bilayer with one or more HERV-derived ENV / glycoproteins (e.g., overexpressed in heVLP-producing cells from an exogenous source, e.g., a plasmid or a stably integrated transgene) (e.g., as shown in Table 1) on the outside, and a human endogenous GAG protein, other cell membrane recruitment domain, and / or biomolecule / chemical cargo located in the core of the heVLP inside the membrane, where the biomolecule cargo may or may not be fused to a human endogenous GAG or other cell membrane recruitment domain (e.g., as shown in Table 6), and the heVLP does not express non-human gag and / or pol proteins, except for gag proteins encoded in the human genome or gag proteins encoded by consensus sequences derived from gag proteins found in the human genome. Human-derived GAG or other cell membrane recruitment domains fused to biomolecule cargo can be overexpressed in heVLP-producing cells from an exogenous source, e.g., a plasmid or a stably integrated transgene.

[0009] In some embodiments, the HERV ENV may be truncated or fused to an scFv or other targeting polypeptide.

[0010] In some embodiments, the HERV GAG may be fused to a cell membrane recruitment domain (eg, as shown in Table 6).

[0011] In other embodiments, the engineered heVLP comprises a membrane comprising a phospholipid bilayer with one or more HERV-derived ENV / glycoproteins (e.g., overexpressed in heVLP-producing cells from an exogenous source, e.g., a plasmid or a stably integrated transgene) (e.g., as shown in Table 1) on the outside; and optionally a cell membrane recruitment domain (e.g., as shown in Table 6) on the inside of the particle; and optionally a biomolecule / chemical cargo.

[0012] Also provided is a method of delivering cargo to a target cell, eg, a cell in vivo or in vitro, by contacting the cell with a heVLP of claim 1, which comprises a biomolecule and / or chemical as cargo.

[0013] Further provided herein are methods for producing heVLPs comprising a biomolecular cargo. The methods include providing cells that express (e.g., engineered to express or overexpress) one or more HERV-derived envelope proteins (e.g., those listed in Table 1) and cargo, where the cells do not express gag and / or pol proteins except for gag proteins encoded in the human genome or gag proteins encoded by consensus sequences derived from gag proteins found in the human genome; and maintaining the cells under conditions such that the cells produce heVLPs. In some embodiments, the methods further include recovering, and optionally purifying and / or concentrating, the produced heVLPs.

[0014] Also provided herein are cells (e.g., isolated cells, preferably mammalian cells, e.g., human cells) that have been induced to express, e.g., overexpress, in combination, one or more HERV-derived envelope proteins (e.g., overexpressed from an exogenous source, e.g., a plasmid or a stably integrated transgene) (e.g., as shown in Table 1) and a cargo fused to a human endogenous or other cell membrane recruitment domain (e.g., as shown in Table 6), wherein the cells do not express gag protein except for gag protein encoded in the human genome or encoded by a consensus sequence derived from gag protein found in the human genome (overexpressed from an exogenous source, e.g., a plasmid or a stably integrated transgene). In some embodiments, the cells are primary or stable human cell lines, e.g., human embryonic kidney (HEK) 293 cells, HEK293 T cells, or BeWo cells. The cells may be used to produce heVLPs as described herein.

[0015] In some embodiments, the method involves using cells that may or may not be engineered to express any foreign protein other than the HERV envelope (e.g., as shown in Table 1) and, optionally, a cell membrane recruitment domain (e.g., as shown in Table 6). In this embodiment, the "empty" particles produced can be loaded with a biological or chemical molecule cargo by using nucleofection, lipid, polymer, or CaCl2 transfection, sonication, freeze-thawing, and / or heat shock of purified particles mixed with the cargo. In all embodiments, the producing cells do not express any human foreign gag protein. This type of loading allows the cargo to be unmodified by fusion with a cell membrane recruitment domain, representing a significant advance over existing VLP technology.

[0016] In other embodiments, the produced and isolated cargo-containing heVLPs can carry additional biomolecular or chemical cargo by using nucleofection, lipid, polymer, or CaCl2 transfection, sonication, freeze-thawing, incubation at various temperatures, and / or heat shock of purified particles mixed with the cargo.

[0017] In some embodiments, the cargo is a therapeutic or diagnostic protein, or a nucleic acid encoding a therapeutic or diagnostic protein.

[0018] In some embodiments, the cargo is a chemical compound or molecule.

[0019] In some embodiments, the chemical molecule is a trigger for protein-protein dimerization or multimerization, such as an A / C heterodimerizer or rapamycin.

[0020] In some embodiments, the chemical compound is a DNA PK inhibitor, such as M3814, NU7026, or NU7441, which potently enhances homology-directed repair gene editing.

[0021] In some embodiments, the biomolecular cargo is a gene editing reagent.

[0022] In some embodiments, the gene editing reagent comprises a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-based genome editing or regulatory protein; a nucleic acid encoding a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-based genome editing or regulatory protein; or a ribonucleoprotein complex (RNP) comprising a CRISPR-based genome editing or regulatory protein.

[0023] In some embodiments, the gene editing reagent is selected from the proteins listed in Tables 2, 3, 4 & 5.

[0024] In some embodiments, the gene editing reagent comprises a CRISPR-based genome editing or regulatory protein, and the heVLP further comprises one or more guide RNAs that bind to and direct the CRISPR-based genome editing or regulatory protein to the target sequence.

[0025] In some embodiments, the cargo comprises a covalent or non-covalent linkage to a human endogenous GAG or other cell membrane recruitment domain, preferably as shown in Table 6. For example, covalent linkages can include direct protein-protein fusions generated from a single reading frame, inteins capable of forming peptide bonds, R-groups, and / or other proteins capable of forming covalent bonds in RNA splicing. Non-covalent linkages can include, for example, DNA / DNA, DNA / RNA, and / or RNA / RNA hybrids (nucleobase pairing to other nucleic acids via hydrogen bonding interactions), protein domains that dimerize or multimerize with or without the need for chemical compounds / molecules to induce protein-protein binding, single-chain variable fragments, nanobodies, affibodies, proteins that bind DNA and / or RNA, proteins with quaternary structure interactions, optogenetic protein domains that can dimerize or multimerize in the presence of specific wavelengths of light, and / or split proteins that spontaneously reconstitute.

[0026] In some embodiments, the cargo comprises a fusion to a dimerization domain or protein-protein binding domain that may or may not require a molecule to cause dimerization or protein-protein binding.

[0027] In some embodiments, the producer cells are FDA-approved cell lines, allogeneic cells, and / or autologous cells derived from a donor.

[0028] In some embodiments, the complete or active peptide domain of human CD47 may be incorporated onto the surface of the heVLP to reduce immunogenicity.

[0029] Examples of AAV proteins included herein are AAV REP52, REP78, and VP1-3. The capsid site into which the protein can be inserted is T138, starting from the VP1 amino acid count. A dimerization domain, for example, can be inserted into the capsid at this point.

[0030] Examples of dimerization domains included herein that may or may not require a small molecule inducer are dDZF1, dDZF2, DmrA, DmrB, DmrC, FKBP, FRB, GCN4 scFv, 10x / 24xGCN4, GFP nanobody, and GFP.

[0031] Examples of split inteins included herein are Npu DnaE, ​​Cfa, Vma, and Ssp DnaE.

[0032] Other examples of split proteins included herein that create a covalent bond together are Spy tag and Spy catcher.

[0033] Examples of RNA binding proteins included herein are MS2, Com, and PP7.

[0034] Examples of synthetic DNA-binding zinc fingers included herein are ZF6 / 10, ZF8 / 7, ZF9, MK10, zinc finger 268, and zinc finger 268 / NRE.

[0035] Examples of proteins that multimerize as a result of quaternary structure included herein are E. coli ferritin and other chimeric forms of ferritin.

[0036] Examples of optogenetic "light-inducible proteins" included herein are Cry2, CIBN, and Lov2-Ja.

[0037] Examples of transduction-enhancing peptides included herein are L17E, Vectofusin, KALA, and various forms of nisin.

[0038] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art can also be used.Materials, methods and examples are only illustrative and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated by reference in their entirety.In the event of any discrepancy, the present specification, including definitions, shall prevail.

