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

Abstract

Human-derived virus-like particles (heVLPs), comprising a membrane comprising a phospholipid bilayer with one or more HERV-derived envelope proteins on the external side; one or more HERV-derived GAG proteins in the heVLP core, and a cargo molecule, e.g., a biomolecule and/or chemical cargo molecule, disposed in the core of the heVLP on the inside of the membrane, wherein the heVLP does not comprise a gag protein, except for gag proteins that are encoded in the human genome or gag proteins that are encoded by a consensus sequence that is derived from gag proteins found in the human genome, and methods of use thereof for delivery of the cargo molecule to cells.

1 . An engineered human-derived virus-like particle (heVLP), comprising: a membrane comprising a phospholipid bilayer with one or more HERV-derived ENV/glycoprotein(s) on the external side; a HERV-derived GAG protein in the heVLP core, and a cargo disposed in the core of the heVLP on the inside of the membrane, wherein the cargo is fused to a human-endogenous GAG or other plasma membrane recruitment domain, and the heVLP does not comprise a non-human gag and/or pol protein. 
     
     
         2 . The heVLP of  claim 1 , wherein the cargo is a therapeutic or diagnostic protein or nucleic acid encoding a therapeutic or diagnostic protein, or a small molecule. 
     
     
         3 . The heVLP of  claim 1 , wherein the cargo is a gene editing reagent. 
     
     
         4 . The heVLP of  claim 1 , wherein the gene editing reagent comprises a zinc finger (ZF), transcription activator-like effector (TALE), and/or CRISPR-based genome editing or modulating protein; a nucleic acid encoding a zinc finger (ZF), transcription activator-like effector (TALE), and/or CRISPR-based genome editing or modulating protein; or a riboucleoprotein complex (RNP) comprising a CRISPR-based genome editing or modulating protein. 
     
     
         5 . The heVLP of  claim 4 , wherein the gene editing reagent is selected from the proteins listed in Tables 2, 3, 4 & 5. 
     
     
         6 . The heVLP of  claim 4 , wherein the gene editing reagent comprises a CRISPR-based genome editing or modulating protein, and the heVLP further comprises one or more guide RNAs that bind to and direct the CRISPR-based genome editing or modulating protein to a target sequence. 
     
     
         7 . The heVLP of  claim 1 , wherein the cargo comprises a fusion to a human-endogenous GAG or other plasma membrane recruitment domain, preferably as shown in Table 6. 
     
     
         8 . A method of delivering a cargo molecule to a target cell, optionally a cell in vivo or in vitro, the method comprising contacting the cell with the heVLP of  claim 1  comprising the cargo molecule, preferably wherein the cargo molecule is a biomolecule and/or chemical. 
     
     
         9 . A method of producing a heVLP comprising one or more cargo molecules, the method comprising:
 providing a cell expressing one or more HERV-derived envelope proteins, one or more HERV-derived GAG proteins, and the one or more cargo molecules, wherein the cell does not express a gag and/or pol protein, except for gag proteins that are encoded in the human genome or gag proteins that are encoded by a consensus sequence that is derived from gag proteins found in the human genome; and   maintaining the cell under conditions such that the cells produce heVLPs.   
     
     
         10 . The method of  claim 9 , further comprising harvesting and optionally purifying and/or concentrating the produced heVLPs. 
     
     
         11 . The method of  claim 9 , wherein the cargo molecule is a therapeutic or diagnostic protein or nucleic acid encoding a therapeutic or diagnostic protein, or a small molecule therapeutic or diagnostic agent. 
     
     
         12 . The method of  claim 9 , wherein the cargo molecule is a gene editing reagent. 
     
     
         13 . The method of  claim 9 , wherein the gene editing reagent comprises a zinc finger (ZF), transcription activator-like effector (TALE), and/or CRISPR-based genome editing or modulating protein; a nucleic acid encoding a zinc finger (ZF), transcription activator-like effector (TALE), and/or CRISPR-based genome editing or modulating protein; or a riboucleoprotein complex (RNP) comprising a CRISPR-based genome editing or modulating protein. 
     
     
         14 . The method of  claim 13 , wherein the gene editing reagent is selected from the proteins listed in Tables 2, 3, 4 & 5. 
     
     
         15 . The method of  claim 13 , wherein the gene editing reagent comprises a CRISPR-based genome editing or modulating protein, and the heVLP further comprises one or more guide RNAs that bind to and direct the CRISPR-based genome editing or modulating protein to a target sequence. 
     
     
         16 . The method of  claim 9 , wherein the cargo molecule comprises a fusion to a human-endogenous GAG or other plasma membrane recruitment domain, preferably as shown in Table 6. 
     
     
         17 . A cell that expresses, in combination:
 one or more HERV-derived envelope proteins;   one or more HERV-derived GAG proteins; and   a cargo molecule, preferably fused to a human endogenous GAG or other plasma membrane recruitment domain,   wherein the cell does not express non-human gag protein.   
     
     
         18 . The cell of  claim 17 , wherein the cargo molecule is a therapeutic or diagnostic protein or nucleic acid encoding a therapeutic or diagnostic protein. 
     
     
         19 . The cell of  claim 17 , wherein the cargo molecule is a gene editing reagent. 
     
     
         20 . The cell of  claim 17 , wherein the gene editing reagent comprises a zinc finger (ZF), transcription activator-like effector (TALE), and/or CRISPR-based genome editing or modulating protein; a nucleic acid encoding a zinc finger (ZF), transcription activator-like effector (TALE), and/or CRISPR-based genome editing or modulating protein; or a riboucleoprotein complex (RNP) comprising a CRISPR-based genome editing or modulating protein. 
     
     
         21 . The cell of  claim 20 , wherein the gene editing reagent is selected from the proteins listed in Tables 2, 3, 4 & 5. 
     
     
         22 . The cell of  claim 20 , wherein the gene editing reagent comprises a CRISPR-based genome editing or modulating protein, and the heVLP further comprises one or more guide RNAs that bind to and direct the CRISPR-based genome editing or modulating protein to a target sequence. 
     
     
         23 . The cells of  claim 17 , wherein the cargo molecule comprises a fusion to a human-endogenous GAG or other plasma membrane recruitment domain, preferably as shown in Table 6. 
     
     
         24 . The cells of  claim 17 , wherein the cells are primary or stable human cell lines. 
     
     
         25 . The cells of  claim 24 , which are Human Embryonic Kidney (HEK) 293 cells, HEK293 T cells, or BeWo cells.