Compositions and methods for effective in vivo delivery

Abstract

In aspects, disclosed herein are compositions, methods, kits, and systems relating to the efficient delivery of a cargo (e.g., a therapeutic cargo) into a cell (e.g., for in vivo delivery). More specifically, the delivery vehicle is a lipid-containing particle comprising (a) a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule; (b) a combinatorial protein comprising a plasma membrane localization protein; and (c) a cargo.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 342,773, filed May 17, 2022, which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

[0003] Retroviruses can be attractive scaffolds for virus-like particles (VLPs). Retroviral capsids generally lack the rigid symmetry requirements of many non-enveloped icosahedral viruses (Zhang et al., 2015), suggesting increased structural flexibility to incorporate non-native protein freight. In addition, the tropism of retroviruses can be modulated by pseudotyped virions with different envelope glycoproteins, which can enable VLP targeting to specific cell types (Cronin et al., 2005). Previous work has demonstrated that fusing a desired protein freight to the C-terminus of the retroviral gag polyprotein is sufficient to direct packaging of that freight protein within retroviral particles (Kaczmarczyk et al., 2011; Voelkel et al., 2010). More recently, a similar strategy has been applied to package Cas9 RNPs within retroviral particles (Hamilton et al., 2021; Mangeot et al., 2019). However, there remains a need for VLPs with in vivo delivery efficiencies at therapeutic levels. SUMMARY OF THE INVENTION

[0004] In some aspects, the present disclosure provides a lipid-containing particle comprising: a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule; a composite protein comprising a plasma membrane-localized protein conjugated to a nuclear export sequence (NES); and a therapeutic freight.

[0005] In some cases, the plasma membrane-localized protein comprises a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a humanized structural protein; a plekstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein. In some cases, the therapeutic cargo comprises a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease (optionally a type IIS restriction enzyme), a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, DNA, RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, a ribozyme, or a small molecule compound, or any combination thereof. In some cases, the combinatorial protein further comprises a therapeutic cargo. In some cases, the combinatorial protein comprises, in order from the N-terminus to the C-terminus, a plasma membrane-localized protein, an NES, and a therapeutic cargo. In some cases, the combinatorial protein further comprises a cleavable linker, optionally wherein the cleavable linker is positioned between the plasma membrane-localized protein and the therapeutic cargo, optionally wherein the cleavable linker is positioned between the NES and the therapeutic cargo, optionally wherein the combinatorial protein further comprises a nuclear localization sequence (NLS) C-terminus of the cleavable linker. In some cases, the lipid-containing particle comprises a cell; a virus-like particle (VLP); a protein-lipid vehicle (PLV); a liposome, optionally a lipid nanoparticle; or an extracellular vesicle, optionally an exosome or an ectosome. In some cases, the combinatorial protein further comprises a cargo, wherein the cargo is a binding partner of the therapeutic cargo.

[0006] In some aspects, the present disclosure provides a composition comprising: a first nucleic acid molecule encoding a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule; and a second nucleic acid molecule encoding a combinatorial protein comprising a plasma membrane-localized protein coupled to a nuclear export sequence (NES) and a cargo, wherein the cargo comprises a therapeutic cargo or a binding partner of the therapeutic cargo.

[0007] In some cases, the plasma membrane-localized protein comprises a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a humanized structural protein; a plekstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein. In some cases, the non-immunogenic plasma membrane recruitment protein comprises Arc, human Arc, an endogenous retrovirus gag protein, or a human endogenous retrovirus gag protein. In some cases, the therapeutic cargo comprises a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease (optionally a type IIS restriction enzyme), a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, DNA, RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, a ribozyme, or a small molecule compound, or any combination thereof. In some cases, the combined protein comprises a plasma membrane-localized protein, an NES, and a therapeutic cargo arranged in sequence from the N-terminus to the C-terminus. In some cases, the combined protein further comprises a cleavable linker, optionally wherein the cleavable linker is positioned between the plasma membrane-localized protein and the therapeutic cargo, optionally wherein the cleavable linker is positioned between the NES and the therapeutic cargo, optionally wherein the combined protein comprises the nuclear localization sequence (NLS) C-terminus of the cleavable linker. In some cases, the composition is a lipid-containing particle, optionally wherein the lipid-containing particle comprises a cell; a virus-like particle (VLP); a protein-lipid vehicle (PLV); a liposome, optionally a lipid nanoparticle; or an extracellular vesicle, optionally an exosome or an ectosome.

[0008] In some aspects, provided herein is a lipid-containing particle comprising: a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule; and a combined protein comprising a plasma membrane-localized protein coupled to a cleavable linker; and a therapeutic cargo.

[0009] In some cases, the plasma membrane-localized protein comprises a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a pleckstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein. In some cases, the therapeutic cargo comprises a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease (optionally a type IIS restriction enzyme), a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, DNA, RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, a ribozyme, or a small molecule compound, or any combination thereof. In some cases, the combined protein further comprises the therapeutic cargo. In some cases, the combined protein comprises, in order from the N-terminus to the C-terminus, a plasma membrane-localized protein, a cleavable linker, and the therapeutic cargo, optionally wherein the combined protein further comprises a nuclear localization sequence (NLS) at the C-terminus of the cleavable linker. In some cases, the lipid-containing particle comprises a cell; a virus-like particle (VLP); a protein-lipid vehicle (PLV); a liposome, optionally a lipid nanoparticle; or an extracellular vesicle, optionally an exosome or an ectosome. In some cases, the combined protein further comprises a cargo, wherein the cargo is a binding partner of the therapeutic cargo.

[0010] In some aspects, the present disclosure provides a composition comprising: a first nucleic acid molecule encoding a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule; and a second nucleic acid molecule encoding a combined protein comprising a plasma membrane-localized protein coupled to a cleavable linker and a cargo, wherein the cargo comprises a therapeutic cargo or a binding partner of the therapeutic cargo.

[0011] In some cases, the plasma membrane-localized protein comprises a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a pleckstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein. In some cases, the non-immunogenic plasma membrane recruitment protein comprises Arc, human Arc, an endogenous retroviral gag protein, or a human endogenous retroviral gag protein.

[0012] In some cases, the plasma membrane-localized protein comprises a PH domain derived from phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), Disc and actin-related protein 1 (Daap1), phosphatidylinositol 1 general receptor (Grp1), oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton's tyrosine kinase (Btk), phosphatidylinositol-4-phosphate adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), switch B cell complex subunit SWAP70 or MAPK-associated protein 1 (MAPKAP1), or a mutant thereof. In some cases, the plasma membrane-localized protein comprises a PH domain derived from a human protein. In some cases, the plasma membrane-localized protein comprises a PH domain derived from human phospholipase Cδ1, human Akt1, human 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), human Daap1, murine Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70 or human MAPKAP1, or a mutant thereof. In some cases, the plasma membrane-localized protein comprises a membrane protein selected from CD9, CD47, CD63, and CD81, and their transmembrane domains. In some cases, the plasma membrane-localized protein comprises a membrane protein selected from human CD9, human CD47, human CD63, and human CD81, and their transmembrane domains. In some cases, the therapeutic cargo comprises a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease (optionally a type IIS restriction enzyme), a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, DNA, RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, a ribozyme, or a small molecule compound, or any combination thereof. In some cases, the combinatorial protein comprises, in order from the N-terminus to the C-terminus, a plasma membrane-localized protein, a cleavable linker, and a therapeutic cargo, optionally wherein the combinatorial protein further comprises a nuclear localization sequence (NLS) at the C-terminus of the cleavable linker. In some cases, the composition is a lipid-containing particle, optionally wherein the lipid-containing particle comprises a cell; a virus-like particle (VLP); a protein-lipid vehicle (PLV); a liposome, optionally a lipid nanoparticle; or an extracellular vesicle, optionally an exosome or an ectosome.

[0013] In some aspects, provided herein is a lipid-containing particle comprising a combinatorial protein, the combinatorial protein comprising i) a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a pleckstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein and ii) a nuclear export sequence (NES).

[0014] In some cases, the lipid-containing particle further comprises a cargo, wherein the cargo is a therapeutic cargo or a binding partner of a therapeutic cargo, optionally wherein the therapeutic cargo comprises a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease (optionally a type IIS restriction enzyme), a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, DNA, RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, a ribozyme, or a small molecule compound, or any combination thereof. In some cases, the combinatorial protein further comprises a therapeutic cargo. In some cases, the combinatorial protein comprises, in order from the N-terminus to the C-terminus, i) a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a plekstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein, ii) an NES, and iii) a therapeutic cargo. In some cases, the combinatorial protein further comprises a cleavable linker, optionally wherein the cleavable linker is positioned between i) a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a plekstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein and ii) a therapeutic cargo, optionally wherein the cleavable linker is between iii) an NES and iv) a therapeutic cargo, optionally wherein the combinatorial protein further comprises a C-terminus of a nuclear localization sequence (NLS) of the cleavable linker. In some cases, the plasma membrane localization protein comprises a PH domain derived from phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), discoidin (Disc) and actin-related protein 1 (Daap1), phosphatidylinositol 1 universal receptor (Grp1), oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton's tyrosine kinase (Btk), phosphatidylinositol tetraphosphate adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), switch B cell complex subunit SWAP70, or MAPK-related protein 1 (MAPKAP1), or a mutant thereof. In some cases, the plasma membrane localization protein comprises a PH domain derived from a human protein. In some cases, the plasma membrane localization protein comprises a PH domain derived from human phospholipase Cδ1, human Akt1, human 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), human Daap1, murine Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, or human MAPKAP1, or a mutant thereof. In some cases, the plasma membrane localization protein comprises a membrane protein selected from CD9, CD47, CD63, and CD81 and their transmembrane domains.In some cases, the plasma membrane-localized protein comprises a membrane protein selected from human CD9, human CD47, human CD63, and human CD81 and their transmembrane domains. In some cases, the plasma membrane-localized protein comprises Arc, human Arc, an endogenous retroviral gag protein, or a human endogenous retroviral gag protein. In some cases, the lipid-containing particle comprises a cell; a virus-like particle (VLP); a protein-lipid vehicle (PLV); a liposome, optionally a lipid nanoparticle; or an extracellular vesicle, optionally an exosome or an ectosome.

[0015] A composition comprising a nucleic acid molecule encoding a combinatorial protein, the combinatorial protein comprising i) a humanized retroviral structural protein; a human endogenous retroviral (HERV) structural protein, optionally HERV gag; a plekstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein, ii) a nuclear export sequence (NES), and iii) a cargo, where the cargo is a therapeutic cargo or a binding partner of a therapeutic cargo.

[0016] In some cases, the therapeutic cargo comprises a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease (optionally a type IIS restriction enzyme), a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, DNA, RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, a ribozyme, or a small molecule compound, or any combination thereof.

[0017] In some cases, the combinatorial protein comprises, in order from the N-terminus to the C-terminus, i) a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a pleckstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein, ii) a NES, and iii) a therapeutic cargo. In some cases, the combinatorial protein further comprises a cleavable linker, optionally wherein the cleavable linker is positioned between i) the humanized retroviral structural protein; the human endogenous retrovirus (HERV) structural protein, optionally HERV gag; the pleckstrin homology (PH) domain; or the non-immunogenic plasma membrane recruitment protein and ii) the therapeutic cargo, optionally wherein the cleavable linker is between iii) the NES and iv) the therapeutic cargo, optionally wherein the combinatorial protein further comprises a nuclear localization sequence (NLS) C-terminus of the cleavable linker. In some cases, the plasma membrane localization protein comprises a PH domain derived from phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), discoidin (Disc) and actin-related protein 1 (Daap1), phosphatidylinositol 1 universal receptor (Grp1), oxysterol binding protein 1 - Homo sapiens (OSBP), Bruton's tyrosine kinase (Btk), phosphatidylinositol phosphate adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), switch B cell complex subunit SWAP70 or MAPK-related protein 1 (MAPKAP1) or a mutant thereof. In some cases, the plasma membrane localization protein comprises a membrane protein selected from CD9, CD47, CD63 and CD81 and their transmembrane domains. In some cases, the plasma membrane localization protein comprises a PH domain derived from a human protein. In some cases, the plasma membrane localization protein comprises a PH domain derived from human phospholipase Cδ1, human Akt1, human 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), human Daap1, murine Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70 or human MAPKAP1 or a mutant thereof. In some cases, the plasma membrane localization protein comprises a membrane protein selected from human CD9, human CD47, human CD63 and human CD81 and their transmembrane domains. In some cases, the plasma membrane localization protein comprises a non-immunogenic plasma membrane recruitment protein comprising Arc, human Arc, an endogenous retroviral gag protein or a human endogenous retroviral gag protein. In some cases, the composition is a lipid-containing particle, optionally wherein the lipid-containing particle comprises a cell; a virus-like particle (VLP); a protein-lipid mediator (PLV); a liposome, optionally a lipid nanoparticle; or an extracellular vesicle, optionally an exosome or a nucleosome.

[0018] In some aspects, disclosed herein is a lipid-containing particle comprising a combinatorial protein comprising i) a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a plekstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein, ii) a cleavable linker, and iii) a cargo, wherein the cargo is a therapeutic cargo or a binding partner of a therapeutic cargo.

[0019] In some instances, the therapeutic cargo comprises a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease (optionally a type IIS restriction enzyme), a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, DNA, RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, a ribozyme, or a small molecule compound, or any combination thereof. In some instances, the cleavable linker is between i) the humanized retroviral structural protein; the human endogenous retrovirus (HERV) structural protein, optionally HERV gag; the plekstrin homology (PH) domain; or the non-immunogenic plasma membrane recruitment protein and ii) the therapeutic cargo.

[0020] In some cases, the combinatorial protein further comprises an NES, optionally wherein the NES is between i) a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a plekstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein and ii) a therapeutic cargo, optionally wherein the NES is between i) a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a plekstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein and ii) a cleavable linker, optionally wherein the combinatorial protein further comprises the C-terminus of the nuclear localization sequence (NLS) of the cleavable linker. In some cases, the plasma membrane localization protein comprises a PH domain derived from phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), Discoidin (Disc) and actin-related protein 1 (Daap1), phosphatidylinositol 1 universal receptor (Grp1), oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton's tyrosine kinase (Btk), tetraspanin adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), switch B cell complex subunit SWAP70 or MAPK-related protein 1 (MAPKAP1) or mutants thereof. In some cases, the plasma membrane localization protein comprises a PH domain derived from a human protein. In some cases, the plasma membrane localization protein comprises a PH domain derived from human phospholipase Cδ1, human Akt1, human 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), human Daap1, murine Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70 or human MAPKAP1 or mutants thereof. In some cases, the plasma membrane localization protein comprises a membrane protein selected from CD9, CD47, CD63 and CD81 and their transmembrane domains. In some cases, the plasma membrane localization protein comprises a membrane protein selected from human CD9, human CD47, human CD63 and human CD81 and their transmembrane domains. In some cases, the plasma membrane localization protein comprises a non-immunogenic plasma membrane recruitment protein comprising Arc, human Arc, an endogenous retroviral gag protein or a human endogenous retroviral gag protein. In some cases, the lipid-containing particle comprises a cell; a virus-like particle (VLP); a protein-lipid vehicle (PLV); a liposome, optionally a lipid nanoparticle; or an extracellular vesicle, optionally an exosome or an ectosome.

[0021] In some aspects, the present disclosure provides a composition comprising a nucleic acid molecule encoding a combinatorial protein, the combinatorial protein comprising i) a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a plekstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein, ii) a cleavable linker, and iii) a cargo, wherein the cargo is a therapeutic cargo or a binding partner of a therapeutic cargo.

[0022] In some cases, the therapeutic cargo comprises a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease (optionally a type IIS restriction enzyme), a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, DNA, RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, a ribozyme, or a small molecule compound, or any combination thereof. In some cases, the cleavable linker is between i) a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a plekstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein and ii) the therapeutic cargo. In some cases, the combinatorial protein further comprises an NES, optionally wherein the NES is between i) a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a plekstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein and ii) the therapeutic cargo, optionally wherein the NES is between i) a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a plekstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein and ii) the cleavable linker, optionally wherein the combinatorial protein further comprises a nuclear localization sequence (NLS) C-terminus of the cleavable linker.

[0023] In some cases, the plasma membrane-localized protein comprises a PH domain derived from phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), Discoidin (Disc) and actin-associated protein 1 (Daap1), phosphatidylinositol 1 general receptor (Grp1), oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton's tyrosine kinase (Btk), four-phosphate adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), switch B cell complex subunit SWAP70 or MAPK-associated protein 1 (MAPKAP1) or a mutant thereof. In some cases, the plasma membrane-localized protein comprises a PH domain derived from a human protein. In some cases, the plasma membrane-localized protein comprises a PH domain derived from human phospholipase Cδ1, human Akt1, human 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), human Daap1, mouse Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70 or human MAPKAP1 or a mutant thereof. In some cases, the plasma membrane-localized protein comprises a membrane protein selected from CD9, CD47, CD63 and CD81 and their transmembrane domains. In some cases, the plasma membrane-localized protein comprises a membrane protein selected from human CD9, human CD47, human CD63 and human CD81 and their transmembrane domains. In some cases, the plasma membrane-localized protein comprises a non-immunogenic plasma membrane recruitment protein, which non-immunogenic plasma membrane recruitment protein comprises Arc, human Arc, endogenous retroviral gag protein or human endogenous retroviral gag protein.

[0024] In some cases, the composition is a lipid-containing particle, optionally wherein the lipid-containing particle comprises a cell; a virus-like particle (VLP); a protein-lipid mediator (PLV); a liposome, optionally a lipid nanoparticle; or an extracellular vesicle, optionally an exosome or an ectosome.

[0025] In some cases, the lipid-containing particle further comprises a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; or a humanized structural protein. In some cases, the composition further comprises a third nucleic acid molecule encoding a human endogenous retrovirus (HERV) structural protein (optionally HERV gag) or a humanized structural protein. In some cases, the percentage of the second nucleic acid molecule in the composition relative to the sum of the second nucleic acid molecule and the third nucleic acid molecule is about, at least, or at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some cases, the composition further comprises a nucleic acid molecule encoding a human endogenous retrovirus (HERV) structural protein (optionally HERV gag) or a humanized structural protein. In some cases, the percentage of the nucleic acid molecule encoding the combined protein relative to the sum of the nucleic acid molecule encoding the combined protein and the nucleic acid molecule encoding a human endogenous retrovirus (HERV) structural protein (optionally HERV gag) or a humanized structural protein is about, at least, or at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0026] In some cases, the lipid-containing particles do not contain a non-human gag protein or a humanized gag protein. In some cases, the lipid-containing particles further comprise an outer lipid layer and one or more immunomodulators in the outer lipid layer. In some cases, the one or more immunomodulators are immunosuppressive molecules. In some cases, the immunosuppressive molecules comprise CTLA-4, B7-1, B7-2, PD-1, PDL-1, PDL-2, VISTA, TIM-3, GAL9, TIGIT, CD155, LAG3, VISTA, BTLA, HVEM, or any combination thereof. In some cases, the immunosuppressive molecules comprise CTLA-4 and PD-L1, CTLA-4 and PD-L2, CTLA-4 and PD-1, CTLA-4 and VISTA, CTLA-4 and anti-CD28, PD-1 and VISTA, B7-1 and PD-L1, B7-1 and PD-L2, B7-l and PD-1, B7-1 and VISTA, B7-1 and anti-CD28, B7-2 and PD-L1, B7-2 and PD-L2, B7-2 and PD-1, B7-2 and VISTA, B7-2 and anti-CD28, PD-1 and VISTA, PD-1 and anti-CD-28, VISTA and anti-CD28, PD-L1 and VISTA, PD-L1 and anti-CD-28, PD-L2 and VISTA, PD-L2 and anti-CD-28, or VISTA and anti-CD28.

[0027] In some cases, the composition does not contain a nucleic acid molecule encoding a non-human gag protein or a humanized gag protein.

[0028] In some aspects, provided herein is a method that includes contacting a cell with a lipid-containing particle provided herein.

[0029] In some aspects, provided herein is a method that includes administering a lipid-containing particle provided herein to a subject in need thereof.

[0030] In some aspects, provided herein is a method of producing a lipid-containing particle provided herein, the method comprising: providing a system that expresses: a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule; and a combined protein comprising a plasma membrane-localized protein conjugated to a nuclear export sequence (NES); and a cargo, wherein the system produces lipid-containing particles; and optionally harvesting and purifying the lipid-containing particles.

[0031] In some aspects, provided herein is a method of producing the lipid-containing particles provided herein, the method comprising: providing a system that expresses: a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule; and a composite protein comprising a plasma membrane-localized protein conjugated to a cleavable linker; and a cargo, wherein the system produces lipid-containing particles; and optionally harvesting and purifying the lipid-containing particles.

[0032] In some aspects, provided herein is a method of producing the lipid-containing particles provided herein, the method comprising: providing a system that expresses a composite protein comprising i) a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a pleckstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein and ii) a nuclear export sequence (NES), wherein the system produces lipid-containing particles; and optionally harvesting and purifying the lipid-containing particles.

[0033] In some aspects, provided herein is a method of producing the lipid-containing particles provided herein, the method comprising: providing a system that expresses a composite protein comprising i) a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a pleckstrin homology (PH) domain; or a non-immunogenic plasma membrane recruitment protein, ii) a cleavable linker, and iii) a cargo; wherein the system produces lipid-containing particles; and optionally harvesting and purifying the lipid-containing particles. In some instances, the system further expresses a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; or a humanized structural protein. In some instances, the system comprises a production cell, a cell-free extract, or a cell lysate.

[0034] Incorporation by Reference

[0035] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description and its accompanying drawings, which set forth illustrative embodiments in which the principles of the present disclosure are utilized, where:

[0037] Figure 1A A schematic base editing virus-like particle (BE-VLP) is shown. As depicted, the base editor protein is fused to the C-terminus of the murine leukemia virus (MLV) gag polyprotein via a linker that is cleaved by the MLV protease after particle maturation.

[0038] Figure 1B Two graphs are shown summarizing the adenine base editing efficiency of version 1 (v1) BE-VLPs. In HEK293T cells, the adenine base editing efficiency of v1 BE-VLPs at two genomic loci, referred to as "HEK2" and "HEK3" respectively. The protospacer positions of the target adenines are indicated by subscripts (i.e., A5 = adenine at position 5), where the protospacer adjacent motif (PAM) is positions 21-23. Data are shown as individual data points and mean ± s.e.m. for n = 3 independent biological replicates.

[0039] Figure 2A A schematic diagram of v1 engineered VLPs (eVLPs) and v2 eVLP is shown. After VLP maturation, more efficient linker cleavage in v2 BE-VLPs can lead to improved cargo release.

[0040] Figure 2B A graph is shown summarizing the adenine base editing efficiency of v1 and v2 BE-eVLPs at position A7 of the BCL11A enhancer locus in HEK293T cells.

[0041] Figure 2C Is a schematic diagram that demonstrates that improved localization of the cargo, as achieved in v3 eVLP in production cells, leads to more efficient incorporation into the eVLP.

[0042] Figure 2D A schematic diagram is shown demonstrating that installing a 3xNES motif upstream of the cleavable linker can promote cytoplasmic localization of gag-3xNES-cargo in production cells, while promoting nuclear localization of the free adenine base editor (ABE) cargo in transduced cells.

[0043] Figure 2E A graph is shown summarizing the adenine base editing efficiency of v2.4 and v3 BE-eVLPs at position A7 of the BCL11A enhancer locus in HEK293T cells.

[0044] Figure 2F A schematic diagram is shown demonstrating that the optimal gag-cargo:gag-pro-pol stoichiometry can balance the amount of cargo protein per particle and the amount of MMLV protease required for efficient particle maturation.

[0045] Figure 2GFigure showing the adenine base editing efficiency of v3.4 eVLP with different gag-ABE:gag-pro-pol stoichiometries at position A7 of the BCL11A enhancer locus in HEK293T cells. The legend indicates the percentage of the gag-ABE plasmid in the total amount of gag-ABE and gag-pro-pol plasmids. As Figure 2B , 2E and shown in 2G, the values and error bars reflect the mean ± s.e.m. of n = 3 independent biological replicates. The data were fit to a four-parameter logistic curve using nonlinear regression.

[0046] Figure 3A Figure quantifying the amount of BE molecules per eVLP by anti-Cas9 and anti-MLV (p30) ELISA. The values and error bars reflect the mean ± s.e.m. of n = 3 independent replicates.

[0047] Figure 3B Figure quantifying the relative sgRNA abundance by RT-qPCR using sgRNA-specific primers, normalized relative to the v1 sgRNA abundance. The values and error bars reflect the mean ± s.e.m. of n = 3 technical replicates.

[0048] Figures 3C - 3D Figure showing a comparison of the editing efficiencies of v1, v2.4, v3.4, and v4 BE-eVLP at the BCL11A enhancer locus in HEK293T cells ( Figure 3C ) and at the Dnmt1 locus in NIH 3T3 cells ( Figure 3D ). The values and error bars reflect the mean ± s.e.m. of n = 3 independent biological replicates. The data were fit to a four-parameter logistic curve using nonlinear regression.

[0049] Figure 3E Figure summarizing the adenine base editing efficiency of single v4 BE-eVLP targeting HEK2 or the BCL11A enhancer locus or multiplex v4 BE-eVLP targeting both loci in HEK293T cells.

[0050] Figure 3F Figure summarizing the adenine base editing efficiency of FuG-B2-pseudotyped v4 BE-eVLP in Neuro-2a cells or 3T3 fibroblasts.

[0051] Figure 3GA graph summarizing the adenine base editing efficiency at three on-target genomic loci and their corresponding Cas-dependent off-target sites in HEK293T cells treated with v4 BE-eVLP or ABE8e plasmids is shown. OT1 = off-target site 1, OT2 = off-target site 2, OT3 = off-target site 3.

[0052] Figure 3H A graph summarizing the Cas-independent off-target editing frequencies at six off-target R-loops in HEK293T cells treated with v4 BE-eVLP or ABE8e plasmids is shown. OTRL = off-target R-loop.

[0053] Figure 3I A graph quantifying the amount of DNA molecules encoding BE per v4 BE-eVLP by detecting the amount of DNA molecules encoding BE in lysed eVLP or in lysis buffer alone by qPCR.

[0054] Figure 3J A graph quantifying the amount of DNA encoding BE in lysates from HEK293T cells treated with v4 BE-eVLP or transfected with plasmids encoding BE by qPCR. As Figures 3E - 3J shown, the data are presented as individual data points and mean ± s.e.m. for n = 3 independent biological replicates.

[0055] Figure 4A A graph summarizing the correction efficiency of the COL7A1 (R185X) mutation in patient-derived primary human fibroblasts.