[0039] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0040] [Figure 1] Illustrated diagram of exemplary T2 heVLP / T4 heVLP production and transduction for RNP / protein delivery. All heVLP expression constructs are stably integrated into the genome of producer cells. Construct 1-0 corresponds to human endogenous GAG (hGAG). Construct 1-1 corresponds to human endogenous GAG or other phospholipid bilayer recruitment domain. 1-2 corresponds to cargo. 2 corresponds to any guide RNA. 1-0, 1-1, and 1-2 are translated in the cytosol, where fusions of 1-1 and 1-2 are complexed with guide RNA and then recruited to the phospholipid bilayer. 3 corresponds to a HERV-derived glycoprotein (hENV). HERV-derived glycoproteins are expressed as transmembrane proteins on the cell membrane. hGAG buds cargo-containing heVLPs from the cell membrane into the extracellular space. These particles can be purified and can fuse with target cells and deliver cargo by interacting with surface receptors on the target cell surface. [Figure 2]Illustrated diagram of purified heVLPs entering target cells and delivering cargo to the cytosol. Importantly, human endogenous GAG or other phospholipid bilayer recruitment domains allow cargo to enter the target cell nucleus, as long as the cargo has a nucleic acid localization sequence. [Figure 3] Exemplary T1 heVLP-delivered spCas9 genome editing in vitro. HEK 293T cells were transduced with T1 heVLPs containing PLC PH fused to spCas9, hGAGKcon fused to spCas9, or human activity-regulated cytoskeleton-associated protein (hArc) fused to spCas9, targeting VEGF site #3. The heVLPs were pseudotyped with either hENVW (left panel) or hENVFRD (right panel). Genetic modifications were measured by amplicon sequencing. [Figure 4] Schematic diagram of T1 heVLP / T3 heVLP production. The plasmid DNA constructs used for transfection encode the cargo, optional guide RNA, hGAG, and HERV-derived glycoproteins. Plasmids, or other types of DNA molecules, are distributed throughout the producer cell, such that constructs localized in the nucleus express heVLP components and cargo, and constructs localized near the plasma membrane or endosomes are packaged into budding heVLPs. [Figure 5] Illustrative diagram of an exemplary heVLP and cargo composition. The particle was produced by a producer cell expressing an envelope protein. The cargo was packaged into the particle either by expressing the cargo in the producer cell or by introducing the cargo into the particle by various particle introduction methods described herein, such as electroporation. [Figure 6] Illustrative diagram of an exemplary heVLP and cargo composition. The particle was generated by a producer cell expressing an envelope protein. The cargo was packaged into the particle either by expressing a cargo-gag fusion in the producer cell or by introducing the cargo into the particle using various particle introduction methods described herein, such as electroporation. [Figure 7]Illustrative diagram of an exemplary heVLP and cargo configuration. The particle was generated by a producer cell expressing an envelope protein. The cargo was packaged into the particle either by expressing a cargo-PH fusion in the producer cell or by introducing the cargo into the particle using various particle introduction methods described herein, such as electroporation. [Figure 8] Illustrative diagram of an exemplary heVLP and cargo composition. The particle was generated by a producer cell expressing the envelope protein. The cargo was packaged into the particle either by expressing a cargo-gag / PH fusion in the producer cell or by introducing the cargo into the particle using various particle introduction methods described herein, such as electroporation. [Figure 9] Illustrative heVLP and cargo configurations. The particles were generated by producer cells expressing envelope proteins. The cargo was packaged into particles either by expressing gag fused to cargo and DmrA or DmrC in the presence of a dimerization molecule (A / C heterodimerization factor) in the producer cells, or by introducing the particles into the particles by various particle introduction methods described herein, such as electroporation. [Figure 10] Illustrative diagram of exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing an envelope protein. The cargo was packaged into the particle by either expressing the cargo and pH fused with DmrA or DmrC in the presence of a dimerization molecule (A / C heterodimerization factor) in the producer cell, or by introducing the cargo into the particle by various particle introduction methods described herein, such as electroporation. [Figure 11]Illustrative heVLP and cargo configurations. The particles were generated by producer cells expressing envelope proteins. The cargo was packaged into particles either by expressing gag / PH fused to cargo and DmrA or DmrC in the presence of a dimerization molecule (A / C heterodimerization factor) in the producer cells, or by introducing the particles into the particles using various particle introduction methods described herein, such as electroporation. [Figure 12] Illustrative heVLP and cargo configurations. This particle was produced by a producer cell expressing an envelope protein. The cargo was packaged into the particle either by expressing an RNA-binding protein (RBP), gag fused with MS2, which binds to the cargo and the MS2 RNA stem-loop (MS2 SL) complexed with the cargo, in the producer cell, or by introducing it into the particle by various particle introduction methods described herein, such as electroporation. [Figure 13] Illustrative heVLP and cargo configuration. This particle was produced by a producer cell expressing an envelope protein. The cargo was packaged into the particle either by expressing an RNA-binding protein (RBP), PH fused to MS2, which binds to the cargo and the RNA stem-loop (MS2 SL) complexed with the cargo, in the producer cell, or by introducing it into the particle by various particle introduction methods described herein, such as electroporation. [Figure 14] Illustrative heVLP and cargo configuration. This particle was produced by a producer cell expressing an envelope protein. The cargo was packaged into the particle either by expressing gag / PH fused to an RNA-binding protein (RBP), MS2, which binds to the cargo and the RNA stem-loop (MS2 SL) complexed with the cargo, in the producer cell, or by introducing it into the particle by various particle introduction methods described herein, such as electroporation. [Figure 15]Illustrative heVLP and cargo configurations. This particle was produced by a producer cell expressing an envelope protein. The cargo was packaged into the particle either by expressing an RNA-binding protein (RBP), MS2, fused with DmrA or DmrC, which binds to the cargo and the RNA stem-loop (MS2 SL) complexed with gag and cargo in the presence of a dimerization molecule (A / C heterodimerization factor), in the producer cell, or by introducing it into the particle by various particle introduction methods described herein, such as electroporation. [Figure 16] Illustrative heVLP and cargo configurations. This particle was produced by a producer cell expressing an envelope protein. The cargo was packaged into the particle either by expressing an RNA-binding protein (RBP), MS2, fused with DmrA or DmrC, which binds to the cargo and PH and the RNA stem-loop (MS2 SL) complexed with the cargo in the presence of a dimerization molecule (A / C heterodimerization factor), or by introducing it into the particle by various particle introduction methods described herein, such as electroporation. [Figure 17] Illustrative diagram of an exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing an envelope protein. The cargo was packaged into the particle either by expressing an RNA-binding protein (RBP), MS2, fused with DmrA or DmrC, which binds to the cargo and the RNA stem-loop (MS2 SL) complexed with gag / PH and cargo in the presence of a dimerization molecule (A / C heterodimerization factor), in the producer cell, or by introducing it into the particle by various particle introduction methods described herein, such as electroporation. [Figure 18]Illustrative heVLP and cargo configurations. The particles were generated by producer cells expressing envelope proteins. The cargo was packaged into the particles either by expressing gag fused to repeated GCN4 domains bound to the cargo and the scFV fused to the cargo in the producer cells, or by introducing the particles into the particles by various particle introduction methods described herein, such as electroporation. [Figure 19] Illustrative heVLP and cargo configurations. The particles were generated by producer cells expressing envelope proteins. The cargo was packaged into the particles either by expressing the PH fused to a repeat GCN4 domain, which is linked to the cargo and the scFV fused to the cargo, in the producer cells, or by introducing the PH into the particles by various particle introduction methods described herein, such as electroporation. [Figure 20] Illustrative heVLP and cargo configurations. The particles were generated by producer cells expressing envelope proteins. The cargo was packaged into the particles either by expressing gag / PH fused to repeated GCN4 domains bound to the cargo and the scFV fused to the cargo in the producer cells, or by introducing the particles by various particle introduction methods described herein, such as electroporation. [Figure 21] Illustrated diagram of exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing an envelope protein. The cargo was packaged into the particle by a producer cell expressing a repeat GCN4 domain fused with gag and DmrA or DmrC in the presence of a dimerization molecule (A / C heterodimerization factor). GCN4 is linked to an scFV fused with the cargo, which is also expressed in the producer cell. Particles can also be introduced by various particle introduction methods described herein, such as electroporation. [Figure 22]Illustrated diagram of exemplary heVLP and cargo configuration. This particle is produced by a producer cell expressing an envelope protein. The cargo is packaged into the particle by a producer cell expressing a repeat GCN4 domain fused with PH and DmrA or DmrC in the presence of a dimerization molecule (A / C heterodimerization factor). GCN4 is linked to the scFV fused with the cargo, which is also expressed in the producer cell. It can also be introduced into the particle by various particle introduction methods described herein, such as electroporation. [Figure 23] Illustrated diagram of exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing an envelope protein. The cargo was packaged into the particle by a producer cell expressing a repeat GCN4 domain fused with gag / PH and DmrA or DmrC in the presence of a dimerization molecule (A / C heterodimerization factor). GCN4 is linked to an scFV fused with the cargo, which is also expressed in the producer cell. Particles can also be introduced by various particle introduction methods described herein, such as electroporation. [Figure 24] Illustrative diagram of an exemplary heVLP and cargo composition. The particle was produced by a producer cell expressing an envelope protein. The cargo (AAV particle) was packaged into the particle either by expressing the cargo in the producer cell or by introducing the cargo into the particle by various particle introduction methods described herein, such as electroporation. [Figure 25] Illustrative diagram of an exemplary heVLP and cargo composition. The particle was produced by a producer cell expressing the envelope protein. The cargo (AAV particle) was packaged into the particle either by expressing the cargo and gag in the producer cell or by introducing the cargo and gag into the particle by various particle introduction methods described herein, such as electroporation. [Figure 26]Illustrative heVLP and cargo configurations. The particles were produced by producer cells expressing envelope proteins. The cargo (AAV particles) was packaged into the particles either by expressing the cargo and PH in the producer cells or by introducing them into the particles using various particle introduction methods described herein, such as electroporation. [Figure 27] Illustrative diagram of an exemplary heVLP and cargo configuration. The particle was produced by a producer cell expressing an envelope protein. The cargo (AAV particle) was packaged into the particle either by expressing the cargo and gag / PH in the producer cell or by introducing the cargo into the particle by various particle introduction methods described herein, such as electroporation. [Figure 28] Illustrative heVLP and cargo configuration. The particles were produced by producer cells expressing envelope proteins. Cargo (AAV particles with DmrB inserted into the capsid protein, VP2) was packaged into particles either by expressing the cargo and gag fused with DmrB in producer cells in the presence of DmrB dimerization molecules, or by introducing the particles into the particles by various particle introduction methods described herein, such as electroporation. [Figure 29] Illustrative heVLP and cargo configurations. The particles were produced by producer cells expressing envelope proteins. The cargo (AAV particles with DmrB inserted into the capsid protein, VP2) was packaged into particles by either expressing PH fused with cargo and DmrB in producer cells in the presence of DmrB dimerization molecules, or by introducing PH into particles by various particle introduction methods described herein, such as electroporation. [Figure 30]Illustrative heVLP and cargo configurations. The particles were produced by producer cells expressing envelope proteins. The cargo (AAV particles with DmrB inserted into the capsid protein, VP2) was packaged into particles either by expressing gag / PH fused to the cargo and DmrB in producer cells in the presence of DmrB dimerization molecules, or by introducing the particles into the particles by various particle introduction methods described herein, such as electroporation. [Figure 31] Illustrative heVLP and cargo configurations. The particles were produced by producer cells expressing envelope proteins. Cargo (AAV particles with DmrB inserted into the capsid protein, VP2) was packaged into particles by either expressing the cargo and gag fused with DmrA, DmrB, or DmrC in producer cells in the presence of DmrB dimerization and A / C heterodimerization molecules, or by introducing the particles into the particles by various particle introduction methods described herein, such as electroporation. [Figure 32] Illustrative heVLP and cargo configurations. This particle was produced by a producer cell expressing an envelope protein. The cargo (an AAV particle with DmrB inserted into the capsid protein, VP2) was packaged into the particle by either expressing the cargo and PH fused with DmrA, DmrB, or DmrC in the producer cell in the presence of DmrB dimerization and A / C heterodimerization molecules, or by introducing the PH into the particle by various particle introduction methods described herein, such as electroporation. [Figure 33]Illustrative heVLP and cargo configurations. The particles were produced by producer cells expressing envelope proteins. Cargo (AAV particles with DmrB inserted into the capsid protein, VP2) was packaged into particles by either expressing gag / PH fused to cargo and DmrA, DmrB, or DmrC in producer cells in the presence of DmrB dimerization and A / C heterodimerization molecules, or by introducing the particles into the particles by various particle introduction methods described herein, such as electroporation. [Figure 34] Illustrative diagram of an exemplary heVLP and cargo configuration. The particle was produced by a producer cell expressing the envelope protein. Cargo (single-stranded DNA) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. [Figure 35] Illustrative diagram of an exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing envelope proteins and gag. Cargo (single-stranded DNA) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. [Figure 36] Illustrative diagram of an exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing envelope proteins and PH. Cargo (single-stranded DNA) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. [Figure 37] Illustrative diagram of an exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing envelope proteins and gag / PH. Cargo (single-stranded DNA) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. [Figure 38]Illustrative diagram of an exemplary heVLP and cargo configuration. The particle was produced by a producer cell expressing the envelope protein. Cargo (double-stranded DNA) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. [Figure 39] Illustrative diagram of an exemplary heVLP and cargo configuration. The particle was produced by a producer cell expressing envelope proteins and gag. Cargo (double-stranded DNA) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. [Figure 40] Illustrative diagram of an exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing envelope protein and PH. Cargo (double-stranded DNA) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. [Figure 41] Illustrative diagram of an exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing envelope proteins and gag / PH. Cargo (double-stranded DNA) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. [Figure 42] Illustrative diagram of an exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing gag fused to an envelope protein and a zinc finger protein (ZFP) that binds to a specific sequence within the cargo. Cargo (double-stranded DNA) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. [Figure 43] Illustrative diagram of an exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing a PH fused to an envelope protein and a zinc finger protein (ZFP) that binds to a specific sequence within the cargo. Cargo (double-stranded DNA) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. [Figure 44] Illustrative diagram of an exemplary heVLP and cargo configuration. This particle was generated by a producer cell expressing gag / PH fused to a zinc finger protein (ZFP) that binds to a specific sequence within the envelope protein and cargo. Cargo (double-stranded DNA) can also be packaged into particles by various particle introduction methods described herein, such as electroporation. [Figure 45] Illustrative diagram of an exemplary heVLP and cargo configuration. The particle was generated by a producer cell expressing a zinc finger protein (ZFP) that binds to a specific sequence within the cargo fused to DmrA or DmrC in the presence of envelope proteins and gag and A / C heterodimerization molecules. Cargo (double-stranded DNA) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. [Figure 46] Illustrative diagram of an exemplary heVLP and cargo configuration. This particle was generated by a producer cell expressing a zinc finger protein (ZFP) that binds to a specific sequence within the cargo fused to DmrA or DmrC in the presence of envelope proteins and PH and A / C heterodimerization molecules. Cargo (double-stranded DNA) can also be packaged into particles by various particle introduction methods described herein, such as electroporation. [Figure 47] Illustrative diagram of an exemplary heVLP and cargo configuration. The particle was generated by a producer cell expressing a zinc finger protein (ZFP) that binds to a specific sequence within the cargo fused to DmrA or DmrC in the presence of envelope proteins and gag / PH and A / C heterodimerization molecules. Cargo (double-stranded DNA) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. [Figure 48]Illustrated diagram of exemplary heVLP and cargo configuration. This particle is produced by a producer cell that expresses gag fused with an envelope protein and a zinc finger protein (ZFP) that binds to a specific sequence within the cargo. Cargo (double-stranded DNA bound to Cas9 RNP-ZFP fusion) can also be packaged into particles by various particle introduction methods described herein, such as electroporation. Alternatively, Cas9 RNP-ZFP fusion can be expressed by a producer cell and introduced into particles by various particle introduction methods described herein, such as electroporation. [Figure 49] Illustrated diagram of exemplary heVLP and cargo configuration. This particle is produced by a producer cell that expresses PH fused with an envelope protein and a zinc finger protein (ZFP) that binds to a specific sequence within the cargo. Cargo (double-stranded DNA with Cas9 RNP-ZFP fusion bound thereto) can also be packaged into particles by various particle introduction methods described herein, such as electroporation. Alternatively, Cas9 RNP-ZFP fusion can be expressed by a producer cell and introduced into particles by various particle introduction methods described herein, such as electroporation. [Figure 50] Illustrated diagram of exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing gag / PH fused with an envelope protein and a zinc finger protein (ZFP) that binds to a specific sequence within the cargo. The cargo (double-stranded DNA bound to a Cas9 RNP-ZFP fusion) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. Alternatively, the Cas9 RNP-ZFP fusion can be expressed by a producer cell and introduced into the particle by various particle introduction methods described herein, such as electroporation. [Figure 51]Illustrated diagram of exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing gag fused to a zinc finger protein (ZFP) fused to DmrA or DmrC, which binds to a specific sequence within the cargo in the presence of an envelope protein and an A / C heterodimerization molecule. The cargo (double-stranded DNA bound to a Cas9 RNP-ZFP fusion) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. Alternatively, the Cas9 RNP-ZFP fusion can be expressed by the producer cell and introduced into the particle by various particle introduction methods described herein, such as electroporation. [Figure 52] Illustrated diagram of exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing a PH fused to a zinc finger protein (ZFP) fused to DmrA or DmrC, which binds to a specific sequence within the cargo in the presence of an envelope protein and an A / C heterodimerization molecule. The cargo (double-stranded DNA bound to a Cas9 RNP-ZFP fusion) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. Alternatively, the Cas9 RNP-ZFP fusion can be expressed by a producer cell and introduced into the particle by various particle introduction methods described herein, such as electroporation. [Figure 53] Illustrated diagram of exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing gag / PH fused to a zinc finger protein (ZFP) fused to DmrA or DmrC, which binds to a specific sequence within the cargo in the presence of an envelope protein and an A / C heterodimerization molecule. The cargo (double-stranded DNA bound to a Cas9 RNP-ZFP fusion) can also be packaged into the particle by various particle introduction methods described herein, such as electroporation. Alternatively, the Cas9 RNP-ZFP fusion can be expressed by a producer cell, and the particle can be introduced into the particle by various particle introduction methods described herein, such as electroporation. [Figure 54] Illustrative diagram of an exemplary heVLP and cargo configuration. The particle was produced by a producer cell expressing an envelope protein. The cargo (RNA) was packaged into the particle either by expressing the cargo in the producer cell or by introducing it into the particle using various particle introduction methods described herein, such as electroporation. [Figure 55] Illustrative heVLP and cargo configuration. The particles were produced by producer cells expressing envelope proteins. The cargo (RNA) was packaged into the particles either by expressing the cargo and gag in the producer cells or by introducing the cargo into the particles by various particle introduction methods described herein, such as electroporation. [Figure 56] Illustrative heVLP and cargo configuration. The particle was produced by a producer cell expressing an envelope protein. The cargo (RNA) was packaged into the particle either by expressing the cargo and PH in the producer cell or by introducing the cargo into the particle by various particle introduction methods described herein, such as electroporation. [Figure 57] Illustrative diagram of an exemplary heVLP and cargo configuration. The particle was produced by a producer cell expressing the envelope protein. The cargo (RNA) was packaged into the particle either by expressing the cargo and gag / PH in the producer cell or by introducing the cargo into the particle by various particle introduction methods described herein, such as electroporation. [Figure 58] Illustrative heVLP and cargo configurations. This particle was produced by a producer cell expressing an envelope protein. Cargo (RNA with an MS2 stem loop) was packaged into the particle by either expressing the cargo and gag fused with MS2 in the producer cell, or by introducing it into the particle by various particle introduction methods described herein, such as electroporation. [Figure 59]Illustrative heVLP and cargo configuration. This particle was produced by a producer cell expressing an envelope protein. The cargo (RNA with an MS2 stem loop) was packaged into the particle by either expressing the cargo and PH fused with MS2 in the producer cell, or by introducing it into the particle by various particle introduction methods described herein, such as electroporation. [Figure 60] Illustrative heVLP and cargo configuration. This particle was produced by a producer cell expressing an envelope protein. The cargo (RNA with an MS2 stem loop) was packaged into the particle by either expressing the cargo and gag / PH fused with MS2 in the producer cell, or by introducing the cargo into the particle using various particle introduction methods described herein, such as electroporation. [Figure 61] Illustrative heVLP and cargo configurations. This particle was produced by a producer cell expressing an envelope protein. The cargo (RNA with an MS2 stem loop) was packaged into the particle either by expressing MS2 fused to DmrA or DmrC in the presence of the cargo and gag and A / C heterodimerization factors in the producer cell, or by introducing MS2 into the particle by various particle introduction methods described herein, such as electroporation. [Figure 62] Illustrative heVLP and cargo configurations. This particle was produced by a producer cell expressing an envelope protein. The cargo (RNA with an MS2 stem loop) was packaged into the particle by either expressing MS2 fused to DmrA or DmrC in the presence of the cargo and PH and A / C heterodimerization factors in the producer cell, or by introducing MS2 into the particle by various particle introduction methods described herein, such as electroporation. [Figure 63]Illustrative heVLP and cargo configurations. This particle was produced by a producer cell expressing an envelope protein. The cargo (RNA with an MS2 stem loop) was packaged into the particle either by expressing MS2 fused to DmrA or DmrC in the presence of the cargo and gag / PH and A / C heterodimerization factors in the producer cell, or by introducing MS2 into the particle by various particle introduction methods described herein, such as electroporation. [Figure 64] Illustrative diagram of exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing an envelope protein. Cargo (RNA with an RBP stem loop) was packaged into the particle by either expressing the cargo fused with an RBP and gag fused with another RBP in the producer cell, or by introducing it into the particle by various particle introduction methods described herein, such as electroporation. [Figure 65] Illustrative heVLP and cargo configuration. This particle was produced by a producer cell expressing an envelope protein. The cargo (RNA with an RBP stem loop) was packaged into the particle by either expressing the cargo fused with an RBP and the PH fused with another RBP in the producer cell, or by introducing the cargo into the particle using various particle introduction methods described herein, such as electroporation. [Figure 66] Illustrative heVLP and cargo configurations. This particle was produced by a producer cell expressing an envelope protein. The cargo (RNA with an RBP stem loop) was packaged into the particle either by expressing the cargo fused to an RBP and gag / PH fused to another RBP in the producer cell, or by introducing the particles into the particle by various particle introduction methods described herein, such as electroporation. [Figure 67]Illustrative heVLP and cargo configurations. This particle was produced by a producer cell expressing an envelope protein. The cargo (RNA with an RBP stem loop) was packaged into the particle by either expressing the cargo fused to the RBP and other RBPs fused to DmrA or DmrC in the presence of gag and A / C heterodimerization molecules in the producer cell, or by introducing the particles into the particle by various particle introduction methods described herein, such as electroporation. [Figure 68] Illustrated diagram of exemplary heVLP and cargo configuration. This particle was produced by a producer cell expressing an envelope protein. Cargo (RNA with an RBP stem loop) was packaged into the particle by either expressing the cargo fused to the RBP and other RBPs fused to DmrA or DmrC in the presence of PH and A / C heterodimerization molecules, or by introducing the particles into the particles by various particle introduction methods described herein, such as electroporation. [Figure 69] Illustrative heVLP and cargo configurations. This particle was produced by a producer cell expressing an envelope protein. The cargo (RNA with an RBP stem loop) was packaged into the particle by either expressing the cargo fused with the RBP and other RBPs fused with gag / PH and DmrA or DmrC in the presence of an A / C heterodimerization molecule in the producer cell, or by introducing the particles into the particle by various particle introduction methods described herein, such as electroporation. DETAILED DESCRIPTION OF THE INVENTION