[0056] Figure 4B A graph summarizing the correction efficiency of the Idua (W392X) mutation in primary mouse fibroblasts. As Figures 4A - 4B shown, the values and error bars reflect the mean ± s.e.m. for n = 3 independent biological replicates. The data were fitted to a four-parameter logistic curve using nonlinear regression.

[0057] Figure 4C A graph summarizing the adenine base editing efficiency at the B2M and CIITA loci in primary human T cells. The data are presented as individual data points and mean ± s.e.m. for n = 3 independent biological replicates.

[0058] Figure 5A A schematic diagram of P0 ICV injection of v4 BE-eVLP is shown. v4BE-eVLP targeting Dnmt1 was co-injected with a lentivirus encoding EGFP-KASH. Tissues were harvested 3 weeks after injection, and the cortex and midbrain were isolated. For each tissue, the nuclei were dissociated and analyzed by high-throughput sequencing as bulk unsorted (all nuclei) or GFP+ nuclei.

[0059] Figure 5B This is a graph summarizing the adenine base editing efficiency at the Dnmt1 locus in a large number of unsorted (all cell nuclei) and GFP+ populations. Data are shown as individual data points and mean ± s.e.m. for n = 4 mice.

[0060] Figure 6A This is a schematic diagram of the systemic injection of BE-eVLP. BE-eVLP targeting Pcsk9 was injected retroorbitally into 6- to 7-week-old C57BL / 6J mice. Organs were harvested one week after injection, and genomic DNA of unsorted cells was sequenced.

[0061] Figure 6B This is a graph summarizing the adenine base editing efficiency at the splice donor of Pcsk9 exon 1 in the mouse liver after systemic injection of v1 BE-VLP or v4 BE-eVLP. Data are shown as individual data points and mean ± s.e.m. for n = 3 mice (4x1011 v1 BE-VLP and 4x1011 v4 BE-eVLP) or n = 4 mice (7x1011 v4 BE-eVLP).

[0062] Figure 6C This is a graph summarizing the adenine base editing efficiency at the splice donor of Pcsk9 exon 1 in the mouse heart, kidney, liver, lung, muscle, and spleen after systemic injection of 7x1011 v4 BE-eVLP. Data are shown as individual data points and mean ± s.e.m. for n = 4 mice (treated) or n = 3 mice (untreated).

[0063] Figure 6D This is a graph summarizing the quantification of DNA sequencing reads containing A·T to G·C mutations within protospacer positions 4-10 of 14 CIRCLE-seq-named off-target loci in the liver from v4 BE-eVLP-treated, AAV-treated, and untreated mice. Data are shown as individual data points and mean ± s.e.m. for n = 4 mice (BE-eVLP), n = 5 mice (AAV), or n = 3 mice (untreated). vg = viral genome.

[0064] Figure 6E This is a graph summarizing the quantification of serum Pcsk9 levels measured by ELISA. Data are shown as individual data points and mean ± s.e.m. for n = 4 mice (treated) or n = 3 mice (untreated).

[0065] Figure 7ASchematic of exon 3 of Rpe65 around the R44X mutation, which can be corrected by an A·T to G·C conversion at position A6 in the protospacer (shaded region, PAM underlined).

[0066] Figure 7B Schematic of subretinal injection. Five weeks after injection, phenotype rescue was evaluated via ERG, and tissues were subsequently harvested for sequencing.

[0067] Figure 7C Graph summarizing the adenine base editing efficiency at positions A3, A6, and A8 in the protospacer of genomic DNA harvested from rd12 mice. Data are shown as individual data points and mean ± s.e.m. for n = 6 mice (two treated groups) or n = 4 mice (untreated).

[0068] Figure 7D Graph summarizing the allele frequency distribution of genomic DNA harvested from treated rd12 mice. Data are shown as mean ± s.e.m. for n = 6 mice. 8e-LV = ABE8e-NG-LV, 8e-eVLP = v4 ABE8e-NG-eVLP.

[0069] Figure 7E Graph summarizing the scotopic a-wave and b-wave amplitudes measured by ERG after overnight dark adaptation. Data are shown as individual data points and mean ± s.e.m. for n = 8 mice (wild type), n = 6 mice (ABE8e-NG-LV and v4 ABE8e-NG-eVLP), or n = 4 mice (untreated).

[0070] Figure 7F Graph summarizing the adenine base editing efficiency at positions A3, A6, and A8 in the protospacer of genomic DNA harvested from rd12 mice. Data are shown as individual data points and mean ± s.e.m. for n = 6 mice (v4ABE7.10-NG-eVLP) or n = 4 mice (ABE7.10-NG-LV and untreated). P values were calculated using a two-tailed t test.

[0071] Figure 7G Graph summarizing the allele frequency distribution of genomic DNA harvested from treated rd12 mice. Data are shown as mean ± s.e.m. for n = 6 mice (v4 ABE7.10-NG-eVLP) or n = 4 mice (ABE7.10-NG-LV and untreated). 7.10-LV = ABE7.10-NG-LV, 7.10-eVLP = v4 ABE7.10-NG-eVLP.

[0072] Figure 7HFigure summarizing scotopic a-wave and b-wave amplitudes measured by ERG after overnight dark adaptation. Data are shown as individual data points and mean ± s.e.m. for n = 8 mice (wild type), n = 7 mice (v4ABE7.10-NG-eVLP), n = 5 mice (ABE7.10-NG-LV), or n = 4 mice (untreated). Two-tailed t-tests were used to calculate p-values.

[0073] Figure 7I Images of western blots of protein extracts from RPE tissues of wild-type, untreated, v4 ABE7.10-NG-eVLP-treated, and ABE7.10-NG-LV-treated mice are shown.

[0074] Figure 7J Representative ERG waveforms from wild-type, untreated, ABE7.10-NG-LV-treated, and v4ABE7.10-NG-eVLP-treated mice are shown.

[0075] Figure 8A Images of immunoblot analysis of proteins from purified BE-VLPs using anti-Cas9, anti-p30, and anti-VSV-G antibodies are shown to verify VLP production.

[0076] Figure 8B Figure summarizing adenine base editing efficiency of v1 BE-VLPs at position A7 in the BCL11A enhancer locus in HEK293T cells. Values and error bars reflect mean ± s.e.m. of n = 3 independent biological replicates. Data were fit to a four-parameter logistic curve using nonlinear regression.

[0077] Figure 8C Schematic of immature BE-VLPs with ABE8e fused to the gag structural protein. Various MMLV protease cleavage sites were inserted between gag and ABE8e to determine the optimal cleavable sequence that promotes release of ABE8e from gag during proteolytic virion maturation. Arrows indicate cleavage sites.

[0078] Figure 8D Representative images of western blots evaluating cleaved ABE8e relative to full-length gag-ABE8e in purified v2 BE-eVLP variants are shown.

[0079] Figure 8E Figure summarizing densitometry-based quantification of the cleaved ABE8e fraction from western blots. Data are shown as individual data points and mean ± s.e.m. of n = 3 technical replicates.

[0080] Figure 9ASchematic diagrams of v2.4 and v3 BE-eVLP constructs are shown. Three HIV NESs were fused to the C-terminus or N-terminus of the gag-ABE chimera. We incorporated a protease-cleavable linker between the ABE and NES sequences such that after proteolytic virion maturation, the final BE cargo would lack the NES.

[0081] Figure 9B Representative immunofluorescence images of producer cells transfected with the v2.4 gag-ABE construct or the v3.4 gag-3xNES-ABE construct are shown. After 48 h of transfection, the cells were fixed in paraformaldehyde and stained with an anti-tubulin antibody to stain the cytoskeleton, with DAPI for nuclear staining, and with an anti-Cas9 antibody to visualize the gag-ABE chimera. The scale bar represents 50 μm.

[0082] Figure 9C A graph summarizing the quantification of the cytoplasmic localization of the v2.4 gag-ABE construct or the v3.4 gag-3xNES-ABE construct based on automated image analysis is shown. Data are presented as individual data points and mean ± s.e.m. of n = 3 technical replicates. A two-tailed t-test was used to calculate the p-value.

[0083] Figure 10A A representative negative-staining transmission electron micrograph (TEM) of v4 BE-eVLP is shown. The scale bar represents 200 nm.

[0084] Figures 10B - 10C Quantification of the protein content of v1, v2.4, v3.4, and v4 BE-eVLP measured by anti-Cas9 or anti-MLV (p30) ELISA is shown. Data are presented as individual data points and mean ± s.e.m. of n = 3 technical replicates.

[0085] Figure 10D A graph comparing the editing efficiency at the BCL11A enhancer locus normalized to the particle number of v1, v2.4, v3.4, and v4 BE-VLPs in HEK293T cells is shown. Data are presented as mean ± s.e.m. of n = 3 biological replicates.

[0086] Figure 10E A graph summarizing the cell viability after treatment with v4BE-eVLP in HEK293T cells and NIH 3T3 fibroblasts is shown. Data are presented as mean ± s.e.m. of n = 3 biological replicates.

[0087] Figure 10FGraph summarizing indel frequencies generated by v1 Cas9-VLPs and v4 Cas9-eVLPs at the EMX1 locus in HEK293T cells. Data are shown as mean ± s.e.m. of n = 3 biological replicates. Data were fit to a four-parameter logistic curve using nonlinear regression.

[0088] Figure 10G Graph showing the adenine base editing efficiency of VSV-G pseudotyped v4 BE-eVLPs in Neuro-2a cells or 3T3 fibroblasts. Data are shown as individual data points and mean ± s.e.m. of n = 3 biological replicates.

[0089] Figure 11A Shows the experimental timeline of the orthogonal R-loop assay.

[0090] Figure 11B Graph summarizing on-target editing controls for the orthogonal R-loop experiment. Data are shown as individual data points and mean ± s.e.m. of n = 3 biological replicates.

[0091] Figure 11C Graph summarizing cell viability after treatment with v4 BE-eVLPs in RDEB fibroblasts. Data are shown as mean ± s.e.m. of n = 3 biological replicates.

[0092] Figure 11D Graph quantifying DNA sequencing reads containing A·T to G·C mutations within protospacer positions 4 - 10 of ten previously identified off-target loci in genomic DNA from v4-BE-eVLP-treated RDEB patient-derived fibroblasts. The grey dashed line represents the highest observed background mutation rate of 0.1%. Data are shown as individual data points and mean ± s.e.m. of n = 3 biological replicates.

[0093] Figures 12A - 12B Shows Figures 5A - 5B Flow cytometry analysis of nuclear sorting from mouse brains after P0 ICV injection related to Figure 12A Shows a representative flow cytometry plot. Singlet nuclei were gated based on the FSC / BSC ratio and DyeCycle Ruby signal. The first row shows the gating strategy on a GFP-negative sample. Bulk nuclei correspond to events passing through gate D of the singlet nuclei. Figure 12B Graph summarizing the percentage of GFP-positive nuclei measured by flow cytometry after P0 ICV injection. Data are shown as mean + s.e.m. of n = 3 biological replicates.

[0094] Figure 13AFigure showing plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels one week after v4 BE-eVLP injection.

[0095] Figures 13B - 13C Showing untreated mice ( Figure 13B ) and v4 BE-eVLP-treated mice ( Figure 13C ) at 1 week after injection, representative images of histopathological evaluation by hematoxylin and eosin staining of the liver. Representative examples of each are shown. Scale bar represents 50 μm.

[0096] Figures 14A - 14C Showing the results of sequencing analysis of RPE cDNA after v4 BE-eVLP or lentivirus treatment. Figure 14A Showing that v4 BE-eVLP and lentivirus treatment result in 50 - 60% A·T to G·C conversion at the target adenine (A6) of the Rpe65 transcript. Data are shown as individual data points and mean ± s.e.m. for n = 6 (ABE8e-NG-LV, ABE8e-NG-eVLP, and ABE7.10-NG-eVLP) or n = 4 (ABE7.10-NG-LV and untreated) replicates. Figures 14B - 14C Showing off-target A to G RNA editing by v4 BE-eVLP and lentivirus as measured by high-throughput sequencing of Mcm3ap ( Figure 14B ) and Perp ( Figure 14C ) transcripts. Data are shown as individual data points and mean ± s.e.m. for n = 6 (ABE8e-NG-LV, ABE8e-NG-eVLP, and ABE7.10-NG-eVLP) or n = 4 (ABE7.10-NG-LV and untreated) replicates.

[0097] Figure 15 Illustrating different configurations of components (such as combinatorial proteins containing a PH domain) that can be delivered by the lipid-containing particles described herein.

[0098] Figure 16 Additional examples illustrating different configurations of components (such as combinatorial proteins containing GAGKcon / Arc / tetraspanin (e.g., CD9)) that can be delivered by the lipid-containing particles described herein.

[0099] Figure 17 Showing analysis of indel formation at the target editing site, which demonstrates the activity of various PH-Cas9 combinatorial proteins and gRNAs targeting VegFs3 delivered via lipid-containing particles to HEK293 cells and K562 cells. Detailed Description

[0100] Unless otherwise indicated, the practice of some of the methods disclosed herein employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (edited by F. M. Ausubel et al.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (edited by M. J. MacPherson, B. D. Hames, and G. R. Taylor (1995)), Antibodies, A Laboratory Manual, edited by Harlow and Lane (1988), and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (edited by R. I. Freshney (2010)).

[0101] Definitions

[0102] As used in this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "chimeric transmembrane receptor polypeptide" includes multiple chimeric transmembrane receptor polypeptides.

[0103] The term "about" or "approximately" means within an acceptable error range of a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, in accordance with practice in the art, "about" can mean within 1 or greater than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold of the value, and more preferably within 2-fold of the value. Where a particular value is described in this application, unless otherwise indicated, the term "about" should be assumed to mean within the acceptable error range of that particular value.

[0104] As used herein, "cell" generally can refer to a biological cell. A cell can be a basic structural, functional, and / or biological unit of a living organism. The cell can be derived from any organism having one or more cells. Some examples include: prokaryotic cells, eukaryotic cells, bacterial cells, archaeal cells, cells of unicellular eukaryotic organisms, protozoan cells, cells from plants (e.g., cells from plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, hay, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, etc.), seaweeds (e.g., kelp), fungal cells (e.g., yeast cells, cells from mushrooms), animal cells, cells from invertebrates (e.g., Drosophila, cnidarians, echinoderms, nematodes, etc.), cells from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals), cells from mammals (e.g., pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, humans, etc.), and so on. Sometimes the cell is not derived from a natural organism (e.g., the cell can be synthetically prepared, sometimes called an artificial cell).

[0105] As used herein, the term "antigen" refers to a molecule or a fragment thereof that can be bound by a selective binding agent. As an example, an antigen can be a ligand that can be bound by a selective binding agent such as a receptor. As another example, an antigen can be an antigenic molecule that can be bound by a selective binding agent such as an immunoprotein (e.g., an antibody). An antigen can also refer to a molecule or a fragment thereof that can be used in an animal to generate an antibody that can bind to the antigen.

[0106] As used herein, the term "antibody" refers to a protein-binding molecule having immunoglobulin-like function. The term antibody includes antibodies (e.g., monoclonal and polyclonal antibodies), as well as derivatives, variants, and fragments thereof. Antibodies include immunoglobulins (Ig) of different classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (such as IgG1, IgG2, etc.). Derivatives, variants, or fragments thereof may refer to functional derivatives or fragments that retain the binding specificity (e.g., fully and / or partially) of the corresponding antibody. Antigen-binding fragments include Fab, Fab’, F(ab’)2, variable fragment (Fv), single-chain variable fragment (scFv), minibody, diabody, and single-domain antibody (“sdAb” or “nanobody” or “camelid antibody”). The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered, or chemically conjugated. Examples of antibodies that have been optimized include affinity matured antibodies. Examples of antibodies that have been engineered include Fc-optimized antibodies (e.g., antibodies optimized in the fragment crystallizable region) and multispecific antibodies (e.g., bispecific antibodies).

[0107] As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotides can include synthetic nucleotides. Nucleotides can include synthetic nucleotide analogs. Nucleotides can be monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance on nucleic acid molecules containing them. As used herein, the term nucleotide can refer to dideoxyribonucleoside triphosphates (ddNTPs) and derivatives thereof. Illustrative examples of dideoxyribonucleoside triphosphates can include ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled by well-known techniques. Labeling can also be performed with quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Fluorescent labels for nucleotides can include fluorescein, 5-carboxyfluorescein (FAM), 2′7′-dimethoxy-4′5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), CascadeBlue, Oregon Green, Texas Red, cyanine, and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides can include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA]ddGTP, and [dROX]ddTTP, available from Perkin Elmer, Foster City, Calif.; FluoroLink deoxynucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5-dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP, available from Amersham, Arlington Heights, Ill.; fluorescein 15-dATP, fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP, available from Boehringer Mannheim, Indianapolis, Ind.; and chromosome-labeled nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, cascade blue-7-UTP, cascade blue-7-dUTP, fluorescein-12-UTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, rhodamine green-5-UTP, rhodamine green-5-dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5-UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP, available from Molecular Probes, Eugene, Oreg. Nucleotides can also be labeled or identified by chemical modification. Chemically modified mononucleotides can be biotin-dNTP. Some examples of biotinylated dNTPs can include biotin-dATP (e.g., bio-N6-ddATP, biotin-14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin-14-dCTP), and biotin-dUTP (e.g., biotin-11-dUTP, biotin-16-dUTP, biotin-20-dUTP).

[0108] The terms “polynucleotide,” “oligonucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, in single-stranded form, double-stranded form, or multi-stranded form. Polynucleotides can be exogenous or endogenous to a cell. Polynucleotides can be present in a cell-free environment. Polynucleotides can be a gene or a fragment thereof. Polynucleotides can be DNA. Polynucleotides can be RNA. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Polynucleotides can contain one or more analogs (e.g., modified backbone, sugar, or nucleobase). If present, the nucleotide structure can be modified before or after polymeric assembly. Some examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein attached to sugar), thiol-containing nucleotides, biotinylated nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, brachidial, and wyosine. Examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides, including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides can be interrupted by non-nucleotide components.

[0109] As used herein, the term "gene" refers to a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) and its corresponding nucleotide sequence that participates in encoding an RNA transcript. The term as used herein with respect to genomic DNA includes intervening non-coding regions as well as regulatory regions and may include 5' and 3' ends. In some uses, the term encompasses transcribed sequences, including 5' untranslated regions and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcribed region will contain an "open reading frame" that encodes a polypeptide. In some uses of the term, "gene" includes only the coding sequence necessary to encode a polypeptide (e.g., the "open reading frame" or "coding region"). In some cases, a gene does not encode a polypeptide, e.g., ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term "gene" includes not only transcribed sequences but also non-transcribed regions, including upstream regulatory regions and downstream regulatory regions, enhancers, and promoters. A gene may refer to an "endogenous gene" or a native gene in its natural location in the genome of an organism. A gene may refer to an "exogenous gene" or a non-native gene. A non-native gene may refer to a gene that is not normally found in a host organism but is introduced into the host organism by gene transfer. A non-native gene may also refer to a gene that is not in its natural location in the genome of an organism. A non-native gene may also refer to a naturally occurring nucleic acid or polypeptide sequence (e.g., a non-native sequence) that contains a mutation, insertion, and / or deletion.

[0110] As used herein, the terms "target polynucleotide", "target nucleic acid", and "target sequence" refer to a nucleic acid or polynucleotide targeted by a cargo of the present disclosure. The target polynucleotide can be DNA (e.g., endogenous or exogenous). DNA can refer to the template that gives rise to an mRNA transcript and / or the various regulatory regions that regulate the transcription of mRNA from the DNA template. The target polynucleotide can be part of a larger polynucleotide, such as a chromosome or a region of a chromosome. The target polynucleotide can refer to an extrachromosomal sequence (e.g., episomal sequence, minicircle sequence, mitochondrial sequence, chloroplast sequence, etc.) or a region of an extrachromosomal sequence. The target polynucleotide can be RNA. The RNA can be, for example, mRNA, which can serve as a template for encoding a protein. The target polynucleotide containing RNA can include the various regulatory regions that regulate the translation of the protein from the mRNA template. The target polynucleotide can encode a gene product (e.g., DNA encoding an RNA transcript or RNA encoding a protein product) or contain regulatory sequences that regulate the expression of the gene product. Generally, the term "target sequence" refers to the nucleic acid sequence on a single strand of the target nucleic acid. The target sequence can be part of a gene, a regulatory sequence, genomic DNA, cell-free nucleic acids including cfDNA and / or cfRNA, cDNA, chimeric genes, and RNA including mRNA, miRNA, rRNA, etc. The target polynucleotide can result in altered gene expression and / or activity when targeted by a cargo. The target polynucleotide can result in an edited nucleic acid sequence when targeted by a cargo. The target nucleic acid can contain a nucleic acid sequence that can be unrelated to any other sequence in a nucleic acid sample by a single nucleotide substitution. The target nucleic acid can contain a nucleic acid sequence that can be unrelated to any other sequence in a nucleic acid sample by 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions. In some embodiments, the substitution does not occur within 5, 10, 15, 20, 25, 30, or 35 nucleotides at the 5' end of the target nucleic acid. In some embodiments, the substitution does not occur within 5, 10, 15, 20, 25, 30, 35 nucleotides at the 3' end of the target nucleic acid.

[0111] The term "expression" refers to one or more processes of transcription of a polynucleotide from a DNA template (such as transcription into mRNA or other RNA transcripts), and / or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide can be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. "Upregulation" with respect to expression generally refers to an increase in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence relative to its expression level in the wild-type state, while "downregulation" generally refers to a decrease in the expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence relative to its expression level in the wild-type state.

[0112] As used herein, the terms "complement", "complements", "complementary" and "complementarity" generally refer to a sequence that is fully complementary to and hybridizable with a given sequence. In some cases, a sequence that hybridizes to a given nucleic acid is referred to as the "complement" or "reverse complement" of the given molecule if the sequence of its bases over a given region is capable of binding complementarily to the sequence of the bases of its binding partner such that base pairs such as A-T, A-U, G-C and G-U are formed. In general, a first sequence that is hybridizable to a second sequence can hybridize specifically or selectively to the second sequence such that during a hybridization reaction, hybridization to the second sequence or group of second sequences is preferred over hybridization to non-target sequences (e.g., thermodynamically more stable under a given set of conditions such as stringent conditions commonly used in the art). Hybridizable sequences can share a degree of sequence complementarity over all or a portion of their corresponding lengths, such as complementarity between 25% and 100%, including at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% sequence complementarity. Sequence identity, such as for the purpose of assessing the percentage of complementarity, can be measured by any suitable alignment algorithm, including the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner with optional default settings available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html), the BLAST algorithm (see, e.g., the BLAST alignment tool with optional default settings available at blast.ncbi.nlm.nih.gov / Blast.cgi) or the Smith-Waterman algorithm (see, e.g., the EMBOSS Water aligner with optional default settings available at www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html). Any suitable parameters of the selected algorithm (including default parameters) can be used to assess the optimal alignment.

[0113] Complementarity can be perfect or substantial / sufficient. Perfect complementarity between two nucleic acids may mean that the two nucleic acids can form a duplex, in which each base in the duplex binds to a complementary base through Watson-Crick pairing. Substantial or sufficient complementarity can mean that the sequence in one strand is not completely and / or not perfectly complementary to the sequence in the opposite strand, but sufficient binding occurs between the bases on the two strands to form a stable hybridization complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequence and standard mathematical calculations to determine the Tm of the hybridizing strands, or by empirical determination of the Tm via the use of conventional methods.

[0114] As used herein, the term "regulate" with respect to expression or activity means to change the level of expression or activity. Regulation can occur at the transcriptional level, post-transcriptional level, translational level, and / or post-translational level.

[0115] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues linked by peptide bonds. The term does not imply a specific length of the polymer and is not intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The term applies to both naturally occurring amino acid polymers and amino acid polymers that contain at least one modified amino acid. In some cases, the polymer may be interrupted by non-amino acids. The term includes amino acid chains of any length, including full-length proteins, and proteins with or without secondary and / or tertiary structure (e.g., domains). The term also encompasses amino acid polymers that have been modified, such as by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component. As used herein, the terms "amino acid" and "amino acids" generally refer to natural and non-natural amino acids, including modified amino acids and amino acid analogs. Modified amino acids can include natural and non-natural amino acids that have been chemically modified to include groups or chemical moieties that do not naturally occur on amino acids. Amino acid analogs can refer to amino acid derivatives. The term "amino acid" includes both D-amino acids and L-amino acids. In some cases, the amino acid sequences provided herein lack an N-terminal methionine. For example, SEQ ID NO: 1-5, 7-11, 15-17, and 22-77 may lack an N-terminal methionine.

[0116] The term "variant", when used herein to refer to a polypeptide, means a polypeptide that is related to, but not identical to, a wild-type polypeptide, e.g., by amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. Compared to the wild-type polypeptide, variants include polypeptides that contain one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof. Variants also include derivatives of the wild-type polypeptide and fragments of the wild-type polypeptide.

[0117] As used herein, the term "percent identity (%)" means the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). For the purpose of determining percent identity, alignment can be achieved by various methods within the skill in the art, e.g., using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. By aligning a test sequence with a comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to the amino acids or nucleotides at the same positions in the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence, the percent identity between the two sequences can be calculated.

[0118] The Cas protein referred to in this article can be a type of protein or polypeptide. The Cas protein can refer to a nuclease. The Cas protein can refer to an endoribonuclease. The Cas protein can refer to any modified (e.g., shortened, mutated, extended) polypeptide sequence or homolog of the Cas protein. The Cas protein can be codon-optimized. The Cas protein can be a codon-optimized homolog of the Cas protein. The Cas protein can be enzymatically inactivated, partially active, constitutively active, fully active, inducibly active, and / or more active (e.g., more active than the wild-type homolog of the protein or polypeptide). The Cas protein can be a type II Cas protein. The Cas protein can be Cas9. The Cas protein can be a type V Cas protein. The Cas protein can be Cpf1 or Cas12a. The Cas protein can be C2c1. The Cas protein can be C2c3. The Cas protein can be a type VI Cas protein. The Cas protein can be C2c2 or Cas13a. The Cas protein can be Cas13b. The Cas protein can be Cas13c. The Cas protein can be Cas13d. The Cas protein can be Cas14. The Cas protein (e.g., variant, mutated, enzymatically inactivated, and / or conditionally enzymatically inactivated site-directed polypeptide) can bind to a target nucleic acid. The Cas protein (e.g., variant, mutated, enzymatically inactivated, and / or conditionally enzymatically inactivated endoribonuclease) can bind to a target RNA or DNA.

[0119] As used herein, the term "crRNA" generally can refer to a nucleic acid having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% sequence identity and / or sequence similarity to a wild-type exemplary crRNA (e.g., a crRNA from Streptococcus pyogenes (S. pyogenes)). The crRNA generally can refer to a nucleic acid having at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% sequence identity and / or sequence similarity to a wild-type exemplary crRNA (e.g., a crRNA from Streptococcus pyogenes, Staphylococcus aureus (S. aureus), etc.). The crRNA can refer to a modified form of the crRNA, which can contain nucleotide changes such as deletions, insertions or substitutions, variants, mutations or chimeras. The crRNA can be a nucleic acid having at least about 60% sequence identity to a wild-type exemplary crRNA (e.g., a crRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.) over a stretch of at least 6 contiguous nucleotides. For example, the crRNA sequence can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% identical to a wild-type exemplary crRNA sequence (e.g., a crRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.) over a stretch of at least 6 contiguous nucleotides.