[0041] Therapeutic proteins and nucleic acids hold great promise, but for many of these, intracellular delivery of large biomolecules remains an obstacle to clinical development. Genome editing reagents, such as zinc finger nucleases (ZFNs) or RNA-guided enzymatically active / inactive DNA-binding proteins, such as Cas9, have rapidly advanced in terms of the specificity and type of editing they can perform, but the obstacle of safe in vivo delivery still prevents effective gene editing therapy. The following details the properties of heVLPs that make them a novel and optimal platform for the delivery of genome editing reagents, which makes them in contrast to classical delivery modes.

[0042] Retroviral particles, such as lentiviruses, have been developed to deliver RNA that is reverse-transcribed into DNA, which may or may not be integrated into genomic DNA. VLPs have also been developed, which mimic viral particles in their self-assembly ability but lack some of the core viral genes and are therefore not infectious. Both lentiviral and VLP vectors are typically produced by transiently transfecting a producer cell line with a plasmid encoding all the components necessary for lentiviral particle or VLP production. One major drawback we have discovered with lentiviral particles and VSVG-based VLPs produced by this traditional transient transfection method is that, in addition to the conventional cargo, these particles also package and deliver the plasmid DNA used in the initial transient transfection. This unintended delivery of plasmid DNA can be immunogenic and can cause undesirable effects, such as integration of the plasmid DNA into genomic DNA. Identifying the types of biomolecules and / or chemicals to be delivered within the particles is important, and heVLPs have been designed to perform this important function.

[0043] The heVLPs described herein can deliver DNA alone, DNA + RNA + protein, or RNA + protein. Importantly, heVLPs are the first VLP delivery modality to utilize components selected from human endogenous retroviruses (HERVs) to create particles for delivering customizable cargo to eukaryotic cells. HeVLPs can control the form of the cargo (DNA, protein, and / or RNA). All other previously reported VLPs and viral particles package and deliver undesired plasmid DNA (or other types of DNA-based gene expression constructs) introduced into particle-producing cells by transient transfection in addition to the intended protein and / or RNA cargo.

[0044] Another non-obvious aspect of heVLPs is the ENV protein on their surface. The ENV protein is responsible for the ability of heVLPs to efficiently deliver cargo into cells. Most retroviral ENV proteins require post-translational modification in the form of proteolytic cleavage of the intracellular domain (ICD) of the ENV protein to activate the fusogenic properties of the ENV protein, which is essential for infectivity. 1 All of the envelope proteins listed in Table 1 are derived from HERVs (or HERV ENV consensus sequences) that are expressed at various levels in healthy human tissues. Some of these sequences have ICD cleavages that have been shown to enhance fusogenicity, but most do not require cleavage.

[0045] heVLP utilizes endogenous human GAG proteins (or HERV GAG consensus sequences) derived from HERVs, and therefore does not require exogenous viral GAGs for particle formation. 1These HERV GAG proteins enable heVLP formation and are expressed at various levels in healthy human tissues. Importantly, heVLPs differ from previously reported virus particles, VLPs, and extracellular vesicles because they are composed of a novel combination of HERV ENV and GAG components and do not contain exogenous viral components. 2、3 Due to the design optimization described above, heVLPs are particularly suitable for the delivery of combinations of biomolecules and / or chemicals, such as DNA, RNA, proteins, or DNA-encoded or RNP-based genome editing reagents.

[0046] Genome-editing reagents, particularly CRISPR-CAS, zinc finger, and TAL nuclease-based reagents, have the potential to become in vivo therapies for the treatment of genetic diseases, but the techniques for delivering genome-editing reagents into cells have severe limitations or are unsafe for patients. Traditional therapeutic monoclonal antibody delivery has been successful using direct protein injection. Unfortunately, direct injection strategies for gene-editing proteins such as Cas9 are hindered by immunogenicity, degradability, poor cell specificity, and an inability to cross the cell membrane or escape endosomes / lysosomes. 4~10 Broader application of protein therapy and gene editing may be achieved by delivering therapeutic protein cargo into cells. For example, Cas9 cannot efficiently cross the phospholipid bilayer to enter cells and has been shown to have innate and adaptive immunogenicity. 4~8 Therefore, it is neither practical nor preferable to deliver Cas9 by direct injection or as an outer / inner conjugate to lipid, protein, or metal-based nanoparticles, which are cytotoxic and immunogenic and often result in low levels of desired genetic modification. 9~20 .

[0047] Cargo-loaded nanoparticles are another delivery strategy that can be used to deliver DNA, proteins, RNA, and RNPs into cells. 9~18Nanoparticles can be engineered for cell specificity and can trigger endocytosis and subsequent endosomal lysis. However, nanoparticles may have varying levels of immunogenicity depending on the artificially derived vehicle shell. 9~20 Many nanoparticles rely on strong opposing charge distributions to maintain the particle's structural integrity, making them electrostatically toxic and unsuitable for many in vivo therapeutic scenarios. 9 Nanoparticles that deliver RNA have been successful in recent clinical trials, but they have mostly been used only to deliver siRNA or shRNA. Toxicity from such nanoparticles remains a major concern. 9 Nanoparticles that deliver mRNA encoding RNPs for genome editing have also been successful in recent years, but they have a higher number of off-target effects compared to protein delivery, and RNA is less stable than proteins. 17 Nanoparticles that deliver genome-editing RNPs and DNA represent a breakthrough because they can utilize both homology-directed repair (HDR) and non-homologous end joining (NHEJ). However, the frequency of gene modification in vitro and in vivo is very low, limiting their in vivo application as gene editing therapies. 15 .

[0048] Currently, the clinical standard vehicle for delivering genome-editing therapies is the adeno-associated virus (AAV). AAV vectors are a promising delivery modality that has successfully delivered DNA into eukaryotic cells. However, AAV cannot efficiently package and deliver DNA constructs larger than 4.5 kb, preventing the delivery of many CRISPR-based gene-editing reagents that require larger DNA expression constructs. While CRISPR-based gene-editing reagents can be split into multiple distinct AAV particles, this strategy significantly reduces delivery and editing efficiency. AAV and adenovirus vectors can have varying levels of immunogenicity depending on the required dosage. Furthermore, inverted terminal repeats (ITRs) in AAV DNA constructs promote spontaneous episome formation, which can prolong expression of genome-editing reagents and increase off-target effects. ITRs can also promote undesired integration of AAV DNA into genomic DNA. 21~24 .

[0049] Recently, VLPs have been utilized to deliver mRNA and protein cargo into the cytosol of cells. 2、3、25~30 VLPs have emerged as an alternative delivery mode for retroviral particles. VLPs lack the ability to incorporate retroviral DNA and can be designed to package and deliver proteins / RNPs / DNA. However, most VLPs known to date, including recently designed VLPs that deliver genome editing reagents, utilize gag-pol protein fusions and viral proteases from HIV or other viruses to generate retrovirus-like particles. 25~27、29、30 Second, some VLPs containing RGN must also package and express guide RNAs from lentiviral DNA transcripts. 27 Third, some VLPs require viral proteases to form functional particles and release the genome-editing cargo. 25~27、29This viral protease recognizes and cleaves multiple amino acid motifs, potentially damaging protein cargoes, which may be dangerous for therapeutic applications. Fourth, most VLP delivery methods for genome editing proteins published to date have shown low in vitro and in vivo gene modification efficiencies due to low packaging and transduction efficiencies. 25~27 Fifth, the complex viral genomes utilized in these VLP constructs have multiple reading frames and employ RNA splicing, which can result in the delivery of spurious fusion protein products. 25~27、29、30 Sixth, the presence of reverse transcriptase, integrase, capsid, and viral envelope proteins in these VLPs makes them unsuitable for most therapeutic applications due to concerns about immunogenicity and off-target editing. Finally, most retroviral particles, such as lentiviral particles, are pseudotyped with VSVG, and nearly all VLPs described to date that deliver genome editing reagents contain and utilize VSVG. 2、3、25~30 We found that VSVG-based particles formed by transiently transfecting producer cells package and deliver transfected DNA. Current versions of VSVG-based VLPs cannot prevent this inadvertent DNA delivery, which prevents the use of VLPs in settings where immunogenicity and off-target effects must be minimized.

[0050] Extracellular vesicles are another delivery modality that can package and deliver cargo within exosomes and ectosomes. 31、32Like VLPs, extracellular vesicles are composed of a phospholipid bilayer derived from mammalian cells. Unlike VLPs, extracellular vesicles lack viral components and therefore have limited immunogenicity. While VLPs have a high ability to enter cells via their external fusogenic glycoproteins (VSVGs), extracellular vesicles primarily rely on cellular uptake via micropinocytosis, which limits their delivery efficiency.