[0120] As used herein, the term "tracrRNA" generally can refer to a nucleic acid having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% sequence identity and / or sequence similarity to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes). The tracrRNA can refer to a nucleic acid having at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% sequence identity and / or sequence similarity to a wild-type exemplary tracrRNA sequence (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.). The tracrRNA can refer to a modified form of tracrRNA, which can contain nucleotide alterations such as deletions, insertions or substitutions, variants, mutations or chimeras. The tracrRNA can refer to a nucleic acid that is at least about 60% identical to a wild-type exemplary tracrRNA (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.) sequence over a segment of at least 6 consecutive nucleotides. For example, the tracrRNA sequence can be at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% identical to a wild-type exemplary tracrRNA (e.g., tracrRNA from Streptococcus pyogenes, Staphylococcus aureus, etc.) over a segment of at least 6 consecutive nucleotides.

[0121] As used herein, "guide nucleic acid" can refer to a nucleic acid that can hybridize to another nucleic acid. The guide nucleic acid can be RNA. The guide nucleic acid can be DNA. The guide nucleic acid can be programmed to bind site-specifically to a sequence of a nucleic acid. The nucleic acid to be targeted or target nucleic acid can contain nucleotides. The guide nucleic acid can contain nucleotides. A portion of the target nucleic acid can be complementary to a portion of the guide nucleic acid. The strand of a double-stranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid can be referred to as the complementary strand. The strand of a double-stranded target polynucleotide that is complementary to the complementary strand and thus may not be complementary to the guide nucleic acid can be referred to as the non-complementary strand. The guide nucleic acid can contain a polynucleotide chain and can be referred to as a "single guide nucleic acid". The single guide nucleic acid can contain crRNA. The single guide nucleic acid can contain crRNA and tracrRNA. The guide nucleic acid can contain two polynucleotide chains and can be referred to as a "dual guide nucleic acid". The dual guide nucleic acid can contain crRNA and tracrRNA. Unless otherwise specified, the term "guide nucleic acid" can be inclusive and refer to both single guide nucleic acids and dual guide nucleic acids.

[0122] A guide nucleic acid can include a segment that can be referred to as a "nucleic acid targeting segment" or "nucleic acid targeting sequence". The nucleic acid targeting segment can include a sub-segment that can be referred to as a "protein binding segment" or "protein binding sequence" or "Cas protein binding segment".

[0123] As used herein, the term "targeting sequence" refers to a nucleotide sequence encoding a targeting polypeptide and the corresponding amino acid sequence, which targeting polypeptide mediates protein localization (or retention) to a subcellular location, such as a given organelle, nucleus, cytosol, mitochondrion, endoplasmic reticulum (ER), Golgi apparatus, chloroplast, apoplast, peroxisome, or the plasma membrane or membrane of another organelle. For example, a targeting sequence can direct a protein (e.g., a receptor polypeptide or an adaptor polypeptide) to the nucleus using a nuclear localization signal (NLS); direct a protein out of the cell nucleus, e.g., to the cytoplasm, using a nuclear export signal (NES); direct a protein to the mitochondrion using a mitochondrial targeting signal; direct a protein to the endoplasmic reticulum (ER) using an ER retention signal; direct a protein to the peroxisome using a peroxisome targeting signal; direct a protein to the plasma membrane using a membrane localization signal; or a combination thereof.

[0124] As used herein, a "nuclear localization domain" can refer to a nuclear localization signal or other sequence or domain capable of crossing the nuclear membrane and entering the nucleus. The nuclear localization domain can be in-frame fused to a polypeptide, in which case the nuclear localization domain can be referred to as a "heterologous nuclear localization domain".

[0125] As used herein, a "nuclear export domain" can refer to a nuclear export signal or other sequence or domain that is present in a protein and is capable of targeting the protein for export from the nucleus to the cytoplasm using nuclear transport through the nuclear pore complex. The nuclear export domain can be in-frame fused to a polypeptide, in which case the nuclear export domain can be referred to as a "heterologous nuclear export domain".

[0126] As used herein, a "fusion" or "chimera" can refer to a protein and / or nucleic acid that contains one or more non-natural sequences (e.g., portions). A chimera or fusion can contain one or more identical non-natural sequences. A chimera or fusion can contain one or more different non-natural sequences. A chimera or fusion can be a chimera. A chimera or fusion can contain a nucleic acid affinity tag. A chimera or fusion can contain a barcode. A fusion can contain a peptide affinity tag. A chimera or fusion can provide subcellular localization of a site-directed polypeptide (e.g., a nuclear localization signal (NLS) for targeting the nucleus, a mitochondrial localization signal for targeting mitochondria, a chloroplast localization signal for targeting chloroplasts, an endoplasmic reticulum (ER) retention signal, etc.). A chimera or fusion can provide a non-natural sequence (e.g., an affinity tag) that can be used for tracking or purification.

[0127] A fusion or chimera can refer to any protein that has a functional effect. For example, a chimeric protein can contain methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity, remodeling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity, or demyristoylation activity. An effector protein can modify a genomic locus.

[0128] As used herein, "non-natural" can refer to a nucleic acid or polypeptide sequence that is not found in a natural nucleic acid or protein. Non-natural can refer to an affinity tag. Non-natural can refer to a chimera or fusion, such as a chimeric protein or chimeric nucleic acid. Non-natural can refer to a naturally occurring nucleic acid or polypeptide sequence that contains a mutation, insertion, and / or deletion. A non-natural sequence can exhibit and / or encode an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.), and this activity can also be exhibited by a nucleic acid and / or polypeptide sequence fused to the non-natural sequence. A non-natural nucleic acid or polypeptide sequence can be linked by genetic engineering to a naturally occurring nucleic acid or polypeptide sequence (or a variant thereof) to produce a chimeric nucleic acid and / or polypeptide sequence encoding a chimeric nucleic acid and / or polypeptide.

[0129] The terms "subject", "individual", and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, such as a human. Mammals include mice, monkeys, humans, farm animals, sports animals, and pets. Also covered are tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro.

[0130] As used herein, the terms "treatment" and "treating" refer to methods for obtaining a beneficial or desired result, including a therapeutic benefit and / or a prophylactic benefit. For example, treatment can include administering the systems or cell populations disclosed herein. A therapeutic benefit means any treatment-related improvement or effect on one or more diseases, conditions, or symptoms in a treatment. For a prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet manifested.

[0131] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition sufficient to result in a desired activity after administration to a subject in need thereof, such as a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and / or NK cells), which comprises the systems of the present disclosure. In the context of the present disclosure, the term "therapeutically effective" refers to an amount of a composition sufficient to delay the manifestation of a disorder treated by the methods of the present disclosure, prevent its progression, alleviate, or reduce at least one symptom of a disorder treated by the methods of the present disclosure.

[0132] Lipid-containing particles

[0133] In some aspects, the present disclosure relates to delivery vehicles for delivering therapeutic cargoes and / or other molecules into cells in vitro, ex vivo, or in vivo. In some cases, the delivery vehicles of the present disclosure have high efficiency for delivering therapeutic cargoes and / or other molecules into the cells of a subject in vivo. In some cases, the delivery vehicles of the present disclosure include lipid-containing particles such as virus-like particles, exosomes, lipid nanoparticles, protein-lipid vehicles, extracellular vesicle mimics, and membrane vesicles. The delivery vehicles (e.g., lipid-containing particles) disclosed herein can deliver cargoes highly effectively in vivo after being administered to a subject. For example, a high percentage of the cargo loaded in the lipid-containing particles is delivered to the cells of the subject and to the desired subcellular location (e.g., the nucleus or cytoplasm) of the cells of the subject. In some cases, lipid-containing particles are used to deliver genome editing systems into the cells of a subject and can have efficient in vivo gene editing performed by the genome editing system. In some cases, lipid-containing particles are used to deliver expression constructs encoding therapeutic proteins (e.g., antibodies, transcription factors, or chimeric antigen receptors (CARs)) into the cells of a subject and can have efficient expression of the therapeutic protein in the subject.

[0134] In some cases, the lipid-containing particles provided herein comprise a lipid-based outer layer surrounding a lumen (e.g., a protein core). The cargo can be loaded into the lipid-containing particles within the protein core. In some cases, the cargo is loaded into the lipid-containing particles by attaching to the lipid-based outer layer. The lipid-based outer layer can be a single lipid layer or a lipid bilayer composed of two layers of lipid molecules. In some cases, the lipid-containing particles have one or more membrane fusion proteins inserted or attached to the exterior of the outer lipid layer. The membrane fusion proteins can assist the lipid-containing particles in fusing with the membrane of the target cell, thereby delivering the cargo loaded in the lipid vesicles to the target cell.

[0135] The size (e.g., diameter) of the lipid-containing particles can be from about 10 nm to about 1000 nm, such as from about 10 nm to 50 nm, 10 nm to 100 nm, 10 nm to 200 nm, 10 nm to 300 nm, 10 nm to 400 nm, 10 nm to 500 nm, 10 nm to 600 nm, 10 nm to 800 nm, 20 nm to 50 nm, 20 nm to 100 nm, 20 nm to 200 nm, 20 nm to 300 nm, 20 nm to 400 nm, 20 nm to 500 nm, 20 nm to 600 nm, 20 nm to 800 nm, 50 nm to 100 nm, 50 nm to 200 nm, 50 nm to 300 nm, 50 nm to 400 nm, 50 nm to 500 nm, 50 nm to 600 nm, 50 nm to 800 nm, 100 nm to 200 nm, 100 nm to 300 nm, 100 nm to 400 nm, 100 nm to 500 nm, 100 nm to 600 nm, 100 nm to 800 nm, 200 nm to 300 nm, 200 nm to 400 nm, 200 nm to 500 nm, 200 nm to 600 nm, 200 nm to 800 nm, 400 nm to 600 nm, 400 nm to 800 nm or 600 nm to 800 nm. In some cases, the lipid-containing particles comprise virus-like particles, lipid nanoparticles or protein-lipid mediators and have a size (e.g., diameter) of from about 10 nm to about 100 nm, such as from about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 40 nm, about 10 nm to about 50 nm, about 10 nm to about 60 nm, about 10 nm to about 80 nm, about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm to about 50 nm, about 20 nm to about 60 nm, about 20 nm to about 80 nm, about 40 nm to about 50 nm, about 40 nm to about 60 nm or about 40 nm to about 80 nm.In some cases, the lipid-containing particles comprise exosomes and have a size of from about 50 nm to about 200 nm, such as from about 50 nm to about 80 nm, from about 50 nm to about 100 nm, from about 50 nm to about 120 nm, from about 50 nm to about 150 nm, from about 50 nm to about 160 nm, from about 50 to about 180 nm, from about 60 nm to about 80 nm, from about 60 nm to about 100 nm, from about 60 nm to about 120 nm, from about 60 nm to about 160 nm, from about 60 nm to about 160 nm, from about 60 nm to about 180 nm, from about 80 nm to about 100 nm, from about 80 nm to about 120 nm, from about 80 nm to about 160 nm, from about 80 nm to about 180 nm, from about 80 nm to about 180 nm, from about 100 nm to about 120 nm, from about 100 nm to about 150 nm, from about 100 nm to about 180 nm, from about 120 nm to about 150 nm, from about 120 nm to about 180 nm, from about 150 nm to about 180 nm or from about 150 nm to about 200 nm.

[0136] In some aspects, provided herein is a lipid-containing particle that comprises a cell fusion molecule or membrane fusion (e.g., a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule); a combinatorial protein comprising a plasma membrane localization protein (e.g., conjugated to a nuclear export sequence (NES)); and a cargo (e.g., a therapeutic cargo or a binding partner of a therapeutic cargo).

[0137] In some aspects, provided herein is a lipid-containing particle that comprises a membrane fusion molecule (e.g., a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule); and a combinatorial protein comprising a plasma membrane localization protein (e.g., conjugated to a cleavable linker); and a cargo (e.g., a therapeutic cargo or a binding partner of a therapeutic cargo).

[0138] In some aspects, provided herein is a lipid-containing particle that comprises a plasma membrane localization molecule (e.g., a humanized retroviral structural protein or a human endogenous retrovirus (HERV) structural protein, such as HERV gag, a plekstrin homology (PH) domain, or a non-immunogenic plasma membrane recruitment protein) and a nuclear export sequence (NES).

[0139] In some aspects, provided herein is a lipid-containing particle that comprises a combinatorial protein that comprises i) a plasma membrane localization molecule (e.g., a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, such as HERV gag; a plekstrin homology (PH) domain, or a non-immunogenic plasma membrane recruitment protein), ii) a cleavable linker, and iii) a cargo (e.g., a therapeutic cargo or a binding partner of a therapeutic cargo).

[0140] In some aspects, provided herein is a lipid-containing particle that comprises i) a plasma membrane-localizing molecule (e.g., a humanized retroviral structural protein; a human endogenous retroviral (HERV) structural protein, such as HERV gag; a pleckstrin homology (PH) domain, or a non-immunogenic plasma membrane-recruiting protein), and ii) a cargo (e.g., a therapeutic cargo or a binding partner of a therapeutic cargo).

[0141] In some aspects, provided herein is a lipid-containing particle that comprises a combinatorial protein that comprises i) a membrane fusion (e.g., a human endogenous retroviral (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule), ii) a plasma membrane-localizing molecule (e.g., a humanized retroviral structural protein; a human endogenous retroviral (HERV) structural protein, such as HERV gag; a pleckstrin homology (PH) domain, or a non-immunogenic plasma membrane-recruiting protein), and iii) a cargo (e.g., a therapeutic cargo or a binding partner of a therapeutic cargo).

[0142] In some aspects, provided herein is a lipid-containing particle that comprises (a) a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule; (b) a combinatorial protein that comprises a plasma membrane-localized protein selected from the group consisting of the pleckstrin homology (PH) domain of human Daap1, the PH domain of murine Grp1, the PH domain of human Grp1, the PH domain of human OSBP, the PH domain of human Btk, the PH domain of human FAPP1, the PH domain of human CERT, the PH domain of human PKD, the PH domain of human PHLPP1, the PH domain of human SWAP70, and the PH domain of human MAPKAP1; and (c) a cargo. In some cases, the combinatorial protein comprises a plasma membrane-localized protein conjugated to the cargo. In some cases, the combinatorial protein comprises a plasma membrane-localized protein conjugated to a nuclear export sequence (NES). In some cases, the combinatorial protein comprises, in order from the N-terminus to the C-terminus of the combinatorial protein, a plasma membrane-localized protein, an NES, and a cargo. In some cases, the combinatorial protein comprises, in order from the N-terminus to the C-terminus of the combinatorial protein, a plasma membrane-localized protein, an NES, a cargo, and a second NES. In some cases, the second NES is the same as the NES. In some cases, the second NES is different from the NES. In some cases, the combinatorial protein further comprises a cleavable linker. In some cases, the cleavable linker is positioned between the plasma membrane-localized protein and the cargo. In some cases, the cleavable linker is positioned between the NES and the cargo. In some cases, the combinatorial protein further comprises the C-terminus of a nuclear localization sequence (NLS) of the cleavable linker. In some cases, the lipid-containing particle comprises a human endogenous retrovirus envelope protein. In some cases, the human endogenous retrovirus envelope protein is from hENVH1, hENVH2, hENVH3, hENVK1, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)1, or hENVKcon. In some cases, the human endogenous retrovirus envelope protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the sequences in Table 2-1. In some cases, the plasma membrane-localized protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 11-21 and 60-66. In some cases, the lipid-containing particle comprises a lipid-containing membrane encapsulating a protein core. In some cases, the lipid-containing membrane comprises a phospholipid bilayer.In some cases, human endogenous retrovirus (HERV) envelope proteins, humanized envelope proteins, or non-immunogenic membrane fusion molecules are attached to a lipid-containing membrane.

[0143] In some aspects, provided herein is a lipid-containing particle comprising (a) a virus-derived glycoprotein selected from RD114, Fug-E, FuG-E(P440E), and MLV 10A1; (b) a combinatorial protein comprising a plasma membrane-localizing protein conjugated to a nuclear export sequence (NES); and (c) a cargo. In some cases, the combinatorial protein further comprises the cargo. In some cases, the combinatorial protein comprises, in order from the N-terminus to the C-terminus of the combinatorial protein, a plasma membrane-localizing protein, an NES, and a cargo. In some cases, the combinatorial protein further comprises a cleavable linker. In some cases, the cleavable linker is positioned between the plasma membrane-localizing protein and the cargo. In some cases, the cleavable linker is positioned between the NES and the cargo. In some cases, the combinatorial protein comprises the C-terminus of a nuclear localization sequence (NLS) of the cleavable linker. In some cases, the plasma membrane-localizing protein comprises: (a) a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; (b) a humanized structural protein; (c) a pleckstrin homology (PH) domain; or (d) a non-immunogenic plasma membrane-recruiting protein. In some cases, the non-immunogenic plasma membrane-recruiting protein comprises Arc, human Arc, an endogenous retrovirus gag protein, or a human endogenous retrovirus gag protein.

[0144] In some aspects, provided herein is a lipid-containing particle comprising (a) a virus-derived glycoprotein selected from RD114, Fug-E, FuG-E(P440E), and MLV 10A1; (b) a combinatorial protein comprising a plasma membrane-localizing protein conjugated to a cleavable linker; and (c) a cargo. In some cases, the combinatorial protein further comprises the cargo. In some cases, the combinatorial protein comprises, in order from the N-terminus to the C-terminus of the combinatorial protein, a plasma membrane-localizing protein, a cleavable linker, and a cargo. In some cases, the combinatorial protein further comprises the C-terminus of a nuclear localization sequence (NLS) of the cleavable linker. In some cases, the plasma membrane-localizing protein comprises: (a) a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; (b) a humanized structural protein; (c) a pleckstrin homology (PH) domain; or (d) a non-immunogenic plasma membrane-recruiting protein. In some cases, the lipid-containing particle comprises a lipid-containing membrane encapsulating a protein core. In some cases, the lipid-containing membrane comprises a phospholipid bilayer. In some cases, the virus-derived glycoprotein is attached to the lipid-containing membrane.

[0145] In any of the foregoing aspects or related aspects, the plasma membrane-localized protein may comprise a PH domain. In some cases, the PH domain comprises the PH domain of phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), Disc and actin-associated protein 1 (Daap1), phosphatidylinositol 1 general receptor (Grp1), oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton's tyrosine kinase (Btk), phosphatidylinositol transfer protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), switching B cell complex subunit SWAP70 or MAPK-associated protein 1 (MAPKAP1) or mutants thereof. In some cases, the PH domain comprises the PH domain of a human protein. In some cases, the PH domain comprises the PH domain of human phospholipase Cδ1, human Akt1, human 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), human Daap1, murine Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70 or human MAPKAP1 or mutants thereof. In some cases, the PH domain is selected from: the PH domain of human Daap1, the PH domain of murine Grp1, the PH domain of human Grp1, the PH domain of human OSBP, the PH domain of human Btk, the PH domain of human FAPP1, the PH domain of human CERT, the PH domain of human PKD, the PH domain of human PHLPP1, the PH domain of human SWAP70 and the PH domain of human MAPKAP1. In some cases, the PH domain comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of the PH domain sequences listed in Table 3.

[0146] In some cases, the plasma membrane-localized protein comprises a membrane protein selected from CD9, CD47, CD63, and CD81 and their transmembrane domains. In some cases, the plasma membrane-localized protein comprises a membrane protein selected from human CD9, human CD47, human CD63, and human CD81 and their transmembrane domains. In some cases, the membrane protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the CD9, CD47, CD63, or CD81 sequences listed in Table 3. In some cases, the plasma membrane-localized protein comprises a non-immunogenic plasma membrane recruitment protein, which non-immunogenic plasma membrane recruitment protein comprises Arc, human Arc, an endogenous retroviral gag protein, or a human endogenous retroviral gag protein. In some cases, the non-immunogenic plasma membrane recruitment protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the hArc or hGAGK con sequences listed in Table 3.

[0147] In any of the foregoing aspects or related aspects, the cargo may further comprise a therapeutic cargo or a binding partner of a therapeutic cargo.

[0148] In any of the foregoing aspects or related aspects, the combined protein may comprise an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from SEQ ID NO:50, 52-55, and 67-77.

[0149] In some aspects, provided herein is a lipid-containing particle that comprises a combinatorial protein, which comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from SEQ ID NO: 50, 52 - 55 and 67 - 77. In some cases, the lipid-containing particle comprises: (a) a human endogenous retrovirus (HERV) envelope protein; optionally wherein the human endogenous retrovirus envelope protein is from hENVH1, hENVH2, hENVH3, hENVK1, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)1 or hENVKcon; and optionally wherein the human endogenous retrovirus envelope protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences in Table 2 - 1; (b) a humanized envelope protein; (c) a non-immunogenic membrane fusion molecule; or (d) a virus-derived glycoprotein; optionally wherein the virus-derived glycoprotein is selected from: BaEVTR, BaEVTRless, FuG-E, FuG-E(P440E), MVL ENV (amphotropic), MVL ENV (ecotropic), MLV 10A1, VSVG, GP64, gp160 and RD114ENV; and optionally wherein the virus-derived glycoprotein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences in Table 1. In some cases, the combinatorial protein further comprises a cleavable linker, a nuclear export sequence (NES), a cargo, or a combination thereof. In some cases, the lipid-containing particle comprises a lipid-containing membrane encapsulating a protein core. In some cases, the lipid-containing membrane comprises a phospholipid bilayer. In some cases, the human endogenous retrovirus (HERV) envelope protein, the humanized envelope protein, the non-immunogenic membrane fusion molecule, or the virus-derived glycoprotein is attached to the lipid-containing membrane.

[0150] In some aspects, the present disclosure provides a combinatorial protein comprising a plasma membrane-localized protein and a heterologous sequence, wherein the plasma membrane-localized protein is selected from the pleckstrin homology (PH) domain of human Daap1, the PH domain of murine Grp1, the PH domain of human Grp1, the PH domain of human OSBP, the PH domain of human Btk, the PH domain of human FAPP1, the PH domain of human CERT, the PH domain of human PKD, the PH domain of human PHLPP1, the PH domain of human SWAP70, and the PH domain of human MAPKAP1. In some cases, the heterologous sequence is an NES, a cleavable linker, or a combination thereof. In some cases, the plasma membrane-localized protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 11-21 and 60-66. In some aspects, the present disclosure also provides a lipid-containing particle comprising the combinatorial protein described herein. In some cases, the lipid-containing particle comprises a lipid-containing membrane encapsulating a protein core. In some cases, the lipid-containing membrane comprises a phospholipid bilayer. In some cases, the lipid-containing particle further comprises a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule. In some cases, the human endogenous retrovirus (HERV) envelope protein, the humanized envelope protein, or the non-immunogenic membrane fusion molecule is attached to the lipid-containing membrane. In some cases, the human endogenous retrovirus envelope protein is from hENVH1, hENVH2, hENVH3, hENVK1, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)1, or hENVKcon. In some cases, the human endogenous retrovirus envelope protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the sequences in Table 2-1. In some cases, the virus-derived glycoprotein is selected from: BaEVTR, BaEVTRless, FuG-E, FuG-E(P440E), MVL ENV (amphotropic), MVL ENV (ecotropic), MLV 10A1, VSVG, GP64, gp160, and RD114ENV; and optionally wherein the virus-derived glycoprotein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the sequences in Table 1.

[0151] In any of the foregoing aspects or related aspects, the protein core may comprise a structural protein that comprises a second plasma membrane-localized protein. In some cases, the structural protein further comprises a retroviral protease (pro)protein. In some cases, the second plasma membrane-localized protein comprises: (a) a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; (b) a humanized structural protein; (c) a pleckstrin homology (PH) domain, optionally wherein the PH domain comprises phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), discoidin (Disc) and actin-related protein 1 (Daap1), phosphatidylinositol 1 universal receptor (Grp1), oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton's tyrosine kinase (Btk), phosphatidylinositol 4-phosphate adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), switch B cell complex subunit SWAP70 or MAPK-related protein 1 (MAPKAP1) or a mutant thereof, optionally wherein the PH domain is from a human, and optionally wherein the PH domain comprises the PH domain of human phospholipase Cδ1, human Akt1, human 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), human Daap1, murine Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70 or human MAPKAP1 or a mutant thereof; or (d) a non-immunogenic plasma membrane recruitment protein, optionally wherein the non-immunogenic plasma membrane recruitment protein comprises a membrane protein selected from CD9, CD47, CD63 and CD81 and their transmembrane domains. In some cases, the second plasma membrane-localized protein comprises a PH domain, wherein the PH domain comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of the PH domain sequences listed in Table 3. In some cases, the second plasma membrane-localized protein comprises a membrane protein that comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of the CD9, CD47, CD63 or CD81 sequences listed in Table 3. In some cases, the second plasma membrane-localized protein comprises a non-immunogenic plasma membrane recruitment protein that comprises Arc, human Arc, an endogenous retroviral gag protein or a human endogenous retroviral gag protein. In some cases, the second plasma membrane-localized protein comprises a non-immunogenic plasma membrane recruitment protein that comprises a protein having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the hArc or hGAGK listed in Table 3 conAn amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of the sequences. In some cases, the combinatorial protein forms part of a protein core. In some cases, the combinatorial protein contains an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from SEQ ID NO: 50, 52 - 55 and 67 - 77. In some cases, the plasma membrane - localizing protein of the combinatorial protein forms part of a protein core. In some cases, the lipid - containing membrane includes an immunomodulator. In some cases, the immunomodulator is in the phospholipid bilayer. In some cases, the immunomodulator is an immunosuppressive molecule.

[0152] In any of the foregoing or related aspects, the lipid - containing particle can comprise: (a) a cell; (b) a virus - like particle (VLP); (c) a protein - lipid vehicle (PLV); (d) a liposome, optionally a lipid nanoparticle; or (e) an extracellular vesicle, optionally an exosome or an ectosome.

[0153] Combinatorial protein

[0154] In various aspects, combinatorial proteins are disclosed herein that are suitable for assembling cargo into lipid - containing particles and delivering the cargo to cells. The combinatorial protein can form at least part of the lumen (e.g., the protein core) of the lipid - containing particle. The lipid - containing particle can comprise two or more combinatorial proteins. The two or more combinatorial proteins can be the same combinatorial protein. The two or more combinatorial proteins can be different combinatorial proteins. The combinatorial protein can include a structural protein. The structural protein can contain a plasma membrane - localizing protein or polypeptide (e.g., a retroviral gag protein, a human endogenous retroviral gag protein or a plekstrin homology domain). The structural protein can be fused to a cargo protein or polypeptide (e.g., a therapeutic cargo). In some cases, the combinatorial protein contains a cargo that is a binding partner for a therapeutic cargo (e.g., the binding partner can directly bind the therapeutic cargo, or, for example, the binding partner can bind to another molecule that is conjugated to or interacts with the therapeutic cargo). For example, the combinatorial protein can contain a plasma membrane - localizing protein (e.g., a retroviral gag protein, a human endogenous retroviral gag protein or a plekstrin homology domain) conjugated to a nucleic acid - binding protein that can bind, for example, a nucleic acid molecule, such as RNA (e.g., mRNA) or DNA. In some cases, the combinatorial protein is suitable for delivery by the lipid - containing particles disclosed herein.

[0155] The plasma membrane - localizing proteins disclosed herein can be derived from viruses, humans or any other suitable source. In some cases, the plasma membrane - localizing protein is a human endogenous protein.

[0156] In some cases, the combinatorial proteins disclosed herein comprise a nuclear localization sequence (NLS). In some cases, the NLS facilitates delivery of the combinatorial protein or cargo released from the combinatorial protein (e.g., released from the combinatorial protein upon cleavage of a cleavable linker) to the nucleus of a target cell. In some cases, the NLS is an endogenous NLS. In some cases, the endogenous NLS is naturally within a portion of the cargo. In some cases, the NLS is an exogenous NLS. In some cases, the exogenous NLS is not naturally within a portion of the cargo. In some cases, the exogenous NLS is engineered to be a portion of the cargo.

[0157] In some cases, the combinatorial proteins disclosed herein comprise at least one NLS sequence, such as 2 or more, 3 or more, 4 or more, or 5 or more NLS sequences. In some cases, one or more NLS sequences (2 or more, 3 or more, 4 or more, or 5 or more NLS sequences) are located at or near the N-terminus and / or C-terminus of the combinatorial protein (e.g., within 50 amino acids thereof). In some cases, one or more NLS sequences (2 or more, 3 or more, 4 or more, or 5 or more NLS sequences) are located at or near the N-terminus of the combinatorial protein (e.g., within 50 amino acids thereof). In some cases, one or more NLS sequences (2 or more, 3 or more, 4 or more, or 5 or more NLS sequences) are located at or near the C-terminus of the combinatorial protein (e.g., within 50 amino acids thereof). In some cases, one or more NLS sequences (3 or more, 4 or more, or 5 or more NLS sequences) are located at or near both the N-terminus and C-terminus of the combinatorial protein (e.g., within 50 amino acids thereof). In some cases, one NLS sequence is located at the N-terminus of the combinatorial protein and one NLS sequence is located at the C-terminus of the combinatorial protein.

[0158] In some cases, the cargo is a protein and is delivered as part of a combinatorial protein disclosed herein, e.g., operably linked to a structural protein (e.g., a human endogenous retrovirus (HERV) structural protein or a plasma membrane recruitment domain). In some embodiments, one or more NLS sequences are positioned at or near one or both ends of the cargo protein sequence of the combinatorial protein. For example, in some cases, one or more NLS sequences (2 or more, 3 or more, 4 or more, or 5 or more NLS sequences) are positioned at or near the N-terminus and / or C-terminus of the cargo protein sequence (e.g., within 50 amino acids thereof). In some cases, one or more NLS sequences (2 or more, 3 or more, 4 or more, or 5 or more NLS sequences) are positioned at or near the N-terminus of the cargo protein sequence (e.g., within 50 amino acids thereof). In some cases, one or more NLS sequences (2 or more, 3 or more, 4 or more, or 5 or more NLS sequences) are positioned at or near the C-terminus of the cargo protein sequence (e.g., within 50 amino acids thereof). In some cases, one or more NLS sequences (3 or more, 4 or more, or 5 or more NLS sequences) are positioned at or near both the N-terminus and C-terminus of the cargo protein sequence (e.g., within 50 amino acids thereof). In some cases, one NLS sequence is positioned at the N-terminus of the cargo protein sequence, and one NLS sequence is positioned at the C-terminus of the cargo protein sequence.

[0159] In some cases, the combinatorial proteins disclosed herein comprise between 1 and 10 NLS sequences (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, or 2-5 NLS sequences). In some cases, the combinatorial protein comprises (is fused to) between 2 and 5 NLS sequences (e.g., 2-4 or 2-3 NLSs). Examples of NLS sequences include NLS sequences derived from: the NLS of the SV40 virus large T antigen, having the amino acid sequence PKKKRKV (SEQ ID NO:129); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO:130)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO:131) or RQRRNELKRSP (SEQ ID NO:132); the hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:133); the sequence RMRIZFKNKGKDT AELRRRRVE V S VELRK AKKDEQILKRRN V (SEQ ID NO:134) from the IBB domain of importin-α; the sequences VSRKRPRP (SEQ ID NO:135) and PPKKARED (SEQ ID NO:136) of myoma T protein; the sequence PQPKKKPL (SEQ ID NO:137) of human p53; the sequence SALIKKKKKMAP (SEQ IDNO:138) of murine c-abl IV; the sequences DRLRR and PKQKKRK of influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO:139) of hepatitis delta antigen; the sequence REKKKFLKRR of murine Mxl protein; the sequence KRKGDE VDGVDEVAKKKS KK (SEQ ID NO:140) of human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO:141) of the steroid hormone receptor (human) glucocorticoid, and sequences having at least 80% identity to the foregoing. In some cases, the NLS comprises the amino acid sequence MDSLLMNRRKFLY QFKNVRWAKGRRETYLC (SEQ ID NO:142).

[0160] Other examples of NLS sequences include KRTADGSEFESPKKKRKV (SEQ ID NO: 143), KKTELQTTNAENKTKKL (SEQ ID NO: 144), KRGINDRNFWRGENGRKTR (SEQ ID NO: 145), RKSGKIAAIVVKRPRK (SEQ ID NO: 146), and MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 147), SPKKKRKVEAS (SEQ ID NO: 148), AGCCCCAAGAAgAAGAGaAAGGTGGAGGCCAGC (SEQ ID NO: 149), GPKKKRKVAAA (SEQ ID NO: 150), and any of those described in: Cokol et al., EMBO Rep., 2000, 1(5):411-415 and Freitas et al., Current Genomics, 2009, 10(8):550-7; Lu, J. et al., Cell Commun Signal 19, 60 (2021); International Publication No. WO / 2001 / 038547 (each of which is incorporated herein by reference in its entirety), and sequences having at least 80% identity to the foregoing.

[0161] In some cases, the combinatorial proteins disclosed herein include a nuclear export sequence (NES). In some cases, the NES promotes the localization of the combinatorial protein in the cytosol of the target cell relative to the nucleus.

[0162] In some cases, the combinatorial proteins disclosed herein comprise at least one NES sequence, such as 2 or more, 3 or more, 4 or more, or 5 or more NES sequences. In some cases, one or more NES sequences (2 or more, 3 or more, 4 or more, or 5 or more NES sequences) are located at or near the N-terminus and / or C-terminus of the combinatorial protein (e.g., within 50 amino acids thereof). In some cases, the combinatorial proteins disclosed herein comprise only one NES sequence. In some cases, the combinatorial proteins disclosed herein comprise three NES sequences. In some cases, one or more NES sequences (2 or more, 3 or more, 4 or more, or 5 or more NES sequences) are located at or near the N-terminus of the combinatorial protein (e.g., within 50 amino acids thereof). In some cases, one or more NES sequences (2 or more, 3 or more, 4 or more, or 5 or more NES sequences) are located at or near the C-terminus of the combinatorial protein (e.g., within 50 amino acids thereof). In some cases, one or more NES sequences (3 or more, 4 or more, or 5 or more NES sequences) are located at or near both the N-terminus and C-terminus of the combinatorial protein (e.g., within 50 amino acids thereof). In some cases, one NES sequence is located at the N-terminus of the combinatorial protein, and one NES sequence is located at the C-terminus of the combinatorial protein.

[0163] In some cases, the cargo is a protein and is delivered as part of a combinatorial protein disclosed herein, e.g., operably linked to a structural protein (e.g., a human endogenous retrovirus structural protein or a plasma membrane recruitment domain). In some embodiments, one or more NES sequences are positioned at or near one or both ends of the cargo protein sequence within the combinatorial protein. For example, in some cases, one or more NES sequences (2 or more, 3 or more, 4 or more, or 5 or more NES sequences) are positioned at or near the N-terminus and / or C-terminus of the cargo protein sequence (e.g., within 50 amino acids thereof). In some cases, one or more NES sequences (2 or more, 3 or more, 4 or more, or 5 or more NES sequences) are positioned at or near the N-terminus of the cargo protein sequence (e.g., within 50 amino acids thereof). In some cases, one or more NES sequences (2 or more, 3 or more, 4 or more, or 5 or more NES sequences) are positioned at or near the C-terminus of the cargo protein sequence (e.g., within 50 amino acids thereof). In some cases, one or more NES sequences (3 or more, 4 or more, or 5 or more NES sequences) are positioned at or near both the N-terminus and C-terminus of the cargo protein sequence (e.g., within 50 amino acids thereof). In some cases, one NES sequence is positioned at the N-terminus of the cargo protein sequence and one NES sequence is positioned at the C-terminus of the cargo protein sequence. In some cases, the combinatorial proteins disclosed herein contain only one NES sequence. In some cases, the combinatorial protein contains only one NES sequence and the NES sequence is positioned at or near the N-terminus of the cargo protein (e.g., within 50 amino acids thereof).

[0164] In some embodiments, the combinatorial protein contains one NES sequence and two NLS sequences. In some cases of these embodiments, the NES sequence, the NLS sequences, and the cargo protein sequence are positioned in the order from the N-terminus to the C-terminus as follows: NES-NLS-cargo protein-NLS. In some embodiments, the combinatorial protein contains two or more NES sequences and two NLS sequences. In some cases of these embodiments, the NES sequence, the NLS sequences, and the cargo protein sequence are positioned in the order from the N-terminus to the C-terminus as follows: n X NES (n >= 2)-NLS-cargo protein-NLS.

[0165] In some cases, the combinatorial proteins disclosed herein contain between 1 and 10 NES sequences (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, or 2-5 NES sequences). In some cases, the combinatorial protein contains (fused to) between 2 and 5 NES sequences (e.g., 2-4 or 2-3 NES).

[0166] In some cases, the NES sequence that can be used to combine proteins includes LQLPPLERLTL (SEQ ID NO: 151) derived from the HIV-1 Rev protein and sequences having at least 80% identity therewith. In some cases, the NSE sequence includes LALKLAGLDI (SEQ ID NO: 152) derived from PKIα and sequences having at least 80% identity therewith. In some cases, the NES sequences disclosed herein include sequences such as those described in T la Cour et al., Nucleic Acids Res. 2003; 31(1): 393-396; and Xu D et al., Mol Biol Cell. September 2012; 23(18): 3673-6 (each of which is incorporated herein by reference in its entirety). Among the NES sequences described in the NES sequence database ( prodata.swmed.edu / LRNes) or (NESbase; services.healthtech.dtu.dk / datasets / NESbase-1.0), any of them can be used in the combined proteins disclosed herein, for example, for the purpose of packaging cargo (e.g., proteins) into lipid-containing particles (e.g., virus-like particles).

[0167] In some cases, the combined protein includes a cleavable linker between two or more components. For example, the combined protein can include a cleavable linker between the cargo protein sequence and the plasma membrane localization protein sequence (e.g., the retroviral gag protein sequence). In some cases, the cleavable linker separates the plasma membrane localization protein sequence from the NLS sequence and / or NES sequence at its N-terminus or C-terminus. The cleavable linker can separate the cargo protein sequence from the plasma membrane localization protein sequence, NLS sequence, and / or NES sequence at its N-terminus or C-terminus. The cleavable linker sequences provided herein can be cleavable sequences recognized and cleaved by viral proteases, bacterial proteases, or eukaryotic proteases (e.g., proteases derived from plants, animals, or fungi). In some cases, the cleavable sequence is recognized by a retroviral protease (pro, e.g., pro derived from Moloney murine leukemia virus (MMLV) or Friend murine leukemia virus (FMLV)). Examples of cleavable linker sequences that can be used in combined proteins include TSTLLMENSS (SEQ ID NO: 153), PRSSLYPALTP (SEQ ID NO: 154), VQALVLTQ (SEQ ID NO: 155), and PLQVLTLNIERR (SEQ ID NO: 156), and sequences having at least 80% identity, at least 90%, at least 95%, or at least 99% identity with the foregoing.

[0168] In some cases, the combinatorial protein comprises a protease between two or more components. In some cases, the protease is a viral protease. In some cases, the protease is a retroviral protease. In some cases, the protease is an MMLV protease. In some cases, the combinatorial protein comprises a plasma membrane-localized protein and a protease. In some cases, the protease can be expressed and delivered by the lipid-containing particles described herein rather than as part of the combinatorial protein.

[0169] In some cases, the combinatorial proteins disclosed herein further comprise one or more non-cleavable linkers that operably link the components together. The non-cleavable linker can be any suitable linker sequence for combinatorial protein construction, such as a peptide linker composed of glycine (Gly) and serine (Ser) residues. In some embodiments, the non-cleavable linker comprises an amino acid sequence selected from: (GS)x, (GGS)x, (GGGGS)x, (GGSG)x, and (SGGG)x, wherein x is an integer from 1 to 50.

[0170] In some cases, the combinatorial protein has one of the following configurations of components positioned in order from the N-terminus to the C-terminus:

[0171] [Plasma membrane-localized protein]-[n*NES]-[Cleavable linker]-[m1*NLS]-[Cargo protein]-[m2*NLS];

[0172] [Plasma membrane-localized protein]-[Cleavable linker]-[m1*NLS]-[Cargo protein]-[m2*NLS]-[n*NES];

[0173] [Plasma membrane-localized protein]-[Cleavable linker 1]-[m1*NLS]-[Cargo protein]-[m2*NLS]-[Cleavable linker 2]-[n*NES]; and

[0174] [Plasma membrane-localized protein]-[Cleavable linker 1]-[m1*NLS]-[Cargo protein]-[m2*NLS];

[0175] [m1*NLS]-[Cargo protein]-[m2*NLS]-[Cleavable linker]-[n*NES]-[Plasma membrane-localized protein];

[0176] [n*NES]-[m1*NLS]-[Cargo protein]-[m2*NLS]-[Cleavable linker]-[Plasma membrane-localized protein];

[0177] [n*NES]-[Cleavable linker 1]-[m1*NLS]-[Cargo protein]-[m2*NLS]-[Cleavable linker 2]-[Plasma membrane-localized protein]; and

[0178] [m1 * NLS] - [Cargo protein] - [m2 * NLS] - [Cleavable linker] - [Plasma membrane localization protein];

[0179] Where n, m1, and m2 are integers in the range of 0 to 10, respectively, and represent the number of repeats of their respective sequences. The non - cleavable linker sequence may or may not be present in any of the aforementioned configurations between any two adjacent components.

[0180] In some cases, the combined protein contains one of the following configurations, where the components are located in the order from the N - terminus to the C - terminus or as Figures 15 - 16 shown in the structure. In some cases, at least two combined proteins each independently contain one of the following configurations, where the components are located in the order from the N - terminus to the C - terminus or as Figures 15 - 16 shown in the structure.

[0181] 1. [Plasma membrane localization protein] - [NES] - [Cargo]

[0182] 2. [Plasma membrane localization protein] - [NES] - [Cargo] - [NES]

[0183] 3. [Plasma membrane localization protein] - [Cargo] - [NES]

[0184] 4. [Plasma membrane localization protein] - [Cleavable linker] - [Cargo]

[0185] 5. [Plasma membrane localization protein] - [NES] - [Cleavable linker] - [Cargo]

[0186] 6. [Plasma membrane localization protein] - [NES] - [Cleavable linker] - [Cargo] - [NES]

[0187] 7. [Plasma membrane localization protein] - [Protease]

[0188] 8. [PH_Akt] - [NES] - [Cas9 / NLS] - [NES]

[0189] 9. [PH_Akt] - [NES] - [MMLV cleavable sequence] - [Cas9 / NLS]

[0190] Figures 15 - 16 The second combinatorial protein of the structure shown. In some cases, the lipid-containing particles described herein comprise a first combinatorial protein having configuration 7 and a second combinatorial protein having any one of configurations 4, 5, 6, or 9. In some cases, the lipid-containing particles described herein comprise a protease (e.g., a retroviral protease) and a combinatorial protein having any one of configurations 1-6 or 8-9.

[0191] Table 3-1. Exemplary combinatorial protein sequences

[0192]

[0193]

[0194]

[0195] In some aspects, the present disclosure provides a combinatorial protein comprising a plasma membrane-localized protein and a heterologous sequence. In some cases, the plasma membrane-localized protein is selected from: the pleckstrin homology (PH) domain of human Daap1, the PH domain of murine Grp1, the PH domain of human Grp1, the PH domain of human OSBP, the PH domain of human Btk, the PH domain of human FAPP1, the PH domain of human CERT, the PH domain of human PKD, the PH domain of human PHLPP1, the PH domain of human SWAP70, and the PH domain of human MAPKAP1. In some cases, the heterologous sequence is an NES, a cleavable linker, or a combination thereof. In some cases, the plasma membrane-localized protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 11-21 and 60-66.

[0196] Plasma membrane-localized protein

[0197] In some cases, the plasma membrane-localized proteins described herein form the basic structure of the lipid-containing particles disclosed herein. In some cases, the plasma membrane-localized proteins described herein form a structural protein that is at least part of the protein core of the lipid-containing particle. In some cases, the plasma membrane-localized proteins described herein also promote the self-assembly of lipid-containing particles (e.g., VLPs). For example, the plasma membrane-localized proteins can promote the targeting to the plasma membrane and the packaging of lipid-containing particles (e.g., virus-like particles) by forming a membrane shell.

[0198] In some cases, the plasma membrane-localized protein is a viral protein, e.g., a viral protein derived from a virus. In some cases, the plasma membrane-localized protein is a mammalian protein, e.g., a mammalian protein derived from a mammal (e.g., a human). In some cases, the plasma membrane-localized protein is a human endogenous protein.

[0199] In some cases, the plasma membrane-localized protein is a polyprotein derived from a virus, its homolog, its fragment, its variant, or any combination thereof. For example, the plasma membrane-localized protein comprises a retroviral gag protein, e.g., a retroviral polyprotein comprising one or more of a matrix (MA) polypeptide, an RNA-binding phosphoprotein polypeptide, a capsid (CA) polypeptide, or a nucleocapsid (NC) polypeptide. In some cases, the gag protein is derived from Friend murine leukemia virus (FMLV). In some cases, the retroviral gag polyprotein is the gag polyprotein of an alpharetrovirus, a betaretrovirus, a gammaretrovirus, a deltaretrovirus, an epsilonretrovirus, or a foamy virus. In some cases, the retroviral gag polyprotein is the gag polyprotein of human immunodeficiency virus.

[0200] Examples of plasma membrane-localized proteins include human papillomavirus (HPV) L1 protein, HPV L2 protein, hepatitis B virus (HBV) core protein, Chikungunya virus (CHIKV) C-E3-E2-6k-E1, human immunodeficiency virus (HIV) gag-pol, HIV gag, respiratory syncytial virus (RSV) M, RSV NP, human metapneumovirus (HMPV) M, influenza M1, Zika virus (ZIKV) C, ZIKV prM / M, dengue virus (DENV) C-prM, West Nile virus (WNV) prME protein, WNV CprME protein, filovirus VP40 or Z protein, baculovirus P1 protein, rotavirus VP7, rotavirus VP2 protein, rotavirus VP6 protein, SARS M protein, SARS E protein, SARS N protein, porcine circovirus type 2 (PCV2) capsid, baculovirus VP2 protein, baculovirus VP5 protein, baculovirus VP3 protein, or baculovirus VP7 protein, hepatitis C virus (HCV) core protein, Ebola nucleocapsid, parvovirus VP1 protein, parvovirus VP2 protein, Newcastle disease virus (NDV) M protein, hepatitis E virus (HeV) M protein, Nipah virus (NIV) M protein, human polyomavirus 2 (JCPyV) VP1 protein, human parainfluenza virus type 3 (HPIV3) M protein, HPIV3N protein, or mumps virus (MuV) M protein, their homologs, their fragments, their variants, or any combination thereof.

[0201] In some cases, the plasma membrane-localized protein sequence comprises a human endogenous retrovirus (HERV) gag protein. In some cases, the plasma membrane-localized protein sequence comprises a plekstrin homology (PH) domain. Examples of the plasma membrane-localized protein sequence can include those described in Table 3. In some cases, the plasma membrane-localized protein comprises an amino acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences in Table 3. In some cases, the plasma membrane-localized protein comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences in Table 3. In some cases, the plasma membrane-localized protein comprises an amino acid sequence having at least about 80% sequence identity to any one of the sequences in Table 3. In some cases, the plasma membrane-localized protein comprises an amino acid sequence having at least about 85% sequence identity to any one of the sequences in Table 3. In some cases, the plasma membrane-localized protein comprises an amino acid sequence having at least about 90% sequence identity to any one of the sequences in Table 3. In some cases, the plasma membrane-localized protein comprises an amino acid sequence having at least about 95% sequence identity to any one of the sequences in Table 3. In some cases, the plasma membrane-localized protein comprises an amino acid sequence having at least about 96% sequence identity to any one of the sequences in Table 3. In some cases, the plasma membrane-localized protein comprises an amino acid sequence having at least about 97% sequence identity to any one of the sequences in Table 3. In some cases, the plasma membrane-localized protein comprises an amino acid sequence having at least about 98% sequence identity to any one of the sequences in Table 3. In some cases, the plasma membrane-localized protein comprises an amino acid sequence having at least about 99% sequence identity to any one of the sequences in Table 3.

[0202] Membrane fusion / envelope protein

[0203] The membrane fusion proteins disclosed herein can refer to proteins that are present on the outer membrane of lipid-containing particles (e.g., inserted into, attached to, or anchored in the lipid layer) and promote the fusion of lipid-containing particles with a membrane (e.g., the target cell membrane). In some cases, the membrane fusion protein mediates the tropism of lipid-containing particles, e.g., the lipid-containing particles preferentially fuse into one or more specific types of cells. In some cases, the membrane fusion protein results in the mixing of lipids in the lipid-containing particles and lipids in the target cell. In some cases, the lipid-containing particles contain human endogenous retrovirus (HERV) envelope proteins, humanized envelope proteins, or non-immunogenic membrane fusion molecules. Examples of HERV envelope proteins can include those described in Table 2 and Table 2-1.

[0204] In some cases, the HERV envelope protein comprises an amino acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences shown in Table 2-1. In some cases, the HERV envelope protein comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequences of the HERV envelope proteins listed in Table 2-1. In some cases, the HERV envelope protein comprises an amino acid sequence having at least about 80% sequence identity with the sequences of the HERV envelope proteins listed in Table 2-1. In some cases, the HERV envelope protein comprises an amino acid sequence having at least about 85% sequence identity with the sequences of the HERV envelope proteins listed in Table 2-1. In some cases, the HERV envelope protein comprises an amino acid sequence having at least about 90% sequence identity with the sequences of the HERV envelope proteins listed in Table 2-1. In some cases, the HERV envelope protein comprises an amino acid sequence having at least about 95% sequence identity with the sequences of the HERV envelope proteins listed in Table 2-1. In some cases, the HERV envelope protein comprises an amino acid sequence having at least about 96% sequence identity with the sequences of the HERV envelope proteins listed in Table 2-1. In some cases, the HERV envelope protein comprises an amino acid sequence having at least about 97% sequence identity with the sequences of the HERV envelope proteins listed in Table 2-1. In some cases, the HERV envelope protein comprises an amino acid sequence having at least about 98% sequence identity with the sequences of the HERV envelope proteins listed in Table 2-1. In some cases, the HERV envelope protein comprises an amino acid sequence having at least about 99% sequence identity with the sequences of the HERV envelope proteins listed in Table 2-1.

[0205] In some cases, the membrane fusion protein comprises a mammalian protein. In some cases, the membrane fusion protein comprises a viral protein. In some embodiments, the membrane fusion protein comprises a mammalian protein or a homolog of a mammalian protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity), a non-mammalian protein such as a viral protein or a homolog of a viral protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity), a native protein or a derivative of a native protein, a synthetic protein, a fragment thereof, a variant thereof, a protein chimera comprising one or more membrane fusion proteins or fragments, and any combination thereof.

[0206] Compared to a protein that is heterologous to a human subject, the non-immunogenic membrane fusion proteins provided herein can have reduced immunogenicity to a human subject. For example, the non-immunogenic membrane fusion protein can be humanized to reduce its immunogenicity to a human subject. In some embodiments, the membrane fusion protein can be modified to reduce immunoreactivity. For example, the membrane fusion protein can be modified with a molecule that reduces immune interactions such as PEG, such as described in Croyle MA et al., J Virol. January 2004;78(2):912-21 (which is incorporated herein by reference in its entirety). Thus, in some embodiments, the envelope protein comprises PEG, such as a polyethylene glycolated polypeptide. Amino acid residues in the membrane fusion protein that are targeted by the immune system can be altered to be unrecognized by the immune system, such as described in Lech PJ et al., Virology. April 2014;454-455:237-46; and Kneissl S et al., PLoS One. 2012;7(10):e46667 (each of which is incorporated herein by reference in its entirety). In some embodiments, the protein sequence of the membrane fusion protein is altered to be similar to the amino acid sequence found in humans (humanized). In some embodiments, the protein sequence of the membrane fusion protein is altered to a protein sequence that binds less strongly to the MHC complex. In some embodiments, the membrane fusion protein is derived from a virus or organism that does not infect humans (and humans have not been vaccinated against it), increasing the insensitivity of the patient's immune system to the membrane fusion protein possibility (e.g., there is a negligible humoral or cell-mediated adaptive immune response against the membrane fusion protein) (doi:10.1006 / mthe.2002.0550, doi:10.1371 / journal.ppat.1005641, doi:10.1038 / gt.2011.209, DOI 10.1182 / blood-2014-02-558163). In some embodiments, the glycosylation of the envelope protein is altered to alter immune interactions or reduce immunoreactivity.

[0207] In some cases, the membrane fusion protein comprises a sequence selected from the group consisting of Nipah virus protein F, measles virus F protein, tree shrew paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, henipavirus F protein, morbillivirus F protein, respiratory virus F protein, Sendai virus F protein, rubella virus F protein, or avian paramyxovirus F protein or a derivative thereof.