[0051] heVLPs seek to leverage the benefits of extracellular vesicles and VLP delivery. HeVLPs are the first VLP format that eliminates all potentially harmful exogenous viral components. The components of heVLPs are known to be involved in the biogenesis of extracellular vesicles and have local immunosuppressive properties, and are expressed in healthy human tissues to minimize the possibility of eliciting an immune response due to central tolerance. 1Compared with previously reported VLPs, extracellular vesicles, AAVs, and nanoparticles, heVLPs are a safer and more effective alternative, particularly for delivering genome-editing reagents. This is because heVLPs are composed entirely of human-derived components and can deliver DNA + RNPs or RNPs alone, whereas other previously reported VLPs cannot prevent the unintended packaging and delivery of DNA for transient transfection. HeVLPs can deliver specialized DNA molecules, whereas previously reported VLPs, nanoparticles, and AAVs cannot or do not. Furthermore, heVLPs can be produced using patient-derived cells (autologous heVLPs) and other FDA-approved cell lines (allogeneic heVLPs) to further reduce the risk of adverse immune reactions. Here, we describe methods and compositions for producing, purifying, and administering heVLPs for genome editing, epigenome modulation, transcriptome editing, and proteome modulation in vitro and in vivo. Desired editing outcomes vary depending on the therapeutic context, requiring different gene-editing reagents. Streptococcus pyogenes Cas9 (spCas9) and Acidaminococcus sp. Cas12a(functionalized) are two of the most common RNA-guided editing enzymes that utilize NHEJ to introduce stop codons or deletions, or HDR to cause insertions. 34~36 Cas9 deaminase fusions, also known as base editors, are the current standard for precisely editing single nucleotides without double-stranded DNA breaks. 37、38Importantly, the present invention provides new methods of packaging and delivering reagents for genome editing, epigenome modulation, transcriptome editing, and proteome modulation applications. Importantly, the present invention also addresses for the first time the phenomenon of inadvertent DNA delivery in VLPs and provides for the first control over the type of biomolecule delivered (DNA, RNA, and / or protein), thereby increasing the variety of therapeutic in vivo genome modifications possible and minimizing adverse off-target effects.

[0052] Section 1: heVLP-mediated delivery of cargo including DNA, proteins, chemical compounds and RNA Conventional VLPs engineered to encapsulate and deliver protein-based cargo typically fuse the cargo to an INT or GAG polyprotein. 25~27、29、30、39、40 After transient transfection of the production plasmid DNA construct, these protein fusions are translated in the cytosol of conventional VLP-producing cell lines, the gag matrix is ​​acetylated and recruited to the plasma membrane, and the gag fusions are encapsulated within the VLPs (along with the unintentional encapsulation of the transient transfection DNA) as they bud out of the membrane.

[0053] In contrast, the heVLPs described herein can package protein-based cargo by integrating all production DNA into the genomic DNA of the production cell line. Once the cell line is created, protein-delivering heVLPs can be produced constitutively or inducibly. Proteins are packaged into heVLPs by fusing selected human endogenous GAG proteins or other cell membrane recruitment domains with the protein-based cargo (e.g., as shown in Table 6). Human endogenous GAG proteins and human pleckstrin homology (PH) domains localize to biological membranes. The PH domain interacts with phosphatidylinositol lipids and proteins in biological membranes, such as PIP2, PIP3, βγ subunits of GPCRs, and PKC. 41、42However, human endogenous GAG proteins, in addition to localizing to the phospholipid bilayer, promote budding and particle formation. 42 The dual function of these human endogenous GAGs allows for cargo packaging and particle budding / formation. One such human endogenous GAG protein used for this purpose is the human Arc protein, which may be fused with a protein-based cargo to recruit the cargo to the cytosolic side of the phospholipid bilayer. 43 These human endogenous GAG phospholipid bilayer recruitment domains can be fused to the N- or C-terminus of protein-based cargoes via polypeptide linkers of variable length, regardless of the location of one or more nuclear localization sequences (NLSs) within the cargo. Preferably, the linker between the protein-based cargo and the human endogenous GAG phospholipid bilayer recruitment domain is a polypeptide linker of 5-20, e.g., 8-12, e.g., 10 amino acids in length, primarily composed of glycine and serine. The human endogenous GAG or other phospholipid bilayer recruitment domain localizes the cargo to the phospholipid bilayer, and this protein cargo is packaged into heVLPs that bud from the producing cell toward the extracellular space (Figure 1). The use of these human endogenous GAGs and other phospholipid bilayer recruitment domains in this application is novel and unique in that these human endogenous GAGs and other proteins facilitate localization of cargo to the cytosolic face of the plasma membrane in heVLP-producing cells and enable localization of cargo to the nucleus of heVLP-transduced cells without the use of exogenous retroviral GAGs or chemical and / or light-based dimerization systems (Figure 2). For example, delivery efficiency is significantly improved with Cas9 heVLPs when fused to a human endogenous GAG protein, a PH plasma membrane recruitment domain, or no fusion at all (Figure 3).

[0054] HeVLPs can also package and deliver combinations of DNA and RNA when produced by transient transfection into producer cell lines. DNA transfected into cells has size-dependent mobility, with a fraction of the transfected DNA remaining in the cytosol, while another fraction of the transfected DNA becomes localized in the nucleus. 44~46 One fraction of the transfected DNA in the nucleus expresses the components necessary to generate heVLPs, while the other fraction in the cytosol / near the cell membrane is encapsulated and delivered within the heVLPs (Figure 4).

[0055] As used herein, heVLP "cargo" may refer to one or more of a chemical substance, such as a small molecule compound, a combination of DNA, RNA, or protein, a combination of RNA and protein, a combination of DNA and protein, or a protein, for example, for therapeutic or diagnostic use, or for genome editing, epigenome regulation, and / or transcriptome regulation. Additionally, endogenous RNA and protein from the producing cell are packaged and / or incorporated into heVLPs. To simplify this distinction, a combination of foreign DNA, foreign RNA, and protein (foreign and / or endogenous protein) is referred to as type 1 cargo (T1 heVLP), a combination of foreign RNA and protein (foreign and / or endogenous protein) is referred to as type 2 cargo (T2 heVLP), a combination of foreign DNA and protein (foreign and / or endogenous protein) is referred to as type 3 cargo (T3 heVLP), and a combination of protein (foreign and / or endogenous protein) is referred to as type 4 cargo (T4 heVLP). Thus, T1 contains DNA, RNA, and / or foreign protein; T2 contains RNA and / or foreign protein; T3 contains DNA and / or foreign protein; and T4 is a particle with or without foreign protein cargo. Therefore, T4, which does not contain foreign protein, is considered an "empty particle" because it lacks "exogenous cargo." "Exogenous cargo" refers to cargo that is not endogenous to the producing cell and can be packaged and / or incorporated into heVLPs. Furthermore, T1-T4 heVLPs can package exogenous chemical molecules in addition to the types of cargo present in the T1-T4 heVLPs. RNA in this context can be, for example, a single-stranded guide RNA (sgRNA), a CRISPR (Clustered Regularly Interspaced Palindromic Repeat) RNA (crRNA), and / or an mRNA encoding the cargo.

[0056] As used herein, "small molecule" refers to a small organic or inorganic molecule having a molecular weight of less than about 3,000 Daltons. Generally, small molecules useful in the present invention have a molecular weight of less than 3,000 Daltons (Da). Small molecules can be, for example, at least about 100 Da to about 3,000 Da (e.g., about 100 to about 3,000 Da, about 100 to about 2,500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1,500, about 500 to about 1,000, about 300 to about 1,000 Da, or about 100 to about 250 Da).

[0057] The cargo is limited by the diameter of the particle, for example, in some embodiments, in the range of 150 nm to 500 nm.

[0058] Cargoes developed for genome editing applications also include nucleases and base editors, such as FokI and AcuI ZFNs, and transcription activator-like effector nucleases (TALENs) and CRISPR-based nucleases or functional derivatives thereof (e.g., as shown in Table 2). (ZFNs are described, for example, in U.S. Patent Application Publication Nos. 20030232410, 20050208489, 20050026157, 20050064474, 20060188987, 20060063231, and WO 07 / 079944.) No. 014275) (TALENs are described, for example, in U.S. Pat. No. 9,393,257 and International Publication No. WO 2014134412) (CRISPR-based nucleases are described, for example, in U.S. Pat. No. 8,697,359, U.S. Patent Application Publication No. 20180208976, and International Publication Nos. 2014093661 and 2017184786) 34~36Base editors described by this work include any CRISPR-based nuclease ortholog (wt, nickase, or catalytically inactive (CI)), or functional derivatives thereof (e.g., as shown in Table 3), fused at the N-terminus to a deaminase, e.g., as shown in Table 2, with or without a C-terminal fusion to one or more uracil glycosylase inhibitors (UGIs) using polypeptide linkers of variable length (base editors are described, e.g., in U.S. Patent Application Publication No. 20150166982, U.S. Patent Application Publication No. 20180312825, U.S. Patent No. 10113163, and WO 2015089406, WO 2018218188, WO 2017070632, WO 2018027078, WO 2018165629). 37、38 Furthermore, prime editing factors are also compatible with heVLP delivery modes (prime editing factors are described, for example, in Anzalone et al., Nature. 2019 Dec;576(7785):149-157).

[0059] The sgRNA is complexed with the genome editing reagent during packaging and co-delivered within the heVLP. To date, this concept has been demonstrated in vitro by experiments demonstrating the delivery of RGN RNPs by T2 heVLPs for site-specific editing of endogenous sites (Figure 3). For example, Cas9 RNPs were delivered into HEK 293T cells using T2 heVLPs to edit endogenous VEGF site #3 (Figure 3).

[0060] Cargos designed for epigenome regulation include a CI CRISPR-based nuclease, zinc finger (ZF), and TALE fused to an epigenome regulator or combination of epigenome regulators, or functional derivatives thereof linked by one or more variable length polypeptide linkers (Tables 2 and 4). T1-T4 cargos designed for transcriptome editing include a CRISPR-based nuclease from Table 5, or any functional derivative thereof, or a CI CRISPR-based nuclease from Table 5, or any functional derivative thereof fused to a deaminase from Table 3 by one or more variable length polypeptide linkers.