[0208] In some cases, the membrane fusion protein comprises a mammalian protein. Examples of mammalian membrane fusion proteins can include SNARE family proteins such as vSNARE and tSNARE, syncytin proteins such as syncytin-1 and syncytin-2, myomaker, myomixer, myomerger, FGFRL1 (fibroblast growth factor receptor-like 1), Minion, isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (e.g., as disclosed in U.S. Patent No. 6,099,857A), gap junction proteins such as connexin 43, connexin 40, connexin 45, connexin 32, or connexin 37 (e.g., as disclosed in US2007 / 0224176), Hap2, any protein capable of inducing syncytium formation between heterologous cells, homologs thereof, fragments thereof, variants thereof, and protein chimeras comprising one or more of the proteins or fragments thereof. In some embodiments, the membrane fusion protein comprises a curvature-generating protein, e.g., Epsin1, dynamin, or a protein comprising a BAR domain, such as those described in Kozlov et al., Curr Op StrucBio August 2015 2015;33:61-67; Zimmerberg et al., Nat Rev Mol Cell Biol. January 2006;7(1):9-19; Richard et al., Biochem J. December 1, 2011;440(Pt 2):185-193 (each of which is incorporated herein by reference in its entirety).

[0209] In some cases, the membrane fusion protein comprises a non-mammalian protein, for example, a viral membrane fusion protein. In some embodiments, the viral membrane fusion protein is a class I viral membrane fusion protein, a class II viral membrane fusion protein, a class III viral membrane fusion protein, a viral membrane fusion protein, or other viral membrane fusion proteins, or homologs thereof, fragments thereof, variants thereof, or protein chimeras comprising one or more proteins or fragments thereof. Examples of class I viral membrane fusion proteins that can be used in the VLPs disclosed herein include baculovirus F proteins, such as F proteins of the genus Nucleopolyhedrovirus (NPV), such as the Spodoptera exigua MNPV (SeMNPV) F protein and the Lymantria dispar MNPV (LdMNPV), influenza virus HA, parainfluenza virus F, HIV Env, Ebola virus GP, hemagglutinin from orthomyxoviruses, F proteins from paramyxoviruses (e.g., measles, (Katoh et al., BMC Biotechnology 2010, 10:37)), ENV proteins from retroviruses, and membrane fusion proteins of filoviruses and coronaviruses. In an embodiment, class II viral membrane fusion proteins (such as dengue E glycoprotein) have a structural feature of a β-sheet that forms an extended ectodomain, which refolds to produce a trimer of hairpins. In an embodiment, class II viral membrane fusion proteins lack a central coiled coil. Examples of class II viral membrane fusion proteins that can be used in the VLPs disclosed herein include tick-borne encephalitis E (TBEV E), Semliki Forest virus E1 / E2, and membrane fusion proteins derived from Sindbis, rubella virus, and dengue virus. In an embodiment, class III viral membrane fusion proteins such as vesicular stomatitis virus G glycoprotein combine the structural features found in class I and class II. In an embodiment, class III viral membrane fusion proteins comprise helices (e.g., like class I viral membrane fusion proteins, forming a six-helix bundle to fold back the protein), and three sheets with an amphipathic membrane fusion peptide at its end, reminiscent of class II viral membrane fusion proteins. Examples of class III viral membrane fusion proteins that can be used in the VLPs disclosed herein include rhabdovirus G (e.g., protein G of vesicular stomatitis virus (VSV-G)), herpesvirus glycoprotein B (e.g., herpes simplex virus 1 (HSV-1) gB), Epstein-Barr virus glycoprotein B (EBV gB), Sogotovirus G, baculovirus gp64 (e.g., Autographa californica multiple NPV (AcMNPV) gp64), and Borna disease virus (BDV) glycoprotein (BDV G).In an embodiment, class IV viral membrane fusion proteins are fusion-associated small transmembrane (FAST) proteins (doi:10.1038 / sj.emboj.7600767, Nesbitt, Rae L., “Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with Multifunctional FAST proteins” (2012). Electronic Thesis and Dissertation Repository. Paper 388), which are encoded by non-enveloped reoviruses. In an embodiment, class IV viral membrane fusion proteins are small enough that they do not form hairpins (doi:10.1146 / annurev-cellbio-101512-122422, doi:10.1016 / j.devcel.2007.12.008).

[0210] Examples of other viral membrane fusion proteins that can be used in the VLPs disclosed herein include viral syncytia proteins such as influenza hemagglutinin (HA) or mutants or chimeric proteins thereof; human immunodeficiency virus type 1 membrane fusion protein (HIV-1 ENV), gp120 from HIV that binds LFA-1 to form a lymphocyte syncytium, HIV gp41, HIV gp160, or HIV trans-activator of transcription (TAT); viral glycoprotein VSV-G, the viral glycoprotein from vesicular stomatitis virus of the family Rhabdoviridae; glycoproteins gB and gH-gL of varicella-zoster virus (VZV); murine leukemia virus (MLV)-10A1; endogenous feline virus RD114 envelope glycoprotein; FuG-B2 envelope glycoprotein; fusion protein of vesicular stomatitis Indiana virus and rabies virus glycoproteins (FuG-E); modified FuG-E (FuG-E(P440E)); gibbon ape leukemia virus glycoprotein (GaLV); G-type glycoproteins in rabies, Mokola, vesicular stomatitis virus, and togaviruses; murine hepatitis virus JHM surface projection protein; porcine respiratory coronavirus spike and membrane glycoproteins; avian infectious bronchitis spike glycoprotein and its precursor; bovine enteric coronavirus spike protein; F and H, HN, or G genes of measles virus; canine distemper virus, Newcastle disease virus, human parainfluenza virus 3, simian virus 41, Sendai virus, and human respiratory syncytial virus; gH of human herpesvirus 1 and simian varicella virus, with chaperone protein gL; gB of human, bovine, and cercopithicine herpesviruses; envelope glycoproteins of Friend murine leukemia virus and Mason Pfizer monkey virus; mumps virus hemagglutinin neuraminidase and glycoproteins F1 and F2; membrane glycoprotein from Venezuelan equine encephalitis; paramyxovirus F protein; SIV gp160 protein; Ebola virus G protein; or Sendai virus membrane fusion protein, or homologs, fragments, variants, or any combination thereof. In some cases, the viral membrane fusion protein comprises measles virus hemagglutinin (HA) protein and / or measles virus membrane fusion glycoprotein, influenza virus neuraminidase (NA) protein, measles virus F protein, influenza virus HA protein, Moloney virus MLV-A protein (amphotropic), Moloney virus MLV-E protein (ecotropic), baboon endogenous retrovirus (BAEV) glycoprotein, or modified baboon endogenous retrovirus glycoprotein (BaEVTRless), Ebola virus glycoprotein, foamy virus membrane fusion protein, or homologs, fragments, variants, or any combination thereof.

[0211] Examples of other viral membrane fusion proteins that can be used in the VLPs disclosed herein include hemagglutinin (HA) or neuraminidase (NA) proteins derived from Orthomyxoviridae - Influenza virus A, the E1 and E2 subunits (both together and separately included in the complex) of the E protein of Togaviridae - CHIV; the S, E, or MN proteins from Coronaviridae - SARS and COVID19; the F or G proteins from Paramyxoviridae - Nipah virus; the GP protein from Filoviridae - Ebola; the E protein from Flaviviridae - Dengue virus; the Gn and Gc proteins (both together and separately included in the complex) from Phenuviridae - Sandfly fever virus; the GP protein from Arenavirida - Lassa virus; the Gn and Gc proteins (both together and separately included in the complex) from Hantaviridae - Hantavirus; the G protein from Bornaviridae - Borna disease virus; the Gn and Gc proteins (both together and separately included in the complex) from Bunyaviridae - Crimean - Congo hemorrhagic fever virus; the S, M, or L proteins from Hepadnaviridae - Hepatitis B virus; the membrane fusion protein from Herpesviridae - Herpes simplex virus 1; the EV protein from Poxviridae - Smallpox virus; the S, L, or M proteins from Hepatitis D; or the glycoprotein from Hepeviridae - Hepatitis E virus, or homologs, fragments, variants thereof, and protein chimeras comprising one or more of the proteins or fragments thereof.

[0212] In some embodiments, the membrane fusion protein is derived from a paramyxovirus. In some embodiments, the membrane fusion protein is Nipah virus F protein, Measles virus F protein, Tupaia paramyxovirus F protein, Paramyxovirus F protein, Hendra virus F protein, Henipavirus F protein, Morbillivirus F protein, Respiratory virus F protein, Sendai virus F protein, Rubella virus F protein, or Avian paramyxovirus F protein.

[0213] In some embodiments, the membrane fusion protein is derived from Poxviridae. Additional exemplary membrane fusion proteins are disclosed in U.S. Patent No. 9,695,446, US2004 / 0028687, U.S. Patent No. 6,416,997, 7,329,807, US2017 / 0112773, US2009 / 0202622, and US 2004 / 0009604, and International Patent Publication Nos. WO 2006 / 027202 and WO2020102709, each of which is incorporated herein by reference in its entirety.

[0214] In some embodiments, the membrane fusion protein comprises EFF-1, AFF-1, connexins, such as connexin (such as Cn43, GAP43, CX43) (DOI: 10.1021 / jacs.6b05191), other tumor connexins, homologs thereof, fragments thereof, variants thereof, and protein fusions comprising one or more of the proteins or fragments thereof.

[0215] The membrane fusion proteins disclosed herein can be retargeted by mutating amino acid residues in the membrane fusion protein (e.g., a hemagglutinin protein). In some embodiments, the envelope protein is randomly mutated. In some embodiments, the envelope protein is rationally mutated. In some embodiments, the envelope protein undergoes directed evolution.

[0216] The membrane fusion proteins disclosed herein can be retargeted by covalently conjugating a targeting moiety. For example, the membrane fusion protein can be covalently conjugated to the targeting moiety by expressing a chimeric protein comprising an envelope protein linked to the targeting moiety. Targets of the targeting moiety include any peptide (e.g., a receptor) displayed on the target cell. In some instances, the target is expressed at a higher level on the target cell than on non-target cells.

[0217] For example, the targeting moiety can be selected to target a specific tissue type, such as muscle, brain, liver, pancreas, and lung, or to target diseased tissue, such as a tumor. In a particularly preferred embodiment of the present disclosure, the exosome targets the brain tissue.

[0218] Specific examples of the targeting moiety include a muscle-specific peptide targeting skeletal muscle discovered by phage display, a 29-amino acid fragment of the rabies virus glycoprotein that binds to the acetylcholine receptor, or a fragment of nerve growth factor that targets its receptor to target neurons, and a secretin peptide that binds to the secretin receptor and can be used to target bile and pancreatic epithelium. As an alternative, immunoglobulins and their derivatives (including scFv antibody fragments) can also be expressed as membrane fusion proteins targeting specific antigens, such as VEGFR for cancer gene therapy. As an alternative, the natural ligand for a receptor can be expressed as a membrane fusion protein to confer specificity, such as NGF that binds to NGFR and confers neuronal-specific targeting.

[0219] The targeting moiety can include, for example, an antibody or an antigen-binding fragment thereof (e.g., Fab, Fab’, F(ab’)2, Fv fragment, scFv antibody fragment, disulfide-linked Fv (sdFv), Fd fragment consisting of VH and CH1 domains, linear antibody, single-domain antibody such as sdAb (VL or VH), nanobody or camelid VHH domain), an antigen-binding fibronectin type III (Fn3) scaffold such as a fibronectin polypeptide minibody, a ligand, a cytokine, a chemokine or a T cell receptor (TCR). The membrane fusion protein can be retargeted by non-covalently conjugating the targeting moiety to the membrane fusion protein or a targeting protein (e.g., a hemagglutinin protein). For example, the membrane fusion protein can be engineered to bind to the Fc region of an antibody that targets an antigen on a target cell, redirecting the membrane fusion activity to the cell presenting the antibody target.

[0220] The targeting moiety can comprise, for example, a humanized antibody molecule, a full-length IgA, IgG, IgE or IgM antibody; a bispecific or multispecific antibody (e.g., etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments and isolated CDRs or combinations thereof; single-chain Fv; polypeptide-Fc chimeras; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., ); small modular immunopharmaceuticals (“SMIPs TM ”); single-chain or tandem diabodies VHH; minibodies; ankyrin repeat proteins or DART; TCR-like antibodies; MicroProteins; and

[0221] In embodiments, the targeting moiety attached to the membrane fusion protein binds to a cell surface marker on the target cell, e.g., a protein, glycoprotein, receptor, cell surface ligand, agonist, lipid, sugar, class I transmembrane protein, class II transmembrane protein or class III transmembrane protein.

[0222] In some cases, the lipid-containing particles (e.g., VLPs, exosomes or lipid nanoparticles) disclosed herein also display a targeting moiety that is not conjugated to a membrane fusion protein or other protein, in order to redirect the fusion activity of the lipid-containing particle to the cell bound by the targeting moiety, or to affect the homing of the lipid-containing particle towards the target cell.

[0223] Virus-like particles

[0224] In some aspects, the present disclosure provides compositions, methods, and systems related to virus-like particles that can be used to deliver cargoes into cells.

[0225] The virus-like particles (VLPs) disclosed herein can comprise one or more virus-derived proteins, such as the structural and envelope proteins of the VLPs. In some cases, the virus-derived proteins are present as part of a combinatorial protein that forms the VLPs.

[0226] In some cases, the loading capacity ratio of the VLPs disclosed herein is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 80-fold, 100-fold higher than that of conventional VLPs.

[0227] Structural proteins of VLPs

[0228] In some cases, the structural proteins described herein form the basic structure of the virus-like particles, e.g., at least a portion of the capsid that encapsulates the protein core of the VLPs. The structural proteins of the virus-like particles can include plasma membrane-localized proteins. In some cases, the plasma membrane-localized proteins described herein also facilitate the self-assembly of the VLPs, e.g., by forming a membrane coat that promotes the localization of the plasma membrane and the packaging of the virus-like particles. In some cases, the structural proteins described herein facilitate the release of the VLPs from the production cells that produce the VLPs.

[0229] In some cases, the structural proteins of the VLPs (e.g., plasma membrane-localized proteins) are viral proteins, e.g., viral proteins derived from a virus. In some cases, the structural proteins of the VLPs are mammalian proteins, e.g., mammalian proteins derived from a mammal (e.g., human). In some cases, the structural proteins of the VLPs are human endogenous proteins.

[0230] In some cases, the structural protein of the VLP (e.g., a plasma membrane-localized protein) is a polyprotein derived from a virus, its homolog, its fragment, its variant, or any combination thereof. For example, the structural protein of the VLP (e.g., a plasma membrane-localized protein) comprises a retroviral gag protein, e.g., a retroviral polyprotein comprising one or more of a matrix (MA) polypeptide, an RNA-binding phosphoprotein polypeptide, a capsid (CA) polypeptide, or a nucleocapsid (NC) polypeptide. In some cases, the gag protein is derived from Friend murine leukemia virus (FMLV). In some cases, the retroviral gag polyprotein is a gag polyprotein of an alpharetrovirus, betaretrovirus, gammaretrovirus, deltaretrovirus, epsilonretrovirus, or spumavirus. In some cases, the retroviral gag polyprotein is a gag polyprotein of human immunodeficiency virus.

[0231] Examples of the structural protein of the VLP (e.g., a plasma membrane-localized protein) include human papillomavirus (HPV) L1 protein, HPV L2 protein, hepatitis B virus (HBV) core protein, chikungunya virus (CHIKV) C-E3-E2-6k-E1, human immunodeficiency virus (HIV) gag-pol, HIV gag, respiratory syncytial virus (RSV) M, RSV NP, human metapneumovirus (HMPV) M, influenza M1, Zika virus (ZIKV) C, ZIKV prM / M, dengue virus (DENV) C-prM, West Nile virus (WNV) prME protein, WNV CprME protein, filovirus VP40 or Z protein, baculovirus P1 protein, rotavirus VP7, rotavirus VP2 protein, rotavirus VP6 protein, SARS M protein, SARS E protein, SARS N protein, porcine circovirus type 2 (PCV2) capsid, baculovirus VP2 protein, baculovirus VP5 protein, baculovirus VP3 protein, or baculovirus VP7 protein, hepatitis C virus (HCV) core protein, Ebola nucleocapsid, parvovirus VP1 protein, parvovirus VP2 protein, Newcastle disease virus (NDV) M protein, hepatitis E virus (HeV) M protein, Nipah virus (NIV) M protein, human polyomavirus 2 (JCPyV) VP1 protein, human parainfluenza virus type 3 (HPIV3) M protein, HPIV3 N protein, or mumps virus (MuV) M protein, its homolog, its fragment, its variant, or any combination thereof.

[0232] Envelope protein

[0233] In some cases, the VLPs disclosed herein comprise a lipid-based outer membrane ("envelope"). In some cases, the envelope comprises a single lipid layer. In some cases, the envelope comprises a lipid bilayer. In some cases, the envelope further comprises a membrane fusion protein (also referred to as an "envelope protein" for VLPs) inserted into, attached to, or anchored in the lipid layer.

[0234] The envelope protein can facilitate the fusion of the VLP with a membrane (e.g., a cell membrane). In some cases, the envelope protein mediates the tropism of the VLP, e.g., the VLP preferentially fuses into one or more specific types of cells. In some cases, the envelope protein causes mixing between the lipids in the VLP and the lipids in the target cell. The envelope protein can be any of the membrane fusion proteins disclosed above. In some cases, the envelope protein can be a chimeric protein comprising a targeting moiety disclosed above.

[0235] In some cases, the envelope protein of the VLP is engineered to pseudotype the VLP for some property (e.g., specific tropism for a selected cell population). In some cases, the envelope protein of the VLP is a viral glycoprotein or a mutant thereof, such as a pseudotyped viral glycoprotein, such as hepatitis B virus (HBV) glycoprotein, hepatitis C virus (HCV) glycoprotein, Marburg virus glycoprotein, Ebola virus glycoprotein, VSV-G glycoprotein or a mutant thereof; and the target cell is a hepatocyte. In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, such as the viral glycoprotein selected from influenza virus hemagglutinin, SARS-CoV glycoprotein, respiratory syncytial virus glycoprotein, human parainfluenza virus glycoprotein, and VSV-G or a mutant thereof; and the target cell is a lung cell. In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, such as the viral glycoprotein is measles virus hemagglutinin and / or measles virus membrane fusion glycoprotein or a mutant thereof, and the target cell is CD34 + cells. In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, such as the viral glycoprotein selected from measles virus hemagglutinin and / or measles virus membrane fusion glycoprotein, HTLV-1 glycoprotein, and VSV-G glycoprotein or a mutant thereof; and the target cell is CD8 +T cells. In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, such as, the viral glycoprotein is selected from HIV-1 envelope, HTLV-1 glycoprotein, measles virus hemagglutinin, and VSV-G glycoprotein or mutants thereof; and the target cell is a CD4+ T cell. In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, such as Ross River virus glycoprotein or VSV-G, or mutants thereof; and the target cell is a skeletal muscle cell. In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, such as, the viral glycoprotein is selected from Ebola virus glycoprotein, Marburg virus glycoprotein, and VSV-G or mutants thereof; and the target cell is an ocular cell (e.g., in retinal cells, photoreceptor cells, etc.). In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, such as, the viral glycoprotein is selected from Ebola virus glycoprotein, Marburg virus glycoprotein, and VSV-G or mutants thereof; and the target cell is an auditory cell (e.g., hair cells, cochlear cells, etc.). In some cases, the envelope protein of the VLP is a pseudotyped viral glycoprotein, such as, the viral glycoprotein is selected from rabies glycoprotein, Mokola virus glycoprotein, Semliki Forest virus glycoprotein, Sindbis virus glycoprotein, Venezuelan equine encephalitis virus glycoprotein, influenza hemagglutinin glycoprotein, and VSV-G or mutants thereof; and wherein the target cell is a central nervous system cell (e.g., neurons (e.g., excitatory and inhibitory neurons)); and glial cells (e.g., oligodendrocytes, astrocytes, and microglia)). In some cases, the envelope protein of the VLP may include those described in Table 1. In some cases, the membrane fusion protein comprises an amino acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence shown in Table 1. In some cases, the membrane fusion protein comprises an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence shown in Table 1. In some cases, the membrane fusion protein comprises an amino acid sequence having at least about 80% sequence identity to the sequence shown in Table 1. In some cases, the membrane fusion protein comprises an amino acid sequence having at least about 85% sequence identity to the sequence shown in Table 1. In some cases, the membrane fusion protein comprises an amino acid sequence having at least about 90% sequence identity to the sequence shown in Table 1. In some cases, the membrane fusion protein comprises an amino acid sequence having at least about 95% sequence identity to the sequence shown in Table 1. In some cases, the membrane fusion protein comprises an amino acid sequence having at least about 96% sequence identity to the sequence shown in Table 1.In some cases, the membrane fusion protein comprises an amino acid sequence having at least about 97% sequence identity to the sequences shown in Table 1. In some cases, the membrane fusion protein comprises an amino acid sequence having at least about 98% sequence identity to the sequences shown in Table 1. In some cases, the membrane fusion protein comprises an amino acid sequence having at least about 99% sequence identity to the sequences shown in Table 1.

[0236] Table 1. Sequences of virus-derived glycoproteins

[0237]

[0238]

[0239]

[0240]

[0241] Human endogenous VLPs and humanized VLPs

[0242] In some aspects, provided herein are virus-like particles having reduced immunogenicity or no immunogenicity in a human subject, e.g., non-viral human endogenous virus-like particles (heVLPs), or humanized VLPs comprising humanized structural proteins (e.g., humanized viral structural proteins) or humanized envelope proteins (e.g., humanized viral envelope proteins). In some embodiments, the humanized envelope proteins disclosed herein are derived from viral envelope proteins, e.g., by mutating or engineering the viral envelope protein such that the protein is non-immunogenic to humans. In some embodiments, the humanized structural proteins disclosed herein are derived from viral structural proteins, e.g., by mutating or engineering a viral structural protein such as a retroviral gag protein such that the protein is non-immunogenic to humans.

[0243] Unlike virus-like particles according to some embodiments of the present disclosure, the heVLPs or humanized VLPs described herein can package protein cargo by integrating all production DNA into the genomic DNA of a production cell line. Once the cell line is created, the protein delivery heVLPs can be produced constitutively or inducibly. The protein cargo is packaged into the heVLPs by fusing a selected human endogenous GAG protein or other plasma membrane-localizing protein (also referred to herein as a "plasma membrane recruitment domain") to the protein-based cargo.

[0244] The heVLP or humanized VLP system described herein has the potential to be simpler, more effective, and safer than conventional, artificially derived lipid / gold nanoparticles and virus particle-based delivery systems because the heVLP or humanized VLP is composed of human-derived or humanized components. The cargo within the particle can be human-derived or non-human-derived, but the heVLP or humanized VLP is derived from humans or contains human endogenous components or synthetic non-immunogenic components.

[0245] "Synthetic" components include surface scFv / nanobodies / darpin peptides, which have been shown to be non-immunostimulatory and can be used to enhance the targeting and cellular uptake of heVLP. This means that the outer surface of the particle lacks components that can significantly immunostimulate, which can minimize the immunogenicity and antibody neutralization of these particles.

[0246] In some cases, in addition to the cargo, the heVLP provided herein does not contain other exogenous viral components inherent to VLP, and this represents a significant and novel technological advancement. Additionally, the heVLP can utilize (but does not require) chemical-based dimers, and the heVLP can have the ability to package and deliver cargo molecules including therapeutic or diagnostic agents, which include biomolecules and chemicals such as specific single-stranded and / or double-stranded DNA molecules (e.g., plasmids, mini circles, end-capped linear DNA, AAV DNA, episomes, phage DNA, homology-directed repair templates, etc.), single-stranded and / or double-stranded RNA molecules (e.g., single-guide RNA, prime editing guide RNA, messenger RNA, transfer RNA, long non-coding RNA, circular RNA, RNA replicons, circular or linear spliced RNA, microRNA, small interfering RNA, short hairpin RNA, piwi-interacting RNA, toehold switch RNA, RNA that can be bound by RNA-binding proteins, phage RNA, RNA containing an internal ribosome entry site, etc.), proteins, chemical compounds, and / or molecules (e.g., small molecules), as well as combinations of the cargo listed above (e.g., AAV particles).

[0247] The heVLP described herein is different from conventional retroviral particles, virus-like particles (VLP), exosomes, and other previously described extracellular vesicles that can be loaded with cargo, at least because the heVLP can be produced by the strategic overexpression of human-derived components in human cells, the heVLP has a wide variety of possible cargoes and loading strategies, the heVLP lacks restrictive DNA / RNA length limitations, the heVLP lacks proteins derived from pol and exogenous gag, and the heVLP has a unique cellular entry mechanism.

[0248] This disclosure describes compositions and methods for cargo delivery that can be used with a variety of protein and nucleic acid molecules, including genome editing, epigenome regulation, transcriptome editing, and proteome regulation reagents suitable for many disease therapies.

[0249] In some aspects, this disclosure provides engineered heVLPs that include a membrane comprising a phospholipid bilayer that has one or more HERV-derived ENV / glycoproteins on the outer side (e.g., overexpressed from an exogenous source (such as a plasmid or a stably integrated transgene) in the heVLP-producing cells) (e.g., as shown in Table 2 or Table 2-1) or other human endogenous envelope proteins; and a biomolecule / chemical cargo that is a human endogenous GAG protein, other plasma membrane-localized proteins (e.g., as shown in Table 3), and / or is disposed in the core of the heVLP on the inner side of the membrane (e.g., disposed in a protein core surrounded by the phospholipid bilayer).

[0250] In some cases, the lipid-containing particles (e.g., VLPs) provided herein include a plasma membrane-localized protein that is a PH domain derived from phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), Discoidin (Disc) and actin-related protein 1 (Daap1), phosphatidylinositol 1 universal receptor (Grp1), oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton's tyrosine kinase (Btk), phosphatidylinositol 4-phosphate adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), switch B cell complex subunit SWAP70, or MAPK-associated protein 1 (MAPKAP1) or a mutant thereof. In some cases, the plasma membrane-localized protein includes a PH domain derived from a human protein. In some cases, the plasma membrane-localized protein includes a PH domain derived from human phospholipase Cδ1, human Akt1, human 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), human Daap1, murine Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, or human MAPKAP1 or a mutant thereof. In some cases, the plasma membrane-localized protein includes a membrane protein selected from CD9, CD47, CD63, and CD81 and their transmembrane domains. In some cases, the plasma membrane-localized protein includes a membrane protein selected from human CD9, human CD47, human CD63, and human CD81 and their transmembrane domains. In some cases, the plasma membrane-localized protein includes a non-immunogenic plasma membrane recruitment protein that includes Arc, human Arc, an endogenous retroviral gag protein, or a human endogenous retroviral gag protein. In some cases, the plasma membrane-localized protein includes any one of the sequences in Table 3.

[0251] In some aspects, provided herein are humanized VLPs comprising a membrane that includes a phospholipid bilayer having on the outer side one or more HERV-derived ENV / glycoproteins (e.g., overexpressed from an exogenous source (such as a plasmid or a stably integrated transgene) in heVLP-producing cells) (e.g., as shown in Table 2 or Table 2-1) or other human endogenous envelope proteins; and viral structural proteins (e.g., retroviral gag proteins) on the inner side of the membrane (e.g., in the protein core surrounded by the phospholipid bilayer).

[0252] In some aspects, provided herein are humanized VLPs comprising a membrane that includes a phospholipid bilayer having one or more of the viral envelope proteins disclosed herein; and human endogenous GAG proteins, other plasma membrane-localized proteins, and / or biomolecule / chemical cargo disposed in the core of the heVLP on the inner side of the membrane (e.g., disposed in the protein core surrounded by the phospholipid bilayer).

[0253] The cargo can be fused to human endogenous GAG or other plasma membrane-localized proteins. In some cases, the cargo is not fused to human endogenous GAG or other plasma membrane-localized proteins. In some cases, the heVLP or humanized VLP does not comprise non-human gag and / or pol proteins. In some cases, the heVLP or humanized VLP does not express gag and / or pol proteins, except for gag proteins encoded in the human genome or gag proteins encoded by a consensus sequence derived from gag proteins found in the human genome. The human-derived GAG or other plasma membrane-localized proteins fused to the cargo can be overexpressed from an exogenous source (such as a plasmid or a stably integrated transgene) in heVLP-producing cells.

[0254] Human endogenous GAG proteins and human pleckstrin homology (PH) domains can be localized to biological membranes. The PH domain can interact with phosphatidylinositol lipids and proteins (such as PIP2, PIP3, the βγ subunits of GPCRs, and PKC) within the biological membrane. However, in addition to being localized to the phospholipid bilayer, human endogenous GAG proteins can also drive budding and granule formation. This dual functionality of human endogenous GAG enables the packaging of cargo and the budding / formation of granules. One such human endogenous GAG protein for this purpose is the human Arc protein, which can be fused to protein-based cargo to recruit the cargo to the cytoplasmic side of the phospholipid bilayer. These human endogenous GAG phospholipid bilayer recruitment domains can be fused to the N-terminus or C-terminus of protein-based cargo via a polypeptide linker of variable length, regardless of the position of one or more nuclear localization sequences (NLS) within the cargo. In some cases, the linker between the protein-based cargo and the human endogenous GAG phospholipid bilayer recruitment domain is a polypeptide linker consisting mainly of glycine and serine, with a length of 5 - 20 (e.g., 8 - 12, e.g., 10) amino acids.

[0255] Table 2. Exemplary HERV envelope proteins

[0256]

[0257] a “+” and “-” refer to the direction within the sequence entry

[0258] *hENVK con is the consensus sequence from 10 proviral ENV sequences. The ENV sequences used to obtain this consensus ENV sequence are from the following HERVs: HERV-K113, HERV-K101, HERV-K102, HERV-K104, HERV-K107, HERV-K108, HERV-K109, HERV-K115, HERV-K11p22, and HERV-K12q13.

[0259] Table 2-1. Sequences of HERV envelope proteins

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266] Table 3. Exemplary plasma membrane recruitment domains

[0267]

[0268]

[0269]

[0270] *hGAGK con is a consensus sequence derived from 10 proviral GAG sequences. The GAG sequences used to obtain this consensus GAG sequence are from the following HERVs: HERV-K113, HERV-K101, HERV-K102, HERV-K104, HERV-K107, HERV-K108, HERV-K109, HERV-K115, HERV-K11p22, and HERV-K12q13.

[0271] Human endogenous GAG or other phospholipid bilayer recruitment domains can localize cargo to the phospholipid bilayer, and this protein cargo is packaged within heVLPs or humanized VLPs that bud from the production cells into the extracellular space. The use of these human endogenous GAGs and other phospholipid bilayer recruitment domains is novel and unique because these human endogenous GAGs and other proteins can facilitate the localization of cargo to the cytoplasmic face of the plasma membrane within the heVLP or humanized VLP production cells. The use of these human endogenous GAGs and other phospholipid bilayer recruitment domains can allow for the localization of cargo to the nucleus of transduced cells without the use of exogenous retroviral GAG or chemical and / or light-based dimerization systems.

[0272] If heVLPs are produced via transient transfection of a production cell line, the heVLPs can also package and deliver a combination of DNA and RNA. The DNA transfected into the cells will have size-dependent mobility such that a portion of the transfected DNA will remain in the cytosol while another portion of the transfected DNA will localize to the nucleus. A portion of the transfected DNA in the nucleus can express components for the production of heVLPs, while another portion near the cytosol / plasma membrane will be encapsulated and delivered within the heVLPs.

[0273] Combinations of exogenous DNA, exogenous RNA, and proteins (exogenous and / or endogenous proteins) will be referred to as type 1 cargo (T1 heVLP), exogenous RNA and proteins (exogenous and / or endogenous proteins) will be referred to as type 2 cargo (T2 heVLP), combinations of exogenous DNA and proteins (exogenous and / or endogenous proteins) will be referred to as type 3 cargo (T3 heVLP), and proteins (exogenous and / or endogenous proteins) will be referred to as type 4 cargo (T4 heVLP). Thus, T1 contains DNA, RNA, + / - exogenous proteins, T2 contains RNA + / - exogenous proteins, T3 contains DNA + / - exogenous proteins, and T4 is a particle that contains or does not contain exogenous protein cargo. Thus, T4 without exogenous protein is considered an "empty particle" because there is no "exogenous cargo". "Exogenous cargo" is cargo that is not endogenous to the production cell and can be packaged and / or incorporated into the heVLP. In addition to the types of cargo present in the T1-T4 heVLPs, the T1-T4 heVLPs can also package exogenous chemical molecules. For example, RNA in this context can be single guide RNA (sgRNA), Clustered Regularly Interspaced Palindromic Repeat (CRISPR) RNA (crRNA), and / or mRNA encoding the cargo. As used herein, a "small molecule" refers to a small organic or inorganic molecule having a molecular weight below about 3,000 daltons. Generally, small molecules used in this disclosure 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., between about 100 and about 3,000 Da, about 100 and about 2500 Da, about 100 and about 2,000 Da, about 100 and about 1,750 Da, about 100 and about 1,500 Da, about 100 and about 1,250 Da, about 100 and about 1,000 Da, about 100 and about 750 Da, about 100 and about 500 Da, about 200 and about 1500, about 500 and about 1000, about 300 and about 1000 Da, or about 100 and about 250 Da).

[0274] Cargo is limited by the diameter of the particle. For example, in some embodiments, the diameter of the particle is in the range of 150 nm to 500 nm.

[0275] Other examples of heVLPs, human endogenous viral structural proteins, and plasma membrane-localized proteins include those described in International Publication No. WO2020 / 252455, which is incorporated herein by reference in its entirety.

[0276] In some embodiments, to effectively recruit cargo into heVLPs or humanized VLPs, the cargo comprises a covalent or non-covalent linkage to a human endogenous GAG or other plasma membrane recruitment domain, such as those shown in Table 3. For example, the covalent linkage can include direct protein-protein chimeras produced from a single open reading frame, inteins that can form peptide bonds, and other proteins that can form covalent linkages at R-groups and / or RNA splice junctions. For example, non-covalent linkages can include DNA / DNA, DNA / RNA, and / or RNA / RNA hybrids (nucleic acids base pair with other nucleic acids via hydrogen bond interactions), protein domains that dimerize or multimerize in the presence or absence of a chemical compound / molecule to induce protein-protein binding (such as DmrA / DmrB / DmrC (Takara Bio), FKBP / FRB, dDZF, and leucine zippers), single-chain variable fragments, nanobodies, affibodies, 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 certain light wavelengths, and / or naturally-occurring shed proteins.

[0277] In some embodiments, the cargo comprises a fusion to a dimerization domain or a protein-protein binding domain, which may or may not require a molecule to trigger dimerization or protein-protein binding.

[0278] In some embodiments, the production cells are FDA-approved cell lines, allogeneic cells, and / or autologous cells derived from a donor. In some embodiments, the intact or active peptide domain of human CD47 can be incorporated into the heVLP surface to reduce immunogenicity. Examples of AAV proteins included herein are AAV REP 52, REP 78, and VP 1-3. The capsid site where the protein can be inserted is T138 starting from the VPl amino acid count. For example, a dimerization domain can be inserted at this site in the capsid. Examples of dimerization domains that may or may not require a small molecule inducer included herein are dDZF1, dDZF2, DmrA (Takara Bio), DmrB (Takara Bio), DmrC (Takara Bio), FKBP, FRB, GCN4scFv, 10x / 24x GCN4, GFP nanobody, and GFP. Examples of split inteins included herein are Npu DnaE, Cfa, Vma, and Ssp DnaE. Examples of other shedding proteins that together form a covalent bond included herein are Spy Tag and SpyCatcher. Examples of RNA-binding proteins included herein are MS2, Com, and PP7. Examples of synthetic DNA-binding zinc fingers included herein are ZF6 / 10, ZF8 / 7, ZF9, MK10, zinc finger 268, and zinc finger 268 / NRE. Examples of proteins that oligomerize due to quaternary structure included herein are Escherichia coli ferritin and other chimeric forms of ferritin. Examples of optogenetic "light-inducible proteins" included herein are Cry2, CIBN, and Lov2-Ja. Examples of peptides that enhance transduction included herein are L17E, Vectofusin-1 (Miltenyi Biotec), KALA, and various forms of nisin.

[0279] In another embodiment, the produced and isolated T1-T4 heVLPs can be loaded with biomolecular or chemical molecular cargo by using nucleofection, electroporation, lipids, polymers, or CaCl2 transfection, sonication, freeze thaw, incubation at various temperatures, and / or heat shock of the purified particles mixed with the cargo. These techniques are adapted from techniques used to load cargo into exosomes for therapeutic or research applications. For example, 100 μg of heVLP or humanized VLP can be resuspended in 200 - 450 μl of 50 mM trehalose in PBS, mixed with the cargo at the desired concentration, and electroporated in a 0.4 cm cuvette at 0.200 kV and 125 uF (GenePulser II electroporation system with a capacitance extender, Bio-Rad, Hercules, CA, USA).

[0280] In some embodiments, heVLPs or humanized VLPs are harvested from the cell culture supernatant 36 - 48 hours after transfection, or when the heVLPs or humanized VLPs are at their maximum concentration in the culture medium of the production cells (the production cells secrete the particles into the culture medium, and at a certain time point, the particle concentration in the culture medium will be optimal for harvesting the particles). The supernatant can be purified by any known method in the art, such as centrifugation, ultracentrifugation, precipitation, ultrafiltration, and / or chromatography. In some embodiments, the supernatant is first filtered, for example, 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, for example, using ultracentrifugation, such as centrifuging at a speed of 80,000 to 100,000 xg for 1 to 2 hours at a temperature between 1°C and 5°C, or centrifuging at a speed of 8,000 to 15,000 g for 10 to 16 hours at a temperature between 1°C and 5°C. After this centrifugation step, the heVLPs or humanized VLPs are concentrated (precipitated) in the form of a centrifugate, which can be resuspended to the desired concentration, mixed with a transduction enhancing reagent, subjected to buffer exchange, or used as is. In some embodiments, the heVLP-containing supernatant or the humanized VLP-containing supernatant can be filtered, precipitated, centrifuged, and resuspended to a concentrated solution. For example, polyethylene glycol (PEG) (such as PEG 8000), or an antibody-bead conjugate that binds to the surface protein or membrane component of the heVLPs or humanized VLPs can be used to precipitate the particles.

[0281] The purified particles are stable and can be stored at 4°C for up to one week or at -80°C for several years without losing significant activity.

[0282] Preferably, the heVLPs or humanized VLPs are resuspended or buffer exchanged such 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).

[0283] Exosomes

[0284] In some aspects, the lipid-containing particles disclosed herein are exosomes. In various aspects, compositions, methods, and systems related to exosomes are disclosed, and the exosomes can be used to deliver cargoes into cells. As used herein, the term "exosome" can refer to small membrane-bound vesicles (30 - 100 nm) of endosomal origin. In some cases, exosomes are released into the extracellular environment after the multivesicular body fuses with the plasma membrane. In some cases, the exosomes described herein are derived from B lymphocytes, dendritic cells (DCs), mesenchymal stromal cells (MSCs), amniotic epithelial (AE) cells, and / or placenta-derived cells.

[0285] Source cells according to the present disclosure can be selected from a wide range of cells, such as mesenchymal stem cells or stromal cells or fibroblasts (obtainable from, for example, bone marrow, adipose tissue, Wharton's jelly of the umbilical cord, perinatal tissues, dental germs, cord blood, skin tissue, etc.), amniotic cells and more specifically amniotic epithelial cells, and myeloid-derived suppressor cells. Generally, both primary cells and cell lines are suitable sources of exosomes. Examples include, for example, the following: human embryonic kidney (HEK) cells, pericytes, endothelial cells, lymphocytes, endothelial and epithelial cells from different organs such as from the trachea, lungs, gastrointestinal tract, urinary tract, etc., dendritic cells (DC) or other cells from the hematopoietic system, such as macrophages, monocytes, B cells or T cells, NK cells, neutrophils, eosinophils, mast cells or basophils, red blood cells or erythrocyte progenitors, platelets and megakaryocytes, etc., cells from different sources, such as placenta-derived cells (e.g., decidul placenta cells), syncytiotrophoblast cells and amniotic epithelial cells, etc., and cells from the CNS and PNS, such as microglial cells, astrocytes, oligodendrocytes and Schwann cells, ependymal cells and nerve cells, etc., adipocytes from brown or white fat, muscle cells from both smooth and skeletal muscle sources, and cardiomyocytes, to name just a few. Generally, exosomes can be substantially derived from any cell source, whether a primary cell source or a cell line. The exosome source cell can be any embryonic, fetal, and adult stem cell type, including induced pluripotent stem cells (iPSC) and other stem cells or progenitor cells derived by any method. When treating neurological diseases, it may be considered to utilize, for example, primary neurons, astrocytes, oligodendrocytes, microglial cells, and neural progenitor cells as source cells. For the patient to be treated, the source cells can essentially be allogeneic, autologous, or even xenogeneic, i.e., the cells can be from the patient himself or from unrelated, matched, or unmatched donors. In some cases, from a medical perspective, allogeneic cells may be preferred because they can provide an immunomodulatory effect, which in some cases cannot be obtained from autologous cells of a patient suffering from a certain indication.

[0286] In some cases, exosomes are produced by many different types of cells, including immune cells such as B lymphocytes, T lymphocytes, dendritic cells (DCs), and most cells. In some cases, for example, exosomes are also produced by glioma cells, platelets, reticulocytes, neurons, intestinal epithelial cells, and tumor cells. In some cases, the exosomes used according to the present application can be derived from any suitable cell, including the cells identified above. Exosomes have also been isolated from physiological fluids such as plasma, urine, amniotic fluid, and malignant effusions.

[0287] In some cases, exosomes are derived from immature DCs. In some cases, exosomes produced by immature DCs do not express MHC-II, MHC-I, or CD86. Thus, such exosomes do not stimulate naive T cells to a great extent and cannot induce a response in a mixed lymphocyte reaction. Therefore, exosomes produced by immature dendritic cells can be ideal candidates for delivering cargo (e.g., therapeutic cargo).

[0288] In some cases, exosomes are obtained from any autologous patient-derived, xenogeneic haplotype-matched, or xenogeneic stem cells in order to reduce or avoid an immune response in the patient to whom the exosomes are delivered. Any cell that produces exosomes can be used for this particular purpose.

[0289] In some cases, exosomes are produced by many different types of cells and have also been isolated from physiological fluids. Thus, according to the present disclosure, exosomes can be obtained from any suitable cell type as discussed above or by isolation from physiological fluids. The methods of the present disclosure can include isolating exosomes from cell culture media or tissue supernatants.

[0290] Exosomes produced by cells can be collected from the culture media by any suitable method. Preparations of exosomes can be prepared from cell cultures or tissue supernatants by centrifugation, filtration, or a combination of these methods. For example, exosomes can be prepared by: differential centrifugation, i.e., centrifugation at low speed (<20000g) to pellet larger particles, followed by high speed (>100000g) centrifugation to pellet exosomes, size filtration with a suitable filter (e.g., a 0.22 μm filter), gradient ultracentrifugation (e.g., with a sucrose gradient), or a combination of these methods.

[0291] In some cases, exosomes are loaded with cargo, such as therapeutic cargo, such as proteins, nucleic acid molecules, or small molecules. In some cases, exosomes are prepared and then loaded with the desired therapeutic cargo for delivery.

[0292] In some aspects, the exosomes disclosed herein are engineered to target desired cell types or tissues. This targeting can be achieved by expressing a targeting moiety on the surface of the exosome that binds to a cell surface moiety expressed on the surface of the cell to be targeted. In some cases, the targeting moiety is a peptide that is expressed as a chimeric protein with a transmembrane protein, which can be expressed on the surface of the exosome.

[0293] In some cases, exosomes target specific cell types or tissues by expressing a targeting moiety, such as a peptide, on their surface. Suitable peptides are those that bind to cell surface moieties, such as receptors or their ligands found on the cell surface of the cell to be targeted. Examples of suitable targeting moieties are short peptides, scFvs, and full proteins, provided that the targeting moiety can be expressed on the surface of the exosome and does not interfere with the insertion of membrane proteins into the exosome. The targeting peptide can be heterologous to the transmembrane exosome protein. The length of the peptide targeting moiety can be less than 100 amino acids, such as less than 50 amino acids, less than 30 amino acids, down to a minimum length of 10, 5, or 3 amino acids.

[0294] The targeting moiety can be selected to target a specific tissue type, such as muscle, brain, liver, pancreas, and lung, for example, or to target diseased tissue, such as a tumor. In a particularly preferred embodiment of the present disclosure, the exosomes are targeted to the brain tissue.

[0295] Specific examples of targeting moieties include muscle-specific peptides targeting skeletal muscle discovered by phage display, a 29-amino acid fragment of the rabies virus glycoprotein that binds to the acetylcholine receptor, or a fragment of nerve growth factor that targets its receptor to target neurons, and secretin peptide that binds to the secretin receptor and can be used to target bile and pancreatic epithelium. As an alternative, immunoglobulins and their derivatives (including scFv antibody fragments) can also be expressed as membrane fusion proteins targeting specific antigens, such as VEGFR for cancer gene therapy. As an alternative, the natural ligand for a receptor can be expressed as a membrane fusion protein to confer specificity, such as NGF that binds to NGFR and confers neuronal-specific targeting.

[0296] The peptide targeting moiety can be expressed on the surface of exosomes by expressing it as a membrane fusion protein with an exosomal transmembrane protein. Many proteins are known to be associated with exosomes; that is, they are incorporated into exosomes when exosomes are formed. In some cases, the targeting moiety comprises or is derived from those that are transmembrane proteins. Examples include Lamp-1, Lamp-2, CD13, CD86, Flotillin, Synaptotagmin-3, CD2, CD36, CD40, CD40L, CD41a, CD44, CD45, ICAM-1, Integrin α4, LiCAM, LFA-1, Mac-1α and Mac-1β, Vti-1A and Vti-1B, CD3ε and CD3ζ, CD9, CD18, CD37, CD53, CD63, CD81, CD82, CXCR4, FcR, GluR2 / 3, HLA-DM (MHC II), immunoglobulins, MHC-I or MHC-II components, TCRβ, and tetraspanins. In a particularly preferred embodiment of the present disclosure, the transmembrane protein is selected from Lamp-1, Lamp-2, CD13, CD86, Flotillin, Synaptotagmin-3. In some cases, the targeting moiety comprises or is derived from variations, alterations, modifications, or derivatizations of the amino acid sequences of the proteins discussed above. It should be understood that such variations, alterations, modifications, or derivatizations of the polypeptides as described herein are subject to the requirement that the polypeptide retain any further activity or characteristics, as may be described in subsequent sections of the present disclosure.

[0297] The targeting moiety can include, for example, an antibody or an antigen-binding fragment thereof (e.g., Fab, Fab’, F(ab’)2, Fv fragment, scFv antibody fragment, disulfide-linked Fv (sdFv), Fd fragment consisting of VH and CH1 domains, linear antibody, single-domain antibody such as sdAb (VL or VH), nanobody, or camelid VHH domain), an antigen-binding fibronectin type III (Fn3) scaffold such as a fibronectin polypeptide minibody, a ligand, a cytokine, a chemokine, or a T cell receptor (TCR). The membrane fusion protein can be retargeted by non-covalently conjugating the targeting moiety to a membrane fusion protein or a targeting protein (e.g., a hemagglutinin protein). For example, the membrane fusion protein can be engineered to bind to the Fc region of an antibody that targets an antigen on a target cell, redirecting the fusion activity to the cell displaying the antibody target.

[0298] The targeting moiety can comprise, for example, a humanized antibody molecule, a complete IgA, IgG, IgE, or IgM antibody; a bispecific antibody or a multispecific antibody (e.g., etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or collections thereof; single-chain Fv; polypeptide-Fc chimeras; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camelid antibodies; masked antibodies (e.g., ); small modular immunopharmaceuticals (“SMIPs TM ”); single-chain or tandem diabodies VHH; minibodies; ankyrin repeat proteins or DART; TCR-like antibodies; MicroProteins; and

[0299] In embodiments, the targeting moiety attached to the membrane protein binds to a cell surface marker on a target cell, e.g., a protein, glycoprotein, receptor, cell surface ligand, agonist, lipid, sugar, class I transmembrane protein, class II transmembrane protein, or class III transmembrane protein.

[0300] In some cases, the targeting moiety is introduced into exosomes by expressing a membrane fusion protein comprising the targeting moiety and an exosome transmembrane protein within the cell used to produce the exosomes. Expression of the membrane fusion protein in the cell allows the membrane fusion protein to be incorporated into the exosomes when produced from the cell.

[0301] In some cases, the targeting moieties suitable for exosomes disclosed herein can also be used for other lipid-containing particles disclosed herein, such as virus-like particles, lipid nanoparticles, and protein-lipid mediators.

[0302] For example, a polynucleotide construct such as a DNA plasmid expressing the membrane fusion protein is transfected into a cell. Any suitable method can be used to introduce the polynucleotide construct into the cell. The polynucleotide construct contains a suitable promoter sequence such that the encoded membrane fusion protein is expressed in the cell. It also contains a signal peptide sequence such that the protein is incorporated into the membrane of the endoplasmic reticulum when produced. The membrane protein is then exported to the exosome / lysosome compartment before being incorporated into the exosomes. The signal sequence can involve the signal peptide sequence for the exosome transmembrane protein.

[0303] In some cases, exosomes produced by the cells can be collected from the culture medium by any suitable method. Preparations of exosomes can be made from cell culture or tissue supernatants by centrifugation, filtration, or a combination of these methods. For example, exosomes can be prepared by: differential centrifugation, i.e., low speed (Centrifuge at <20000g) to precipitate larger particles, and then centrifuge at high speed (>100000g) to precipitate exosomes, perform size filtration using a suitable filter (e.g., 0.22 μm filter), gradient ultracentrifugation (e.g., using a sucrose gradient), or a combination of these methods.

[0304] In some cases, it is not necessary to include a specific targeting moiety in the exosomes. For example, exosomes can be directly administered to the site in need of therapy. Alternatively, for example, in the case where exosomes contain genetic material encoding an immunogen, in some cases, it is not necessary to directly target a specific site, and delivery (e.g., intradermal or intramuscular delivery) may be sufficient to generate the desired immune response without targeting the exosomes to any specific cell type. In some cases, no targeting moiety is included on the surface of the exosomes. However, the exosomes are selected such that they are more likely to target a specific tissue type. For example, exosomes derived from different cells can have a natural affinity for specific cell subtypes according to the requirements of their physiological functions, such as the well-established affinity of exosomes derived from mature dendritic cells for T cells. This affinity can be used to specifically deliver the above-mentioned cargo to the tissue.

[0305] In some cases, exosomes are produced by cells that are modified to express a chimeric polypeptide receptor (e.g., a chimeric antigen receptor (CAR)). In some cases, exosomes are produced from cells that are genetically modified to produce a chimeric polypeptide receptor that comprises (i) an extracellular recognition domain, (ii) at least one protease cleavage site, and (iii) an intracellular transcription factor, wherein binding of the extracellular recognition domain to its target induces proteolytic cleavage of at least one protease cleavage site and endogenous transcription of the intracellular transcription factor by at least one polynucleotide that encodes a gene product comprising at least one exosomal polypeptide. In some cases, the gene product further comprises a protein of interest, e.g., an antibody, a single-chain antibody, or any other antibody derivative, a bispecific T cell engager (BiTE), a receptor, a cytokine such as interleukin, an enzyme such as a cysteine protease, granzyme, Cas, Cas9, a checkpoint inhibitor, a co-stimulatory inhibitor, an RNA-binding protein, a membrane transporter such as NPC-1, a splicing factor, a protein associated with an organelle, a lysosomal enzyme, a transcription factor, a mitochondrial protein, an intracellular protein, an antiviral protein, an antibacterial protein. In some cases, when the protein of interest is an RNA-binding protein, the cells from which the exosomes are produced are further genetically modified to comprise an RNA cargo molecule selected from mRNA, sgRNA, shRNA, miRNA, shRNA, siRNA, lncRNA, ncRNA, piRNA, piwiRNA, circRNA, tRNA, rRNA, crRNA, and any combination thereof. In some cases, the genetic modification is an in vitro or ex vivo genetic modification. In some cases, the cells from which the exosomes are produced are effector immune cells, such as T cells, cytotoxic CD8+ T cells, CD4+ T cells, regulatory T cells, natural killer (NK) cells, B cells, plasma cells, dendritic cells (DCs), macrophages, monocytes, neutrophils, epithelial cells, endothelial cells, microglial cells, astrocytes, neurons, stem cells, bone marrow-derived mesenchymal stromal cells, Wharton's jelly-derived umbilical cord MSCs, or any other cell type. In some cases, the extracellular recognition domain of the chimeric polypeptide receptor is an antibody, an antibody derivative, a single-chain fragment, a single-chain antibody, a nanobody, a peptide, a ligand for a receptor, an adhesion molecule, a receptor, an interleukin receptor, an extracellular matrix component, or any combination thereof. In some cases, at least one protease cleavage site is at least one of the S1, S2, and / or S3 cleavage sites.In some cases, the membrane fusion polypeptide is a chimeric Notch polypeptide that covalently comprises, from the N-terminus to the C-terminus: (i) an extracellular recognition domain that does not naturally occur in the Notch receptor polypeptide; (ii) a Notch regulatory region that comprises Lin 12-Notch repeats, an S2 proteolytic cleavage site, and a transmembrane domain that comprises an S3 proteolytic cleavage site; and (iii) an intracellular transcription factor that is heterologous to the Notch regulatory region, wherein binding of the extracellular recognition domain to its target induces cleavage at the S2 and S3 protease cleavage sites, thereby releasing the intracellular transcription factor that activates transcription of the polynucleotide. In some cases, the Notch regulatory region further comprises a heterodimerization domain that comprises the S2 proteolytic cleavage site. In some cases, the S1 proteolytic cleavage site is a furin-like protease cleavage site that comprises the amino acid sequence Arg-X-(Arg / Lys)-Arg, where X is any amino acid. In some cases, the membrane fusion polypeptide comprises at least one linker. In some cases, the polynucleotide further comprises a transcriptional control element that is operably linked to the coding sequence and is responsive to the transcription factor. In some cases, the cell is genetically modified to produce at least two types of membrane fusion polypeptides, wherein at least one of (i) the extracellular recognition domain, (ii) the protease cleavage site, and (iii) the intracellular transcription factor is different between the membrane fusion polypeptides. In some cases, the extracellular recognition domains of the membrane fusion polypeptides are different from each other.