[0061] The cargo may also comprise any therapeutically or diagnostically useful protein, DNA, RNP, or a combination of DNA, protein, and / or RNP.See, for example, International Publication No. 2014005219, US Patent No. 10137206, US Patent Application Publication No. 20180339166, US Patent No. 5892020, European Patent No. 2134841, and International Publication No. 2007020965.For example, cargo encoding or composed of nuclease or base-editing protein or RNP or its derivative can be delivered to retinal cells to correct splicing site defects that cause Lieber's congenital ecchymosis type 10. In the mammalian inner ear, heVLP delivery of base editing reagents or HDR-promoting cargoes to sensory cells such as cochlear supporting cells and hair cells may help restore hearing loss by editing β-catenin (β-catenin Ser33 is edited to Tyr, Pro, or Cys) to make it more stable.

[0062] In other applications, heVLP delivery of RNA editing or proteome-perturbing reagents can cause a transient reduction in cellular levels of one or more specific proteins of interest (potentially at the systemic level, in specific subsets of cells, such as in specific organs or tumors), thereby creating a therapeutically actionable window in which a secondary agent can be administered (which secondary agent is more effective when the protein of interest is absent or present at low levels). For example, heVLP delivery of RNA editing or proteome-perturbing reagents can cause targeted degradation of MAPK and PI3K / AKT proteins and associated mRNAs in vemurafenib / dabrafenib-resistant BRAF-driven tumor cells, temporarily reversing BRAF inhibitor resistance (the MAPK / PI3K / AKT pathway-based resistance mechanism is temporarily downregulated by the heVLP cargo), thereby opening a window for vemurafenib / dabrafenib administration. This example is particularly relevant when combined with heVLPs that are antigen-inducing and therefore tumor cell specific.

[0063] In other applications, heVLPs can deliver the Yamanaka factors Oct3 / 4, Sox2, Klf4, and c-Myc to human or mouse fibroblasts to generate induced pluripotent stem cells.

[0064] In other applications, heVLPs may deliver dominant negative forms of proteins to elicit a therapeutic effect.

[0065] Antigen-specific heVLPs can be targeted to cancer cells to deliver the pro-apoptotic proteins BIM, BID, PUMA, NOXA, BAD, BIK, BAX, BAK, and / or HRK, causing apoptosis of the cancer cells.

[0066] Ninety percent of patients with pancreatic cancer present with unresectable disease. Approximately 30% of patients with unresectable pancreatic tumors die from local disease progression. Therefore, it is desirable to treat locally advanced pancreatic tumors by ablating them with radiation; however, the intestinal tract cannot tolerate the high doses of radiation required to cause tumor resection. Selective radioprotection of the intestinal tract would allow for ablative radiotherapy of pancreatic tumors while minimizing damage to the surrounding gastrointestinal tract. To this end, dCas9 fused with guide RNAs targeting the transcriptional repressors KRAB and EGLN can be introduced into heVLPs. Inhibition of EGLN has been shown to significantly reduce gastrointestinal toxicity from ablative radiation treatments, as it results in selective radioprotection of the gastrointestinal tract but not the pancreatic tumor. 47 .

[0067] Unbound steroid receptors reside in the cytosol. After binding to ligand, these receptors translocate to the nucleus and initiate transcription of response genes. heVLPs can deliver single-chain variable fragment (scFv) antibodies into the cytosol of cells that bind to and destroy cytosolic steroid receptors. For example, scFvs can bind to the glucocorticoid receptor and block dexamethasone binding, thereby blocking transcription of response genes such as metallothionein 1E, which is associated with tumorigenesis. 48 .

[0068] HeVLPs can be adapted for treatments involving targeted destruction of proteins. For example, heVLPs can be used to target and destroy proteins in the cytosol of cells by delivering antibodies / scFvs to the cytosol of cells. Previously, it was well known that delivering antibodies to the cytosol of cells through the cell membrane was difficult and inefficient. This method of inhibiting proteins is similar to the way targeted small molecules bind to and destroy proteins in the cytosol, and may be useful in treating a variety of diseases. 49~51 .

[0069] Furthermore, targeting of small molecules is limited to proteins of a certain size that contain binding pockets associated with catalytic function or protein-protein interactions. scFvs are not hindered by these limitations because they can be generated to bind to many different sites on proteins to inhibit catalytic function and interactions with other proteins. For example, RAS oncoproteins are involved in many cancer subtypes, and RAS is one of the most frequently observed oncogenes in cancer. For example, the International Cancer Genome Consortium found that KRAS is mutated in 95% of pancreatic adenocarcinoma samples. RAS isoforms are known to activate various pathways that are dysregulated in human cancers, such as the PI3K and MAPK pathways. Despite the aberrant role of RAS in cancer, effective pharmacological direct or indirect small molecule inhibitors of RAS have not been developed or approved for clinical use. One strategy for targeting RAS could be heVLPs, which can specifically deliver scFvs that bind to multiple RAS isoforms and inhibit their function to cancer cells. 49~51 .

[0070] Figures 5-69 show non-limiting examples of exemplary heVLP constructs and cargo molecules.

[0071] Section 2: heVLP composition, production, purification and applications HeVLPs are produced from producer cell lines that are transiently transfected with at least one plasmid or stably express a construct integrated into the genomic DNA of the producer cell line. In some embodiments, for T1 and T3 heVLPs, if a single plasmid is used for transfection, it should contain sequences encoding one or more HERV-derived glycoproteins (e.g., as shown in Table 1) fused with a human endogenous GAG or other cell membrane recruitment domain (e.g., as shown in Table 6), one or more HERV-derived GAG proteins, cargo (e.g., a therapeutic protein or gene editing reagent, such as a zinc finger, transcription activator-like effector (TALE), and / or CRISPR-based genome editing / regulatory protein and / or RNP, such as others listed in Tables 2, 3, 4, and 5), and, optionally, a guide RNA. Preferably, two to three plasmids are used for transfection. These two to three plasmids can include the following (any two or more can be combined in one plasmid): 1. A plasmid comprising a sequence encoding a therapeutic protein or genome editing reagent fused to a human endogenous GAG or other cell membrane recruitment domain. 2. A plasmid containing one or more HERV-derived glycoproteins (e.g., those listed in Table 1). 3. A plasmid containing one or more HERV-derived GAG proteins. 4. If the genome editing reagent from Plasmid 1 requires one or more guide RNAs, a plasmid containing one or more guide RNAs appropriate for the genome editing reagent in Plasmid 1.

[0072] If it is desired to deliver DNA molecules other than plasmids, the transfection methods described above can be performed using double-stranded, closed-circular, linear DNA, episomes, minicircles, double-stranded oligonucleotides, and / or other specialized DNA molecules. Alternatively, for T2 and T4 heVLPs, producer cell lines can be generated that stably express the constructs (1-3) described in the transfection methods above.

[0073] A plasmid, or other type of specialized DNA molecule as described above, will also preferably include other elements to drive expression or translation of the encoded sequence, such as a promoter sequence, an enhancer sequence, e.g., a 5' untranslated region (UTR) or a 3' UTR, a polyadenylation site, an insulator sequence, or other sequences that increase or regulate expression (e.g., an inducible promoter element).

[0074] Preferably, suitable producer cell lines are primary or stable human cell lines that are resistant to the effects of transfection reagents and glycoprotein-induced fusion. Examples of suitable cell lines include human embryonic kidney (HEK) 293 cells, HEK293 T / 17 SF cells, kidney-derived Phoenix-AMPHO cells, and placenta-derived BeWo cells. For example, such cells can be selected for their ability to grow as adherent or suspension cells. In some embodiments, producer cells can be cultured in classic DMEM under serum, serum-free, or exosome-free serum conditions. T1 and T3 heVLPs can be produced from patient-derived cells (autologous heVLPs) and other FDA-approved cell lines (allogeneic heVLPs), as long as these cells can be transfected with DNA constructs encoding the above-mentioned heVLP production components using various techniques known in the art.

[0075] Furthermore, if desired, two or more genome editing reagents can be included in the transfection. The DNA construct can be designed to overexpress a protein in the production cell line. The plasmid backbone used for transfection can be one well known to those skilled in the art, such as a pCDNA3 backbone using a CMV promoter for RNA polymerase II transcription or a U6 promoter for RNA polymerase III transcription. Various techniques known in the art can be employed to introduce nucleic acid molecules into production cells. Such techniques include chemically promoted transfection using compounds such as calcium phosphate, cationic lipids, and cationic polymers; cationic liposomes such as LIPOFECTAMINE (LIPOFECTAMINE 2000 or 3000, and TransIT-X2); liposome-mediated transfection using polyethyleneimine; and non-chemical methods such as electroporation, particle bombardment, and microinjection.

[0076] Human producer cell lines that stably express the necessary heVLP components constitutively and / or inducibly can be used to produce T2 and T4 heVLPs. T2 and T4 heVLPs can be produced from patient-derived cells (autologous heVLPs) and other FDA-approved cell lines (allogeneic heVLPs) when these cells are converted into stable cell lines expressing the aforementioned heVLP components.

[0077] Also provided herein are the production cells themselves.

[0078] In some embodiments, for efficient recruitment of cargo to heVLPs, the cargo preferably comprises a covalent or non-covalent linkage to a human endogenous GAG or other cell membrane recruitment domain, as shown in Table 6. Covalent linkages can include, for example, direct protein-protein fusions generated from a single reading frame, inteins capable of forming peptide bonds, R-groups, and / or other proteins capable of forming covalent bonds upon RNA splicing.52~54 Non-covalent bonds include, for example, DNA / DNA, DNA / RNA, and / or RNA / RNA hybrids (where nucleic acids base pair with other nucleic acids through hydrogen bond interactions), protein domains that dimerize or multimerize with or without the need for chemical compounds / molecules that induce protein-protein binding (e.g., DmrA / DmrB / DmrC (Takara Bio), FKBP / FRB 55 , dDZF 56 , and leucine zipper 57 etc.), single-chain variable fragments 58 , nanobody 59 , Affibody 60 , proteins that bind to DNA and / or RNA, proteins with quaternary structure interactions, optogenetic protein domains that can dimerize or multimerize in the presence of specific light wavelengths. 61 , and / or naturally reconstituted split proteins 62 Examples include:

[0079] In some embodiments, the cargo comprises a fusion with a dimerization domain or a protein-protein binding domain that may or may not require a molecule to cause dimerization or protein-protein binding.