[0306] In some cases, loading of the protein cargo for exosomes disclosed herein is achieved by expressing a three-domain polypeptide construct in the source cell that produces exosomes. In some cases, such a polypeptide construct comprises (i) at least one protein of interest (POI), (ii) at least one multimerization domain, and (iii) at least one exosome sorting domain. The design of the three-domain polypeptide construct enables highly efficient loading of the POI into exosomes and also drives an increase in the yield of exosomes from the source cell.

[0307] The multimerization polypeptide domain can play a role in increasing the loading of the resulting exosomes, and such multimerization domains can interestingly be selected from a large number of different species and can also exhibit relatively different mechanisms of action (e.g., it can be a heterodimerization domain, or it can be a homotrimerization domain, or a pentamerization domain, etc.). In some cases, the multimerization domain is a homomultimerization domain because these domains can enable a simple design of the membrane fusion protein and can support the controlled loading of a single type of membrane fusion polypeptide construct into exosomes (as opposed to multiple membrane fusion constructs). The multimerization domain can be a dimerization domain, a trimerization domain, a tetramerization domain, or essentially any higher-order multimerization domain as long as the domain is capable of promoting the interaction of at least two domains (and the polypeptides of which they form a part). For example, the list of multimerization domains includes the following domains: the leucine zipper homodimerization domain of GCN4 from Saccharomyces cerevisiae, the reverse leucine zipper homodimerization domain of GCN4 from Saccharomyces cerevisiae, the coiled coil homodimerization domain of fibritin (from bacteriophage T4), the fragment X heterodimerization domain of phosphoprotein (from human respiratory syncytial virus A), the human alpha-helical coiled coil oligomerization domain of the collagen superfamily, the leucine zipper heterodimerization domain of Fos and Jun (human), the transmembrane homotrimerization domain of cardiac phospholamban (human), the homodimerization domain of parathyroid hormone (human), the transmembrane homodimerization domain of glycophorin A (human), the trimerization domain of Gp41 (from HIV), the C-terminal homodimerization domain of oncoprotein E7 (from HPV 45), and the EVH2 homotetramerization domain of vasodilator-stimulated phosphoprotein (human), the mitochondrial antiviral signaling protein CARD filaments and / or any combination thereof.

[0308] The multimerization domain can be placed in several different positions within the polypeptide construct. For example, the multimerization domain can be placed between the POI sequence and the exosome sorting domain sequence, within or near the exosome sorting domain sequence, and / or within or near the POI sequence. Generally speaking, the design of the three-domain polypeptide construct (both with respect to the choice of the multimerization domain and its position within the construct, and with respect to the choice of the exosome sorting domain and its position within the construct) can play a role in determining where the polypeptide ends up in exosomes after being produced in the exosome source cell. By selecting, for example, a tetraspanin exosome sorting protein (e.g., CD9, CD63 or CD81) or any other exosome membrane protein (such as Lamp2b), the POI can be enriched on the surface of exosomes. In contrast, selecting an exosome sorting protein that can be present in the exosome protein core, such as ALIX or syntenin, enables enrichment of the polypeptide construct (and thus the POI) substantially inside the exosome. Naturally, the polypeptide construct can be present both outside and inside the exosome, as well as in the exosome membrane. Additionally, in a preferred embodiment, the membrane fusion polypeptide construct can comprise various types of linkers between the different domains, i.e., between at least one POI, at least one multimerization domain and at least one exosome sorting domain. The linker can be, for example, a GS (i.e., glycine-serine) linker, i.e., a linker comprising the amino acids glycine and serine, or any other type of suitable linker domain that ensures that the activities of the different domains are not restricted when they are present in the membrane fusion polypeptide construct.

[0309] The three-domain membrane fusion polypeptide construct according to the present disclosure can be schematically described as follows (the following symbols should not be construed as indicating any C- and / or N-terminal orientation, which is only intended for illustrative purposes):

[0310] POI - multimerization domain - exosome sorting domain

[0311] The exosome sorting domain of the present disclosure may be selected from any one of the following proteins: CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, endodomin-1, endodomin-2, Lamp2b, TSPAN8, TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d / ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18 / ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Fc receptor, interleukin receptor, immunoglobulin, MHC-I or MHC-II component, CD2, CD3ε, CD3ζ, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD86, CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD274, CD362, COL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, L1CAM, LAMB1, LAMC1, LFA-1, LGALS3BP, Mac-1α, Mac-1β, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B, and any combination thereof.

[0312] In some cases, exosomes are loaded by means of cell-penetrating peptides, such as those described in U.S. Patent Publication No. US20190388347, which is incorporated herein by reference in its entirety.

[0313] Examples of exosomes, source cells that produce exosomes, cargoes that can be delivered in exosomes, methods of loading exosomes with cargoes, and methods of producing exosomes include those described in U.S. Patent Publication Nos. US20070298118, US20180177727, US20200062813, US20200206360, US20200023012, US20160137716, US20170173113, US20130053426, US20190167810, US20190388347, US20190224331, US20160137716, US20210188903, US20210069254, and US20200407418, each of which is incorporated herein by reference in its entirety).

[0314] Lipid nanoparticles or protein-lipid mediators

[0315] In some aspects, the present disclosure provides compositions, methods, and systems related to lipid nanoparticles that can be used to deliver cargoes to cells. In some aspects, the present disclosure provides compositions, methods, and systems related to protein-lipid mediators that can be used to deliver cargoes to cells.

[0316] Lipid nanoparticles can provide a biocompatible and biodegradable delivery system for the therapeutic cargoes disclosed herein. In some cases, the lipid nanoparticles disclosed herein comprise nanostructured lipid carriers (NLCs), polymer nanoparticles (PNPs), or lipid-polymer nanoparticles (PLNs). NLCs are modified solid lipid nanoparticles (SLNs) that retain the properties of SLNs, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) can play a role in therapeutic delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a new type of carrier that combines liposomes and polymers, can also be employed. These nanoparticles have the complementary advantages of PNPs and liposomes. PLNs consist of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell provides good biocompatibility. Thus, these two components increase drug encapsulation efficiency, facilitate surface modification, and prevent leakage of water-soluble drugs.

[0317] Examples of the lipid nanoparticles disclosed herein include those described in: JA Zuris et al., Nat Biotechnol. October 30, 2014; 33(1):73-80; Hou et al., Lipid nanoparticles for mRNA delivery. Nat Rev Mater (2021); US Patent Publication Nos. US20230140670, US20190136231, US20160311759, US20180290965, US20210078936, US20160106842, US20140303232, US20210371858; International Patent Publication Nos. WO2019 / 067992, WO / 2017 / 173054, WO2015 / 095340, WO2014 / 136086, and WO2019217941, each of which is incorporated herein by reference in its entirety.

[0318] Freight

[0319] The freight can include a therapeutic freight and / or a binding partner of the therapeutic freight. As used herein, "freight" can refer to one or more chemical agents, e.g., a combination of small molecule compounds, DNA, RNA, and proteins, a combination of RNA and proteins, a combination of DNA and proteins, or a protein, for, e.g., therapeutic or diagnostic use, or for applications in genome editing, epigenome regulation, and / or transcriptome regulation. In addition, endogenous RNA and proteins from the production cells can be packaged and / or incorporated into lipid-containing particles (e.g., VLPs, e.g., heVLPs or humanized VLPs). In some cases, the lipid-containing particles disclosed herein are capable of packaging and delivering multiple freights, e.g., biomolecules including nucleic acids (DNA, RNA) or proteins, chemical compounds including small molecules and / or other molecules, and any combination thereof, into eukaryotic cells. In some cases, the term "freight" can be used interchangeably with "cargo".

[0320] In some embodiments, the cargo comprised in and to be delivered by the lipid-containing particles disclosed herein comprises a polypeptide, e.g., a nuclear transport polypeptide, a nucleic acid-binding polypeptide, a reprogramming polypeptide, a DNA editing polypeptide, a DNA repair polypeptide, a DNA recombination polypeptide, a transposase polypeptide, a DNA integration polypeptide, a targeted endonuclease (e.g., a zinc finger nuclease (ZFN), a transcription activator-like nuclease (TALEN), Cas9 or homologs thereof), a recombinase, an enzyme, a structural polypeptide, a signaling polypeptide, a regulatory polypeptide, a transport polypeptide, a sensory polypeptide, a motility polypeptide, a defense polypeptide, a storage polypeptide, a transcription factor, an antibody, a cytokine, a hormone, a catabolic polypeptide, an anabolic polypeptide, a proteolytic polypeptide, a metabolic polypeptide, a kinase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, an enzyme modulator polypeptide, a protein-binding polypeptide, a lipid-binding polypeptide, a membrane fusion polypeptide, a cell differentiation polypeptide, an epigenetic polypeptide, a cell death polypeptide, or any combination thereof. In some embodiments, the cargo comprised in the lipid-containing particles disclosed herein comprises a protein for degrading a protein targeted in a cell. In some cases, the cargo comprised in the lipid-containing particles disclosed herein comprises a chimeric antigen receptor (CAR), an antibody, a T cell receptor, or a functional fragment thereof, or any combination thereof.

[0321] In some embodiments, the cargo comprised in and to be delivered by the lipid-containing particles disclosed herein comprises a polynucleotide, e.g., a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule. In some cases, the polynucleotide encodes a polypeptide, such as those polypeptides described in the previous paragraph. In some cases, the polynucleotide comprises a napR / DNAbp-programmed nucleic acid molecule described below.

[0322] In some embodiments, the cargo comprised in and to be delivered by the lipid-containing particles disclosed herein comprises a ribonucleoprotein (RNP) complex formed between one or more proteins and one or more polynucleotides. For example, the cargo can comprise an RNP complex formed by a nucleic acid-programmable R / DNA-binding protein (napR / DNAbp) and a napR / DNAbp-programmed nucleic acid molecule (e.g., a Cas protein and a guide RNA) described below.

[0323] In some embodiments, the cargo comprised in and to be delivered by the lipid-containing particles disclosed herein comprises other therapeutic molecules, such as ribozymes, aptamers, aptazyme, peptides, oligonucleotides, antibody mimetics, peptidomimetics, antibody-drug conjugates, antibiotics, carbohydrates, ribosomes, mitochondria, and small molecule compounds.

[0324] In some embodiments, the cargo comprised within and to be delivered by the lipid-containing particles disclosed herein comprises a polypeptide, e.g., an enzyme, a structural polypeptide, a signaling polypeptide, a regulatory polypeptide, a transport polypeptide, a sensory polypeptide, a motility polypeptide, a defensive polypeptide, a storage polypeptide, a transcription factor, an antibody, a cytokine, a hormone, a catabolic polypeptide, an anabolic polypeptide, a proteolytic polypeptide, a metabolic polypeptide, a kinase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, an enzyme modulator polypeptide, a protein-binding polypeptide, a lipid-binding polypeptide, a membrane fusion polypeptide, a cell differentiation polypeptide, an epigenetic polypeptide, a cell death polypeptide, a nuclear transport polypeptide, a nucleic acid-binding polypeptide, a reprogramming polypeptide, a DNA editing polypeptide, a DNA repair polypeptide, a DNA recombination polypeptide, a transposase polypeptide, a DNA integration polypeptide, a targeted endonuclease (e.g., a zinc finger nuclease, a transcription activator-like nuclease (TALEN), cas9 and its homologs), a recombinase, and any combination thereof. In some embodiments, the protein targets a protein in a cell for degradation. In some embodiments, the protein targets a protein in a cell for degradation by targeting the protein to the proteasome. In some embodiments, the protein is a wild-type protein. In some embodiments, the protein is a mutant protein. In some embodiments, the protein is a fusion protein or a chimeric protein.

[0325] In some cases, the cargo comprised within and to be delivered by the lipid-containing particles disclosed herein comprises a decoy protein for binding to a target protein that causes a disease; a peptide or protein for inducing endosomal escape, such as HA2; a peptide or protein for targeting exosomes to a tissue or organ or cell type of interest; an antibody, an intrabody, a single-chain variable fragment (scFv), an affibody, a bispecific or multispecific antibody or conjugate, a receptor, etc.; an enzyme for enzyme replacement therapy, such as α-glucosidase and / or glucocerebrosidase; a transport protein, such as NPC1 or cystinosin; a peptide or protein for optimizing the in vivo behavior of exosomes (e.g., its circulation time or immune system recognition), such as CD47 and / or CD55 or a portion of these proteins; a cytokine or chemokine; a targeting peptide or protein, such as an RVG peptide, a VSV-G peptide, a p-selectin binding peptide, or an e-selectin binding peptide; a cell-penetrating peptide (CPP) (e.g., Tat, penetratin, TP10, CADY); or a tumor suppressor.

[0326] In some cases, the cargo comprised within and to be delivered by the lipid-containing particles disclosed herein comprises an immunogenic molecule, such as a vaccine. The vaccine can be a peptide antigen, an RNA (e.g., an mRNA or a circRNA), a DNA (e.g., a DNA molecule encoding an antigen). The cargo can also comprise an adjuvant that enhances the immunogenicity of the vaccine composition.

[0327] In some cases, the cargo is a protein loaded in lipid-containing particles, and its function is to bind to another cargo molecule to be delivered by the lipid-containing particles (e.g., nucleic acid molecules, proteins, RNPs, etc.).

[0328] In some embodiments, the cargo contained in the lipid-containing particles disclosed herein and to be delivered by the lipid-containing particles includes small molecules, such as ions (e.g., Ca 2+ , Cl - , Fe 2+ ), carbohydrates, lipids, reactive oxygen species, reactive nitrogen species, isoprenoids, signaling molecules, heme, polypeptide cofactors, electron-accepting compounds, electron-donating compounds, metabolites, ligands, and any combination thereof. In some embodiments, the small molecule is a pharmaceutical agent that interacts with a target in a cell. In some embodiments, the small molecule targets a protein in a cell for degradation. In some embodiments, the small molecule targets a protein in a cell for degradation by targeting the protein to the proteasome. In some embodiments, the small molecule is a proteolysis-targeting chimera molecule (PROTAC).

[0329] In some embodiments, the cargo contained in the lipid-containing particles disclosed herein and to be delivered by the lipid-containing particles includes a mixture of proteins, nucleic acids, or metabolites, such as multiple polypeptides, multiple nucleic acids, multiple small molecules; combinations of nucleic acids, polypeptides, and small molecules; ribonucleoprotein complexes (e.g., Cas9-gRNA complexes); multiple transcription factors, multiple epigenetic factors, reprogramming factors (e.g., Oct4, Sox2, cMyc, and KLF4); multiple regulatory RNAs; and any combination thereof.

[0330] In some embodiments, the cargo contained in the lipid-containing particles disclosed herein and to be delivered by the lipid-containing particles includes one or more organelles, such as chondriosome, mitochondria, lysosomes, nuclei, cell membranes, cytoplasm, endoplasmic reticulum, ribosomes, vacuoles, endosomes, spliceosomes, polymerases, capsids, acrosomes, autophagosomes, centrioles, glycolysosomes, glyoxysomes, hydrogenosomes, melanosomes, spindle remnants, myofibrils, nematocysts, peroxisomes, proteasomes, vesicles, stress granules, networks of organelles, and any combination thereof.

[0331] In some cases, the cargo contained in the lipid-containing particles disclosed herein and to be delivered by the lipid-containing particles includes one or more of the following: RNA (viral or heterologous), DNA (single-stranded, double-stranded), green fluorescent protein, nucleases, iron oxide NPs (IONPs), paclitaxel, Alexa 488, porphyrin, doxorubicin, fluorescein, DOTA chelator, RNA (messenger, micro, small interfering), ricin A chain, HIV-1 Tat peptide, alkaline phosphatase, green fluorescent protein, quantum dot 585, methacrylate (monomer, polymer), CpG DNA, fluorescent protein, luciferase, nickel, biotin, fluorescein polymethacrylate, gadolinium diethylenetriaminepentaacetic acid polymethacrylate, CRISPR (Cas9 and guide RNA), green fluorescent protein or mCherry, CellB protein, [NiFe] hydrogenase, ziconotide peptide, three-enzyme cascade (genetically linked), alcohol dehydrogenase, polystyrene sulfonate, RNA, green or blue-green fluorescent protein, Pseudozyma antarctica lipase B, horseradish peroxidase, DOTAC10 micelle (which contains Gd(III) or Zn(II)), Gd(DOTA), fluorescent probe, doxorubicin, DAPI, acridine orange, propidium iodide, diaminoacridine, iron oxide NP, Gd(III) or Tb(III). In some cases, the cargo contained in the lipid-containing particles disclosed herein includes those described in Rohovie, M.J. et al., Bioengineering & Translational Medicine, 2:43-57 (which is incorporated herein by reference in its entirety).

[0332] In some cases, the cargo contained in the lipid-containing particles of the present disclosure and to be delivered by the lipid-containing particles comprises a polypeptide having the following lengths: at least 10 amino acids (aa), at least 20 aa, at least 30 aa, at least 50 aa, at least 80 aa, at least 100 aa, at least 150 aa, at least 200 aa, at least 250 aa, at least 300 aa, at least 350 aa, at least 400 aa, at least 500 aa, at least 600 aa, at least 700 aa, at least 800 aa, at least 900 aa, at least 1000 aa, at least 1200 aa, at least 1400 aa, at least 1500 aa, at least 1800 aa, at least 2000 aa, at least 2500 aa, at least 3000 aa, at least 4000 aa or at least 5000 aa. In some cases, the cargo contained in the lipid-containing particles of the present disclosure comprises a polypeptide having the following lengths: about 20 aa, about 30 aa, about 50 aa, about 80 aa, about 100 aa, about 150 aa, about 200 aa, about 250 aa, about 300 aa, about 350 aa, about 400 aa, about 500 aa, about 600 aa, about 700 aa, about 800 aa, about 900 aa, about 1000 aa, about 1200 aa, about 1400 aa, about 1500 aa, about 1800 aa, about 2000 aa, about 2500 aa, about 3000 aa, about 4000 aa or about 5000 aa.

[0333] In some cases, the cargo comprised in the lipid-containing particles disclosed herein and to be delivered by the lipid-containing particles comprises polynucleotides encoding polypeptides having the following lengths: at least 20 aa, at least 30 aa, at least 50 aa, at least 80 aa, at least 100 aa, at least 150 aa, at least 200 aa, at least 250 aa, at least 300 aa, at least 350 aa, at least 400 aa, at least 500 aa, at least 600 aa, at least 700 aa, at least 800 aa, at least 900 aa, at least 1000 aa, at least 1200 aa, at least 1400 aa, at least 1500 aa, at least 1800 aa, at least 2000 aa, at least 2500 aa, at least 3000 aa, at least 4000 aa or at least 5000 aa. In some cases, the cargo comprised in the lipid-containing particles of the present disclosure comprises polynucleotides encoding polypeptides having the following lengths: about 20 aa, about 30 aa, about 50 aa, about 80 aa, about 100 aa, about 150 aa, about 200 aa, about 250 aa, about 300 aa, about 350 aa, about 400 aa, about 500 aa, about 600 aa, about 700 aa, about 800 aa, about 900 aa, about 1000 aa, about 1200 aa, about 1400 aa, about 1500 aa, about 1800 aa, about 2000 aa, about 2500 aa, about 3000 aa, about 4000 aa or about 5000 aa.

[0334] In some cases, the polypeptide contained in the lipid-containing particles disclosed herein and to be delivered by the lipid-containing particles forms a protein that is at least 1 kDa, at least 2 kDa, at least 5 kDa, at least 10 kDa, at least 15 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 35 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 100 kDa, at least 120 kDa, at least 150 kDa, at least 180 kDa, at least 200 kDa, at least 220 kDa, at least 250 kDa, at least 280 kDa, at least 300 kDa, at least 320 kDa, at least 350 kDa, at least 400 kDa, at least 500 kDa, at least 600 kDa, at least 700 kDa, at least 800 kDa, at least 900 kDa or at least 1000 kDa. In some cases, the polypeptide contained in the lipid-containing particles disclosed herein and to be delivered by the lipid-containing particles forms a protein that is about 1 kDa, about 2 kDa, about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 100 kDa, about 120 kDa, about 150 kDa, about 180 kDa, about 200 kDa, about 220 kDa, about 250 kDa, about 280 kDa, about 300 kDa, about 320 kDa, about 350 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, about 800 kDa, about 900 kDa or about 1000 kDa.

[0335] In some cases, the cargo contained in the lipid-containing particles disclosed herein and to be delivered by the lipid-containing particles comprises single-stranded polynucleotides having the following lengths: at least 50 nucleotides, at least 80 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, at least 1000 nucleotides, at least 1200 nucleotides, at least 1400 nucleotides, at least 1500 nucleotides, at least 1800 nucleotides, at least 2000 nucleotides, at least 2500 nucleotides, at least 3000 nucleotides, at least 4000 nucleotides, at least 5000 nucleotides, at least 6000 nucleotides, at least 8000 nucleotides, at least 10000 nucleotides, at least 12000 nucleotides, at least 14000 nucleotides or at least 15000 nucleotides. In some cases, the cargo contained in the lipid-containing particles of the present disclosure comprises single-stranded polynucleotides encoding polypeptides having the following lengths: about 20 nucleotides, about 30 nucleotides, about 50 nucleotides, about 70 nucleotides, about 80 nucleotides, about 100 nucleotides, about 120 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, about 1200 nucleotides, about 1400 nucleotides, about 1500 nucleotides, about 1800 nucleotides, about 2000 nucleotides, about 2500 nucleotides, about 3000 nucleotides, about 4000 nucleotides, about 5000 nucleotides, about 6000 nucleotides, about 8000 nucleotides, about 10000 nucleotides, about 12000 nucleotides, about 14000 nucleotides or about 15000 nucleotides.

[0336] In some cases, the cargo included in the lipid-containing particles disclosed herein and to be delivered by the lipid-containing particles comprises double-stranded polynucleotides having the following lengths: at least 50 nucleotides, at least 80 nucleotides, at least 100 base pairs (bp), at least 150 bp, at least 200 bp, at least 250 bp, at least 300 bp, at least 350 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1000 bp, at least 1200 bp, at least 1400 bp, at least 1500 bp, at least 1800 bp, at least 2000 bp, at least 2500 bp, at least 3000 bp, at least 4000 bp, at least 5000 bp, at least 6000 bp, at least 8000 bp, at least 10000 bp, at least 12000 bp, at least 14000 bp, or at least 15000 bp. In some cases, the cargo included in the lipid-containing particles of the present disclosure comprises double-stranded polynucleotides encoding polypeptides having the following lengths: about 20 bp, about 30 bp, about 50 bp, about 70 bp, about 80 bp, about 100 bp, about 120 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1200 bp, about 1400 bp, about 1500 bp, about 1800 bp, about 2000 bp, about 2500 bp, about 3000 bp, about 4000 bp, about 5000 bp, about 6000 bp, about 8000 bp, about 10000 bp, about 12000 bp, about 14000 bp, or about 15000 bp.

[0337] Nuclease

[0338] Any suitable nuclease can be delivered by the lipid-containing particles disclosed herein, which contain the nuclease or a polynucleotide encoding the nuclease. Suitable nucleases include CRISPR-associated (Cas) proteins or Cas nucleases, including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides (e.g., Cas9 or Cas14), type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides (e.g., Cpf1 / Cas12a, C2c1 or c2c3), and type VI CRISPR-associated (Cas) polypeptides (e.g., C2c2 / Cas13a, Cas13b, Cas13c, Cas13d); zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA-binding proteins (RBPs); CRISPR-associated RNA-binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), and eukaryotic Argonaute (eAgo)); any derivatives thereof; any variants thereof; and any fragments thereof.

[0339] In some embodiments, the cargo in the lipid-containing particles disclosed herein comprises or encodes a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated (Cas) protein or Cas nuclease that functions in a non-naturally occurring CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) / Cas (CRISPR-associated) system. In bacteria, the system can provide adaptive immunity against foreign DNA (Barrangou, R. et al., “CRISPR provides acquired resistance against viruses in prokaryotes,” Science (2007) 315:1709-1712; Makarova, K.S. et al., “Evolution and classification of the CRISPR-Cas systems,” Nat Rev Microbiol (2011) 9:467-477; Garneau, J.E. et al., “The CRISPR / Cas bacterial immune system cleaves bacteriophage and plasmid DNA,” Nature (2010) 468:67-71; Sapranauskas, R. et al., “The Streptococcus thermophilus CRISPR / Cas system provides immunity in Escherichia coli,” Nucleic Acids Res (2011) 39:9275-9282).

[0340] One or more components (e.g., modified and / or unmodified) of the CRISPR / Cas system delivered by lipid-containing particles disclosed herein can be used as genome engineering tools in a variety of organisms, including a variety of mammals, animals, plants, and yeast. The CRISPR / Cas system can comprise a guide nucleic acid complexed with a Cas protein, such as guide RNA (gRNA) for targeted regulation of gene expression and / or activity or nucleic acid editing. The RNA-guided Cas protein (e.g., Cas nuclease, such as Cas9 nuclease) can specifically bind to a target polynucleotide (e.g., DNA) in a sequence-dependent manner.Cas proteins, if having nuclease activity, can cleave DNA (Gasiunas, G. et al., “Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria,” Proc Natl Acad Sci USA (2012) 109:E2579-E286; Jinek, M. et al., “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science (2012) 337:816-821; Sternberg, S.H. et al., “DNA interrogation by the CRISPR RNA-guided endonuclease Cas9,” Nature (2014) 507:62; Deltcheva, E. et al., “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III,” Nature (2011) 471:602-607), and have been widely used for programmable genome editing in a variety of organisms and model systems (Cong, L. et al., “Multiplex genome engineering using CRISPR Cas systems,” Science (2013) 339:819-823; Jiang, W. et al., “RNA-guided editing of bacterial genomes using CRISPR-Cas systems,” Nat. Biotechnol. (2013) 31:233-239; Sander, J.D. & Joung, J.K, “CRISPR-Cas systems for editing, regulating and targeting genomes,” Nature Biotechnol. (2014) 32:347-355).