[0080] In some embodiments, the producer cells are FDA-approved cell lines, allogeneic cells, and / or autologous cells derived from a donor.

[0081] In some embodiments, the complete or active peptide domain of human CD47 may be incorporated onto the surface of the heVLP to reduce immunogenicity.

[0082] Examples of AAV proteins included herein are AAV REP52, REP78, and VP1-3. The capsid site into which the protein can be inserted is T138, starting from the amino acid count of VP1. 63 A dimerization domain, for example, can be inserted at this point in the capsid.

[0083] Examples of dimerization domains included herein that may or may not require a small molecule inducer are dDZF1 56 , dDZF2 56 , DmrA (Takara Bio), DmrB (Takara Bio), DmrC (Takara Bio), FKBP 55 , Fed. 55 , GCN4 scFv 58 , 10x / 24x GCN4 58 , GFP nanobody 59 , and GFP 64 is.

[0084] Examples of split inteins included herein are Npu DnaE, ​​Cfa, Vma, and Ssp DnaE. 52 .

[0085] Other examples of split proteins that create covalent bonds together that are included herein are Spy Tag and Spy Catcher. 53 .

[0086] Examples of RNA-binding proteins included herein are MS2, Com, and PP7. 65 .

[0087] Examples of synthetic DNA-binding zinc fingers included herein are ZF6 / 10, ZF8 / 7, ZF9, MK10, zinc finger 268, and zinc finger 268 / NRE. 66、67 .

[0088] Examples of proteins that multimerize as a result of quaternary structure included herein are E. coli ferritin and other chimeric forms of ferritin. 68、69 .

[0089] Examples of optogenetic "light-inducible proteins" included herein are Cry2, CIBN, and Lov2-Ja. 61 .

[0090] Examples of transduction-enhancing peptides included herein include L17E 70 , Vectofusin-1 (Miltenyi Biotec), KALA 71 , and various forms of nisin 72 is.

[0091] In other embodiments, the produced and isolated T1-T4 heVLPs can be loaded with biological or chemical molecular cargo using nucleofection, lipid, polymer, or CaCl transfection, sonication, freeze-thawing, incubation at various temperatures, and / or heat shock of purified particles mixed with the cargo. These techniques are adapted from techniques used to load cargo into exosomes for therapeutic or research applications. 73~75 For example, 100 µg of heVLPs can be resuspended in 200–450 µl of 50 mM trehalose in PBS, mixed with the desired concentration of cargo, and electroporated (GenePulser II Electroporation System with capacitance extender, Bio-Rad, Hercules, CA, USA) in a 0.4 cm cuvette at 0.200 kV and 125 µF.

[0092] Production of cargo-loaded heVLPs and compositions Preferably, heVLPs are harvested from the cell culture supernatant 36-48 hours after transfection, or when the heVLPs reach maximum concentration in the producer cell medium (the producer cells excrete particles into the medium, and at a certain point the particle concentration in the medium is optimal for particle harvesting). The supernatant may be purified by any method known in the art, such as centrifugation, ultracentrifugation, precipitation, ultrafiltration, and / or chromatography. In some embodiments, the supernatant is first filtered, e.g., through a 0.45 μm pore size polyvinylidene fluoride hydrophilic membrane (Millipore Millex-HV) or a 0.8 μm pore size mixed cellulose ester hydrophilic membrane (Millipore Millex-AA), to remove particles larger than 1 μm. After filtration, the supernatant can be further purified and concentrated using, for example, ultracentrifugation, e.g., at 80,000-100,000 x g for 1-2 hours at 1-5°C, or at 8,000-15,000 x g for 10-16 hours at 1-5°C. After this centrifugation step, the heVLPs are concentrated in the form of a centrifugal pellet, which can be resuspended to a desired concentration, mixed with a transduction-enhancing reagent, subjected to buffer exchange, or used directly. In some embodiments, the heVLP-containing supernatant can be filtered, precipitated, centrifuged, and resuspended in a concentrated solution. For example, polyethylene glycol (PEG), e.g., PEG 8000, or antibody-bead conjugates that bind to heVLP surface proteins or membrane components can be used to precipitate the particles. The purified particles are stable and can be stored at 4°C for up to a week or at -80°C for several years without losing appreciable activity.

[0093] Preferably, the heVLPs are resuspended or subjected to buffer exchange so that the particles are suspended in a suitable carrier. In some embodiments, buffer exchange can be performed by ultrafiltration (Sartorius Vivaspin 500 MWCO 100,000). An exemplary suitable carrier for heVLPs used in in vitro applications would preferably be a cell culture medium suitable for the cells to be transduced by the heVLPs. Transduction-enhancing reagents that can be mixed with purified and concentrated heVLP solutions for in vitro applications include reagents known to those skilled in the art (e.g., Miltenyl Biotec Vectofusin-1, Millipore Polybrene, Takara Retronectin, and Sigma Protamine Sulfate). After applying the heVLPs in a suitable carrier to the cells to be transduced, centrifugation can further increase the transduction efficiency. Preferably, the plate containing the heVLPs applied to the cells can be centrifuged at room temperature at a speed of 1,150 g for 30 minutes. After centrifugation, return the cells to an appropriate cell culture incubator (humidified incubator at 37°C and 5% CO2).

[0094] Suitable carriers for heVLPs administered to mammals, particularly humans, will preferably be pharmaceutically acceptable compositions. A "pharmaceutically acceptable composition" refers to any type of non-toxic semi-solid, liquid, or aerosolized filler, diluent, encapsulating material, colloidal suspension, or formulation auxiliary. Preferably, the composition is suitable for injection. It may be, in particular, an isotonic sterile saline solution (monosodium or disodium phosphate, sodium, potassium, calcium, magnesium chloride, and similar solutions or mixtures of such salts), or a dried, particularly lyophilized, composition that can be optionally supplemented with sterile water or saline to constitute an injectable solution. Another suitable pharmaceutical form may be aerosolized particles for administration via intranasal inhalation or endotracheal intubation.

[0095] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or suspensions. The solutions or suspensions may contain additives that are compatible with the heVLPs and do not interfere with the entry of the heVLPs into target cells. In all cases, the form must be sterile and fluid enough to allow the form to be administered via syringe. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. An example of a suitable solution is a buffer such as phosphate-buffered saline.

[0096] Methods for formulating suitable pharmaceutical compositions are known in the art; see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005 and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, and phosphates; tonicity adjusters such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials.

[0097] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough for easy syringability. It must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal.In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin in the composition.

[0098] Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent with one or more of the above-listed components, and then optionally filtration sterilization.Generally, dispersion is prepared by incorporating active compound into sterile vehicle, and this comprises basic dispersion medium and other components that are required from above-listed.For the aseptic powder that is used to prepare aseptic injectable solution, the preferred preparation method is vacuum drying and freeze-drying, and these methods can obtain the powder of active ingredient plus any additional desired components from the solution that has been previously sterilized and filtered.

[0099] The compositions comprising cargo-transfected heVLPs may be included in a container, pack, or dispenser together with instructions for administration. [Example]

[0100] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0101] method HeVLP particles were produced in HEK293T cells using transfection of polyethyleneimine (PEI)-based plasmids. PEI is a 25 kD linear polyethyleneimine (Polysciences #23966-2). To make a "PEI MAX" stock solution, 1 g of PEI was added to 1 L of endotoxin-free dH2O preheated to approximately 80°C and cooled to room temperature. The mixture was neutralized to pH 7.1 by the addition of 10 N NaOH and filter-sterilized through 0.22 μm polyethersulfone (PES). PEI MAX was stored at -20°C.

[0102] HEK293T cells are split to be 70%-90% confluent at the time of transfection and cultured in DMEM medium with 10% FBS. A cargo vector, e.g., a vector encoding a CMV promoter driving expression of a hPLCδ1 PH fusion to a codon-optimized Cas9, is transfected into a U6 promoter-sgRNA-encoding plasmid, hERVK. con GAG(hGAGK con The transfection reaction was assembled in reduced serum medium (Opti-MEM, GIBCO #31985-070). For heVLP particle production in a 10 cm plate, 5 μg of PH-Cas9 expression plasmid, 5 μg of sgRNA expression plasmid, 5 μg of hERVK expression plasmid, and 5 μg of hENVK expression plasmid were co-transfected. conThe GAG ​​expression plasmid and 5 μg of syncytin-1 expression plasmid were mixed in 1 mL of Opti-MEM, followed by the addition of 27.5 μl of PEI MAX. After incubation at room temperature for 20–30 minutes, the transfection reaction mixture was dispensed dropwise onto HEK293T cells.

[0103] HeVLPs were harvested 48–72 h after transfection. The heVLP supernatant was filtered using a 0.8 μm mixed cellulose ester membrane filter and transferred to polypropylene Beckman ultracentrifuge tubes for use with an SW28 rotor (Beckman Coulter #326823). Each ultracentrifuge tube was filled with heVLP-containing supernatant from three 10 cm plates to a final volume of approximately 35–37.5 ml. The heVLP supernatant was ultracentrifuged at approximately 100,000 x g or 25,000 rpm for 2 h at 4°C. After ultracentrifugation, the supernatant was decanted and the heVLP pellet was resuspended in DMEM 10% FBS medium to a concentration approximately 1,000-fold higher than that before ultracentrifugation. The heVLPs were added dropwise to cells seeded in 24-well plates 24 h before transduction. If necessary, supplement with Polybrene (5-10 μg / mL in cell culture medium; Sigma-Aldrich #TR-1003-G) to enhance transduction efficiency. If necessary, add Vectofusin-1 (10 μg / mL in cell culture medium; Miltenyi Biotec #130-111-163) to enhance transduction efficiency. If necessary, immediately after adding heVLPs, centrifuge the 24-well plate at 1,150 x g for 30 min at room temperature to enhance transduction efficiency.

[0104] [Example 1] HEK 293T cells were transfected with VEGF site #3-targeted PLC PH fused with spCas9, hGAGK fused with spCas9, and conThe cells were transduced with T1 heVLPs containing hArc fused to spCas9 or hENVW (left panel) or hENVFRD (right panel). Genetic modification was measured by amplicon sequencing. Particle purification and concentration were performed by PVDF filtration and ultracentrifugation at 100,000 x g for 2 hours. The results are shown in Figure 3. Importantly, the HERV-derived GAG (hGAGK) con ) has not been overexpressed alone in producing cells, and efficient delivery was not achieved.

[0105] [Table 1]

[0106] [Table 2]

[0107] [Table 3]

[0108] [Table 4]

[0109] [Table 5]

[0110] [Table 6] Related protein sequences:

[0111] [ka]

[0112] [ka]

[0113]

change

[0114]

change

[0115]

change

[0116]

change

[0117]

change

[0118]

change

[0119]

change

[0120]

change

[0121]

change

[0122]

change

[0123]

change

[0124]

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[0125]

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[0126]

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[0127]

change

[0128]

change

[0129] References

[0130] Table 7-1

[0131] Table 7-2

[0132] Table 7-3

[0133] Table 7-4

[0134] [Table 7-5]

[0135] [Table 7-6]

[0136] [Table 7-7]

[0137] Other embodiments While the present invention has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to illustrate, but not to limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. The inventions described in the original claims of this application are listed below. [Invention 1] An engineered human virus-like particle (heVLP), wherein the heVLP comprises a membrane comprising a phospholipid bilayer with one or more HERV-derived ENV / glycoproteins on the outside; a HERV-derived GAG protein in the heVLP core on the inside of the membrane; and a cargo disposed in the core of the heVLP, wherein the cargo is fused to a human endogenous GAG or other cell membrane recruitment domain, and the heVLP does not contain non-human gag and / or pol proteins. [Invention 2] The heVLP according to invention 1, wherein the cargo is a therapeutic or diagnostic protein, or a nucleic acid encoding a therapeutic or diagnostic protein, or a small molecule. [Invention 3] The heVLP of Invention 1, wherein the cargo is a gene editing reagent. [Invention 4] The heVLP of Invention 1, wherein the gene editing reagent comprises a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-based genome editing or regulatory protein; a nucleic acid encoding a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-based genome editing or regulatory protein; or a ribonucleoprotein complex (RNP) comprising a CRISPR-based genome editing or regulatory protein. [Invention 5] The heVLP of Invention 4, wherein the gene editing reagent is selected from the proteins listed in Tables 2, 3, 4 and 5. [Invention 6] The heVLP of Invention 4, wherein the gene editing reagent comprises a CRISPR-based genome editing or regulatory protein, and the heVLP further comprises one or more guide RNAs that bind to and guide the CRISPR-based genome editing or regulatory protein to a target sequence. [Invention 7] The heVLP of any one of inventions 1 to 6, wherein the cargo comprises a fusion with a human endogenous GAG or other cell membrane recruitment domain, preferably as shown in Table 6. [Invention 8] A method for delivering a cargo molecule to a target cell, optionally a cell in vivo or in vitro, comprising contacting the cell with a heVLP described in invention 1, comprising the cargo molecule, preferably wherein the cargo molecule is a biological molecule and / or a chemical. [Invention 9] 1. A method for producing heVLPs comprising one or more cargo molecules, comprising: Providing a cell that expresses one or more HERV-derived envelope proteins, one or more HERV-derived GAG proteins, and one or more cargo molecules, wherein the cell does not express gag and / or pol proteins except for gag proteins encoded in the human genome or encoded by a consensus sequence derived from gag proteins found in the human genome; and Maintaining the cells under conditions that allow the cells to produce heVLPs The method comprising: [Invention 10] 10. The method of claim 9, further comprising recovering and optionally purifying and / or concentrating the produced heVLPs. [Invention 11] 10. The method of claim 9, wherein said cargo molecule is a therapeutic or diagnostic protein, or a nucleic acid encoding a therapeutic or diagnostic protein, or a small molecule therapeutic or diagnostic agent. [Invention 12] The method of claim 9, wherein the cargo molecule is a gene editing reagent. [Invention 13] The method of invention 9, wherein the gene editing reagent comprises a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-based genome editing or regulatory protein; a nucleic acid encoding a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-based genome editing or regulatory protein; or a ribonucleoprotein complex (RNP) comprising a CRISPR-based genome editing or regulatory protein. [Invention 14] 14. The method of claim 13, wherein the gene editing reagent is selected from the proteins listed in Tables 2, 3, 4, and 5. [Invention 15] The method of Invention 13, wherein the gene editing reagent comprises a CRISPR-based genome editing or regulatory protein, and the heVLP further comprises one or more guide RNAs that bind to and guide the CRISPR-based genome editing or regulatory protein to a target sequence. [Invention 16] 16. The method according to any one of inventions 9 to 15, wherein the cargo molecule comprises a fusion with a human endogenous GAG or other cell membrane recruitment domain, preferably as shown in Table 6. [Invention 17] envelope proteins from one or more HERVs; one or more HERV-derived GAG proteins, and cargo molecules, preferably fused to a human endogenous GAG or other cell membrane recruitment domain. are expressed by combining Cells that do not express non-human GAG proteins. [Invention 18] 18. The cell according to invention 17, wherein said cargo molecule is a therapeutic or diagnostic protein or a nucleic acid encoding a therapeutic or diagnostic protein. [Invention 19] 18. The cell of claim 17, wherein the cargo molecule is a gene editing reagent. [Invention 20] The cell of invention 17, wherein the gene editing reagent comprises a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-based genome editing or regulatory protein; a nucleic acid encoding a zinc finger (ZF), a transcription activator-like effector (TALE), and / or a CRISPR-based genome editing or regulatory protein; or a ribonucleoprotein complex (RNP) comprising a CRISPR-based genome editing or regulatory protein. [Invention 21] 21. The cell of claim 20, wherein the gene editing reagent is selected from the proteins listed in Tables 2, 3, 4, and 5. [Invention 22] 21. The cell of claim 20, wherein the gene editing reagent comprises a CRISPR-based genome editing or regulatory protein, and the heVLP further comprises one or more guide RNAs that bind to and guide the CRISPR-based genome editing or regulatory protein to a target sequence. [Invention 23] 23. The cell according to any one of claims 17 to 22, wherein the cargo molecule comprises a fusion with a human endogenous GAG or other cell membrane recruitment domain, preferably as shown in Table 6. [Invention 24] 24. The cell according to any one of claims 17 to 23, which is a primary or stable human cell line. [Invention 25] 25. The cell according to claim 24, which is a human embryonic kidney (HEK) 293 cell, an HEK293 T cell, or a BeWo cell.

Claims

1. A particle, the particle comprising: (a) a phospholipid bilayer membrane comprising one or more envelope (ENV) proteins from a human endogenous retrovirus (HERV); and (b) a particle core comprising the gag protein from HERV; and (c) a therapeutic cargo disposed in the particle core inside the phospholipid bilayer membrane. Including, wherein the therapeutic cargo is fused to a cell membrane recruitment domain; The particles do not contain any exogenous viral GAG proteins. The particles.

2. The particle of claim 1, wherein the ENV protein derived from HERV is selected from the group consisting of hENVH1, hENVH2, hENVH3, hENVK1, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVF(c)2, hENVF(c)1, and hENVKcon.

3. The particle according to claim 1 or 2, wherein the HERV-derived ENV protein comprises an amino acid sequence shown in any one of SEQ ID NOs: 2 to 18.

4. The particle of any one of claims 1 to 3, wherein the ENV protein derived from HERV is fused to a targeting polypeptide.

5. The particle of claim 4 , wherein the targeting polypeptide comprises a single-chain variable fragment (scFv).

6. 10. The particle of claim 1, wherein the therapeutic cargo is a gene editing reagent.

7. The particle of claim 6, wherein the gene editing reagent comprises a zinc finger (ZF), a transcription activator-like effector (TALE), or a CRISPR-based genome editing or regulatory protein; a nucleic acid encoding a zinc finger (ZF), a transcription activator-like effector (TALE), or a CRISPR-based genome editing or regulatory protein; or a ribonucleoprotein complex (RNP) comprising a CRISPR-based genome editing or regulatory protein.

8. 8. The particle of claim 6 or 7, wherein the gene editing reagent comprises a CRISPR-based genome editing or regulatory protein, and the particle further comprises one or more guide RNAs that bind to and guide the CRISPR-based genome editing or regulatory protein to a target sequence.

9. The particle of claim 1 , wherein the cell membrane recruitment domain is a human endogenous GAG protein, and the human endogenous GAG protein is a gag protein derived from the HERV.

10. The particle of claim 9, wherein the HERV-derived gag protein comprises a HERV GAG consensus sequence.

11. The particle described in claim 9, wherein the HERV-derived gag protein is encoded by the sequence of SEQ ID NO:

19.

12. The particle described in claim 9, wherein the HERV-derived gag protein comprises a GAG sequence derived from HERV-K113, HERV-K101, HERV-K102, HERV-K104, HERV-K107, HERV-K108, HERV-K109, HERV-K115, HERV-K11p22 or HERV-K12q13 protein.

13. The particle of claim 1 , wherein the cell membrane recruitment domain is a pleckstrin homology (PH) domain.

14. The particle of claim 13, wherein the PH domain is selected from the group consisting of the PH domain of phospholipase Cδ1 (PLCδ1), the PH domain of Akt1 or a variant thereof, and the PH domain of PDPK1.

15. The particle of claim 9, wherein the cell membrane recruitment domain is selected from the group consisting of the PH domain of phospholipase Cδ1 (PLCδ1), the PH domain of Akt1, a mutant PH domain of human Akt1, human Arc, hGAGKcon, the PH domain of PDPK1, human CD9, human CD47, human CD63, and human CD81, and the mutant PH domain of human Akt1 comprises the amino acid substitution E17K compared to the corresponding wild-type PH domain of Akt1.

16. The particle of any one of claims 13 to 15, wherein the cell membrane recruitment domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 19 and 24 to 32.

17. A pharmaceutical composition for use in a method for delivering a therapeutic cargo to a target cell, the method comprising contacting the cell with the particle described in claim 1.

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