[0341] In some cases, the Cas protein delivered by the lipid-containing particles of the present disclosure is mutated and / or modified to produce a nuclease-deficient protein or a protein having reduced nuclease activity relative to the wild-type Cas protein. The nuclease-deficient protein can retain the ability to bind DNA, but can lack or have reduced nucleic acid cleavage activity. The cargo protein or the protein encoded by the cargo nucleic acid molecule comprising a Cas nuclease (e.g., retaining wild-type nuclease activity, having reduced nuclease activity, and / or lacking nuclease activity) can function in the CRISPR / Cas system to regulate the level and / or activity of a target gene or protein (e.g., reduce, increase, or eliminate). The Cas protein can bind to the target polynucleotide and prevent transcription by a physical barrier or edit the nucleic acid sequence to produce a non-functional gene product.

[0342] In some embodiments, the cargo in the lipid-containing particles disclosed herein comprises or encodes a Cas protein that forms a complex with a guide nucleic acid such as a guide RNA (gRNA). In some embodiments, the cargo in the lipid-containing particles disclosed herein comprises or encodes a Cas protein that forms a complex with a single guide nucleic acid such as a single guide RNA (sgRNA). In some embodiments, the cargo in the lipid-containing particles disclosed herein comprises or encodes an RNA binding protein (RBP) that is optionally complexed with a guide nucleic acid such as a guide RNA (e.g., sgRNA) that is capable of forming a complex with a Cas protein.

[0343] One or more components of any suitable CRISPR / Cas system can be delivered by the lipid-containing particles of the present disclosure. CRISPR / Cas systems can be referred to using a variety of naming systems. Exemplary naming systems are provided in Makarova, K.S. et al., “An updated evolutionary classification of CRISPR-Cas systems,” Nat Rev Microbiol (2015) 13:722-736 and Shmakov, S. et al., “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems,” Mol Cell (2015) 60:1-13. The CRISPR / Cas system can be a type I, II, III, IV, V, VI system or any other suitable CRISPR / Cas system. As used herein, a CRISPR / Cas system can be a class 1, class 2 or any other appropriately classified CRISPR / Cas system. The determination of class 1 or class 2 can be based on the genes encoding the effector modules. Class 1 systems typically have a multi-subunit crRNA-effector complex, while class 2 systems typically have a single protein, such as Cas9, Cpf1, C2c1, C2c2, C2c3 or a crRNA-effector complex. Class 1 CRISPR / Cas systems can use a complex of multiple Cas proteins to effect regulation. Class 1 CRISPR / Cas systems can include, for example, type I (e.g., I, IA, IB, IC, ID, IE, IF, IU), type III (e.g., III, IIIA, IIIB, IIIC, IIID) and type IV (e.g., IV, IVA, IVB) CRISPR / Cas types. Class 2 CRISPR / Cas systems can use a single large Cas protein to effect regulation. Class 2 CRISPR / Cas systems can include, for example, type II (e.g., II, IIA, IIB) and type V CRISPR / Cas types. CRISPR systems can be complementary to each other, and / or can provide functional units in trans to facilitate CRISPR locus targeting.

[0344] Cargo delivered by the lipid-containing particles of the present disclosure can comprise or encode a type 1 or type 2 Cas protein. The Cas protein can be a type I, II, III, IV, V or VI Cas protein. The Cas protein can comprise one or more domains. Examples of domains include: guide nucleic acid recognition and / or binding domains, nuclease domains (e.g., DNase or RNase domains, RuvC, HNH), DNA binding domains, RNA binding domains, helicase domains, protein-protein interaction domains, and dimerization domains. The guide nucleic acid recognition and / or binding domain can interact with a guide nucleic acid. The nuclease domain can comprise catalytic activity for nucleic acid cleavage. The nuclease domain may lack catalytic activity that blocks nucleic acid cleavage. The Cas protein can be a chimeric Cas protein fused to other proteins or polypeptides. The Cas protein can be a chimera of various Cas proteins, e.g., comprising domains from different Cas proteins.

[0345] Examples of Cas proteins that can be delivered by the lipid-containing particles of the present disclosure include c2c1, Cas13a (formerly C2c2), Cas13b, Cas13c, Cas13d, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), Cas10, Cas10d, Cas14, Cas10, Cas10d, CasF, CasG, CasH, Cas12a (formerly Cpf1), Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4 and Cul966 and their homologs or modified versions. Examples of mutant Cas9 proteins or Cas9 variants include SpG, SpRY, eSpCas9(1.1), SpCas9-HF1, nSpCas9, SpCas9(H840A), dSpCas9, SpCas9(N863A), SpCas9(D839A), SpCas9(H983A), and other substances described in Chuang CK et al., Int J Mol Sci. September 13, 2021; 22(18):9872 (which is incorporated herein by reference in its entirety).

[0346] Another example of a Cas protein that can be delivered by the lipid-containing particles of the present disclosure includes Cas14. A Cas14 protein or polypeptide (also referred to as a "CasZ" protein or polypeptide) can bind and / or modify (e.g., cut, nick, methylate, demethylate, etc.) a target nucleic acid and / or a polypeptide associated with the target nucleic acid (e.g., methylation or acetylation of a histone tail) (e.g., in some cases, the CasZ protein contains an active chimeric ligand, and in some cases, the CasZ protein provides nuclease activity). In some cases, the Cas14 protein or polypeptide is a naturally occurring protein (e.g., naturally occurring in a prokaryotic cell) (e.g., the CasZ protein). In other cases, the Cas14 protein or polypeptide is not a naturally occurring polypeptide (e.g., the Cas14 protein is a variant Cas14 protein, a chimeric protein, etc.). The Cas14 protein contains three partial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains), which are not contiguous with respect to the primary amino acid sequence of the Cas14 protein but form the RuvC domain once the protein is produced and folded. Naturally occurring Cas14 proteins act as endonucleases that catalyze cleavage at specific sequences in a targeted nucleic acid (e.g., double-stranded DNA (dsDNA)). Sequence specificity is provided by an associated guide RNA that hybridizes to a target sequence within the target DNA. Naturally occurring Cas14 guide RNAs are crRNAs, where the crRNA includes (i) a guide sequence that hybridizes to the target sequence in the target DNA, and (ii) a protein-binding segment that binds to the Cas14 protein. Examples of Cas14 proteins include those described in U.S. Patent Publication Nos. US20200172886 and US20210214697, Harrington LB et al., Science. November 16, 2018; 362(6416):839-842; Aquino-Jarquin G. Nanomedicine. June 2019; 18:428-431 (each of which is incorporated herein by reference in its entirety). In some cases, the cargoes disclosed herein contain a Cas14 polypeptide or a nucleic acid molecule encoding a Cas14 polypeptide. In some cases, the cargoes disclosed herein contain a Cas14a polypeptide or a nucleic acid molecule encoding a Cas14a polypeptide. In some cases, the cargoes disclosed herein contain a Cas14b polypeptide or a nucleic acid molecule encoding a Cas14b polypeptide. In some cases, the cargoes disclosed herein contain a Cas14c polypeptide or a nucleic acid molecule encoding a Cas14c polypeptide.

[0347] The Cas protein can be from any suitable organism. Examples include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp.) Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp.) Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, Leptotrichia wadeii, Leptotrichia wadeii F0279, Rhodobacter capsulatus SB1003, Rhodobacter capsulatus R121, Rhodobacter capsulatus DE442, Lachnospiraceae bacterium NK4A179, Lachnospiraceae bacterium MA2020, Clostridium aminophilum DSM 10710, Paludibacter propionicigenes WB4, Carnobacterium gallinarum DMS4847, Carnobacterium gallinarum DSM4847, and Francisella novicida. In some aspects, the organism is Streptococcus pyogenes (S. pyogenes). In some aspects, the organism is Staphylococcus aureus (S. aureus). In some aspects, the organism is Streptococcus thermophilus (S. thermophilus).

[0348] Cas proteins can be derived from multiple bacterial species, including Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Listeria seeligeri, Listeria weihenstephanensis FSL R90317, Listeria weihenstephanensis FSL M60635, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp.Torquens), Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp. Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobacter eiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella succinogenes, Campylobacter jejuni subsp.Campylobacter jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.

[0349] The Cas proteins disclosed herein can be wild-type or modified forms of Cas proteins. The Cas proteins can be active variants, inactive variants, or fragments of wild-type or modified Cas proteins. The Cas proteins can contain amino acid alterations relative to the wild-type version of the Cas protein, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof. The Cas proteins can be polypeptides having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a wild-type exemplary Cas protein. The Cas proteins can be polypeptides having at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas protein. The variants or fragments can contain at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to the wild-type or modified Cas protein or a portion thereof. The variants or fragments can be targeted to a nucleic acid locus for complexing with a guide nucleic acid while lacking nucleic acid cleavage activity.

[0350] Cas proteins can comprise one or more nuclease domains, such as a DNase domain. For example, the Cas9 protein can comprise a RuvC-like nuclease domain and / or an HNH-like nuclease domain. The RuvC domain and the HNH domain can each cleave a different strand of double-stranded DNA to create a double-strand break in the DNA. A Cas protein can comprise only one nuclease domain (e.g., Cpf1 comprises a RuvC domain but lacks an HNH domain).

[0351] A Cas protein can comprise an amino acid sequence having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a nuclease domain of a wild-type Cas protein (e.g., a RuvC domain, an HNH domain).

[0352] A Cas protein can be modified to optimize the regulation of gene expression. A Cas protein can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, and / or enzymatic activity. A Cas protein can also be modified to alter any other activity or property of the protein, such as stability. For example, one or more nuclease domains of a Cas protein can be modified, deleted, or inactivated, or the Cas protein can be truncated to remove domains that are not essential for the function of the protein, or to optimize (e.g., enhance or decrease) the activity of the Cas protein for regulating gene expression.

[0353] In some embodiments, the cargo delivered by the lipid-containing particles of the present disclosure comprises a nuclease-null DNA binding protein derived from a DNA nuclease, which can induce transcriptional activation or repression of a target DNA sequence. In some embodiments, the cargo comprises or encodes a nuclease-null RNA binding protein derived from an RNA nuclease, which can induce transcriptional activation or repression of a target RNA sequence. For example, the cargo can comprise or encode a Cas protein lacking cleavage activity.

[0354] A Cas protein can be a chimeric protein. For example, a Cas protein can be fused to a heterologous functional domain. The heterologous functional domain can comprise a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. A Cas protein can also be fused to a heterologous polypeptide to provide enhanced or decreased stability. The fusion domain or heterologous polypeptide can be located at the N-terminus, C-terminus, or internally of the Cas protein.

[0355] The regulation of a gene can be any gene of interest. Genetic homologs of the genes described herein are expected to be covered. For example, a gene can exhibit a certain identity and / or homology to the genes disclosed herein. Thus, genes that show or exhibit about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology (at the nucleic acid or protein level) can be modified. Genes that show or exhibit about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity (at the nucleic acid or protein level) are also expected to be modified.

[0356] The Cas protein can be provided in any form. For example, the Cas protein can be provided in the form of a protein, such as a single Cas protein or a Cas protein complexed with a guide nucleic acid. The Cas protein can be provided in the form of a nucleic acid encoding the Cas protein, such as RNA (e.g., messenger RNA (mRNA)) or DNA.

[0357] The nucleic acid encoding the Cas protein delivered by the lipid-containing particles of the present disclosure can be codon-optimized for efficient translation into a protein in a specific cell or organism.

[0358] In some embodiments, the Cas protein is a dead Cas protein. A dead Cas protein can be a protein lacking nucleic acid cleavage activity.

[0359] The Cas protein can include a modified form of a wild-type Cas protein. The modified form of the wild-type Cas protein can include amino acid alterations (e.g., deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the Cas protein. For example, the modified form of the Cas protein can have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of the wild-type Cas protein (e.g., Cas9 from Streptococcus pyogenes). The modified form of the Cas protein may have no substantial nucleic acid cleavage activity. When the Cas protein is in a modified form with no substantial nucleic acid cleavage activity, it can be referred to as enzymatically inactivated and / or “dead” (abbreviated “d”). The dead Cas protein (e.g., dCas, dCas9) can bind to the target polynucleotide but cannot cleave the target polynucleotide. In some aspects, the dead Cas protein is a dead Cas9 protein.

[0360] The dCas9 polypeptide can bind to a single guide RNA (sgRNA) to activate or repress transcription of the target DNA. The sgRNA can be introduced into cells that express an engineered chimeric receptor polypeptide. In some cases, such cells contain one or more different sgRNAs that target the same nucleic acid. In other cases, the sgRNAs target different nucleic acids in the cell. The nucleic acid targeted by the guide RNA can be any nucleic acid expressed in a cell such as an immune cell. The targeted nucleic acid can be a gene involved in immune cell regulation. In some embodiments, the nucleic acid is related to cancer. The nucleic acid related to cancer can be a cell cycle gene, a cell response gene, an apoptosis gene, or a phagocytosis gene. The recombinant guide RNA can be recognized by a CRISPR protein, a nuclease-inactive CRISPR protein, its variants, its derivatives, or its fragments.

[0361] Enzymatically inactivated can refer to a polypeptide that can bind to a nucleic acid sequence in a polynucleotide in a sequence-specific manner but cannot cleave the target polynucleotide. The enzymatically inactivated site-directed polypeptide can include an enzymatically inactivated domain (e.g., nuclease domain). Enzymatically inactivated can refer to inactive. Enzymatically inactivated can refer to substantially inactive. Enzymatically inactivated can refer to essentially inactive. Enzymatically inactivated can refer to an activity that is less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% of the activity compared to the wild-type exemplary activity (e.g., nucleic acid cleavage activity, wild-type Cas9 activity).

[0362] One or more nuclease domains of a Cas protein (e.g., RuvC, HNH) can be deleted or mutated such that they no longer have function or contain reduced nuclease activity (e.g., inactivated or dead Cas, i.e., "dCas"). For example, in a Cas protein that contains at least two nuclease domains (e.g., Cas9), if one of the nuclease domains is deleted or mutated, the resulting Cas protein (referred to as a nickase) can create a single-strand break at the CRISPR RNA (crRNA) recognition sequence within double-stranded DNA, but not a double-strand break. Such a nickase can cut either the complementary or non-complementary strand, but not both. If all nuclease domains of a Cas protein (e.g., both the RuvC nuclease domain and the HNH nuclease domain in a Cas9 protein; the RuvC nuclease domain in a Cpf1 protein) are deleted or mutated, the resulting Cas protein can have a reduced ability or no ability to cut both strands of double-stranded DNA. Examples of mutations that can convert a Cas9 protein into a nickase are the D10A (aspartic acid to alanine at position 10 of Cas9) mutation in the RuvC domain of Cas9 from Streptococcus pyogenes. The H939A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) in the HNH domain of Cas9 from Streptococcus pyogenes can convert Cas9 into a nickase. Examples of mutations that can convert a Cas9 protein into dead Cas9 are the D10A (aspartic acid to alanine at position 10 of Cas9) mutation in the RuvC domain of Cas9 from Streptococcus pyogenes and the H939A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) in the HNH domain.

[0363] Relative to the wild-type version of the protein, the dead Cas protein can comprise one or more mutations. The mutations can result in nucleic acid cleavage activity that is less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% in one or more of the multiple nucleic acid cleavage domains of the wild-type Cas protein. The mutations can result in one or more of the multiple nucleic acid cleavage domains retaining the ability to cleave the complementary strand of the target nucleic acid, but reducing its ability to cleave the non-complementary strand of the target nucleic acid. The mutations can result in one or more of the multiple nucleic acid cleavage domains retaining the ability to cleave the non-complementary strand of the target nucleic acid, but reducing its ability to cleave the complementary strand of the target nucleic acid. The mutations can result in one or more of the multiple nucleic acid cleavage domains lacking the ability to cleave the complementary and non-complementary strands of the target nucleic acid. The residues to be mutated in the nuclease domain can correspond to one or more catalytic residues of the nuclease. For example, residues (such as Asp10, His840, Asn854, and Asn856) in the wild-type exemplary Streptococcus pyogenes Cas9 polypeptide can be mutated to inactivate one or more of the multiple nucleic acid cleavage domains (e.g., the nuclease domain). The residues to be mutated in the nuclease domain of the Cas protein can correspond to the residues Asp10, His840, Asn854, and Asn856 in the wild-type Streptococcus pyogenes Cas9 polypeptide, as determined, for example, by sequence and / or structural alignment.

[0364] As an example, the residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 (or corresponding mutations of any Cas protein) can be mutated. For example, for example, D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A. Mutations other than alanine substitutions can be suitable.

[0365] The D10A mutation can be combined with one or more of the H840A, N854A, or N856A mutations to produce a Cas9 protein (e.g., a dead Cas9 protein) that substantially lacks DNA cleavage activity. The H840A mutation can be combined with one or more of the D10A, N854A, or N856A mutations to produce a site-directed polypeptide that substantially lacks DNA cleavage activity. The N854A mutation can be combined with one or more of the H840A, D10A, or N856A mutations to produce a site-directed polypeptide that substantially lacks DNA cleavage activity. The N856A mutation can be combined with one or more of the H840A, N854A, or D10A mutations to produce a site-directed polypeptide that substantially lacks DNA cleavage activity.

[0366] In some embodiments, the Cas protein is a type II Cas protein. In some embodiments, the Cas protein is a type II Cas protein. In some embodiments, the Cas protein is a Cas9 protein, a modified version of the Cas9 protein, or is derived from the Cas9 protein. For example, a Cas9 protein lacking cleavage activity. In some embodiments, the Cas9 protein is the Cas9 protein from Streptococcus pyogenes (e.g., SwissProt accession number Q99ZW2). In some embodiments, the Cas9 protein is the Cas9 from Staphylococcus aureus (e.g., SwissProt accession number J7RUA5). In some embodiments, the Cas9 protein is a modified version of the Cas9 protein from Streptococcus pyogenes or Staphylococcus aureus. In some embodiments, the Cas9 protein is derived from the Cas9 protein from Streptococcus pyogenes or Staphylococcus aureus. For example, a Streptococcus pyogenes or Staphylococcus aureus Cas9 protein lacking cleavage activity.

[0367] Cas9 can generally refer to a polypeptide having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from Streptococcus pyogenes). Cas9 can refer to a polypeptide having at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity to a wild-type exemplary Cas9 polypeptide (e.g., from Streptococcus pyogenes). Cas9 can refer to the wild-type or modified form of the Cas9 protein, which can contain amino acid alterations such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0368] In some embodiments, the cargo comprises or encodes a "zinc finger nuclease" or "ZFN". A ZFN refers to a chimera between a cleavage domain (such as the cleavage domain of FokI) and at least one zinc finger motif (e.g., at least 2, 3, 4, or 5 zinc finger motifs), and the zinc finger motifs can bind to polynucleotides (such as DNA and RNA). The heterodimerization of two separate ZFNs in a certain orientation and spacing at a certain position in a polynucleotide can result in the cleavage of the polynucleotide. For example, a ZFN that binds to DNA can induce a double-strand break in the DNA. To allow the dimerization of the two cleavage domains and the cleavage of DNA, two separate ZFNs can bind to opposite strands of the DNA, with their C-termini separated by a certain distance. In some cases, the linker sequence between the zinc finger domain and the cleavage domain may require the 5' edge of each binding site to be separated by about 5 - 7 base pairs. In some cases, the cleavage domain is fused to the C-terminus of each zinc finger domain. Exemplary ZFNs include those described in Urnov et al., Nature Reviews Genetics, 2010, 11:636 - 646; Gaj et al., Nat Methods, 2012, 9(8):805 - 7; U.S. Patent Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; 6,979,539; 7,013,219; 7,030,215; 7,220,719; 7,241,573; 7,241,574; 7,585,849; 7,595,376; 6,903,185; 6,479,626; and U.S. Application Publication Nos. 2003 / 0232410 and 2009 / 0203140.

[0369] In some embodiments, a cargo protein comprising a ZFN or a protein encoded by a cargo nucleic acid molecule can create a double-strand break in a target polynucleotide, such as DNA. The double-strand break in the DNA can lead to DNA break repair, which allows for the introduction of gene modifications (e.g., nucleic acid editing). The DNA break repair can occur via non-homologous end joining (NHEJ) or homology-directed repair (HDR). In HDR, a donor DNA repair template can be provided, which comprises homology arms flanking the site of the target DNA. In some embodiments, the ZFN is a zinc finger nickase that induces a site-specific single-strand DNA break or nick, resulting in HDR. Descriptions of zinc finger nickases can be found, for example, in Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7):1327-33. In some embodiments, the ZFN binds to a polynucleotide (e.g., DNA and / or RNA), but is unable to cleave the polynucleotide.

[0370] In some embodiments, the cleavage domain of a cargo protein comprising a ZFN or a protein encoded by a cargo nucleic acid molecule comprises a modified form of a wild-type cleavage domain. The modified form of the cleavage domain can include amino acid alterations (e.g., deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the cleavage domain. For example, the modified form of the cleavage domain can have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of the wild-type cleavage domain. The modified form of the cleavage domain can have no substantial nucleic acid cleavage activity. In some embodiments, the cleavage domain is enzymatically inactivated.

[0371] In some embodiments, the cargo protein or the protein encoded by the cargo nucleic acid molecule comprises a "TALEN" or "TAL effector nuclease". A TALEN refers to an engineered transcriptional activator-like effector nuclease that typically comprises a central domain of DNA-binding tandem repeats and a cleavage domain. A TALEN can be generated by fusing a TAL effector DNA-binding domain and a DNA cleavage domain. In some cases, the DNA-binding tandem repeats have a length of 33-35 amino acids and contain two hypervariable amino acid residues at positions 12 and 13, which can recognize at least one specific DNA base pair. The transcriptional activator-like effector (TALE) protein can be fused to a nuclease, such as a wild-type or mutant FokI endonuclease or the catalytic domain of FokI. For its use in TALENs, several mutations have been made to FokI, for example, these mutations improve cleavage specificity or activity. Such TALENs can be engineered to bind to any desired DNA sequence. TALENs can be used to generate gene modifications (e.g., nucleic acid sequence editing) by creating a double-strand break in a target DNA sequence, which then undergoes NHEJ or HDR. In some cases, a single-stranded donor DNA repair template is provided to facilitate HDR. A detailed description of TALENs and their use in gene editing can be found, for example, in U.S. Patent Nos. 8,440,431; 8,440,432; 8,450,471; 8,586,363; and 8,697,853; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Beurdeley et al., Nat Commun, 2013, 4:1762; and Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(1):49-55.

[0372] In some embodiments, the TALEN is engineered for reduced nuclease activity. In some embodiments, the nuclease domain of the TALEN comprises a modified form of the wild-type nuclease domain. The modified form of the nuclease domain can include amino acid alterations (e.g., deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the nuclease domain. For example, the modified form of the nuclease domain can have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of the wild-type nuclease domain. The modified form of the nuclease domain can have no substantial nucleic acid cleavage activity. In some embodiments, the nuclease domain is enzymatically inactivated.

[0373] In some embodiments, transcription activator-like effector (TALE) proteins are fused to domains that can regulate transcription and that do not contain a nuclease. In some embodiments, transcription activator-like effector (TALE) proteins are designed to act as transcriptional activators. In some embodiments, transcription activator-like effector (TALE) proteins are designed to act as transcriptional repressors. For example, the DNA-binding domain of a transcription activator-like effector (TALE) protein can be fused (e.g., linked) to one or more transcriptional activation domains, or one or more transcriptional repression domains. Examples of transcriptional activation domains include the herpes simplex virus VP16 activation domain and tetrameric repeats of the VP16 activation domain, such as the VP64 activation domain. Other examples include VP16, VP32, VP64, VPR, p65, RTA, KRAB, or P65HSF1. Examples of transcriptional repression domains include the Krüppel-associated box domain.

[0374] In some embodiments, the cargo protein or the protein encoded by the cargo nucleic acid molecule comprises a meganuclease. Meganucleases generally refer to rare-cutting endonucleases or homing endonucleases, which can be highly specific. Meganucleases can recognize DNA target sites with a length range of at least 12 base pairs, such as DNA target sites with a length of 12 to 40 base pairs, 12 to 50 base pairs, or 12 to 60 base pairs. Meganucleases can be modular DNA-binding nucleases, such as any chimeric protein that comprises at least one catalytic domain of an endonuclease and at least one DNA-binding domain or a protein that specifies a nucleic acid target sequence. The DNA-binding domain can comprise at least one motif that recognizes single-stranded or double-stranded DNA. Meganucleases can be monomeric or dimeric. In some embodiments, the meganuclease is naturally occurring (found in nature) or wild-type, and in other cases, the meganuclease is non-natural, artificial, engineered, synthetic, rationally designed, or man-made. In some embodiments, the meganucleases of the present disclosure include I-CreI meganuclease, I-CeuI meganuclease, I-MsoI meganuclease, I-SceI meganuclease, variants thereof, derivatives thereof, and fragments thereof. A detailed description of useful meganucleases and their applications in gene editing can be found, for example, in Silva et al., Curr GeneTher, 2011, 11(1):11-27; Zaslavoskiy et al., BMC Bioinformatics, 2014, 15:191; Takeuchi et al., Proc Natl Acad Sci USA, 2014, 111(11):4061-4066, as well as U.S. Patent Nos. 7,842,489; 7,897,372; 8,021,867; 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,36; and 8,129,134.

[0375] In some embodiments, the nuclease domain of the meganuclease comprises a modified form of the wild-type nuclease domain. The modified form of the nuclease domain can include amino acid alterations (e.g., deletions, insertions, or substitutions) that reduce the nucleic acid cleavage activity of the nuclease domain. For example, the modified form of the nuclease domain can have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid cleavage activity of the wild-type nuclease domain. The modified form of the nuclease domain can have no substantial nucleic acid cleavage activity. In some embodiments, the nuclease domain is enzymatically inactivated. In some embodiments, the meganuclease can bind DNA but cannot cleave DNA.

[0376] Targetable 3'-overhang nuclease

[0377] In some cases, the cargo delivered by the lipid-containing particles of the present disclosure includes a nuclease that generates a 3'-overhang double-strand break in DNA, such as a type IIS restriction enzyme or a functional domain of a type IIS restriction enzyme. As used herein, the term "type IIS restriction enzyme" is a restriction enzyme that recognizes an asymmetric DNA sequence and cuts outside of its recognition sequence. In one embodiment, the restriction enzyme is Acul.

[0378] In some cases, the cargo includes a targetable nuclease chimeric protein that includes a dimerization-dependent nuclease domain, such as a type IIS restriction enzyme domain. For example, the targetable nuclease chimeric protein includes a dimerization-dependent nuclease domain that generates a 3'-overhang double-strand break in DNA; and a DNA-binding domain (DBD). In some cases, the dimerization-dependent nuclease domain is a type IIS restriction enzyme nuclease domain, such as an Acul nuclease domain.

[0379] In some cases, the DBD is a protein or protein domain that binds to its target nucleic acid in a sequence-dependent manner. In some cases, the DBDs disclosed herein are zinc finger arrays or dCas9.

[0380] In some cases, the nuclease chimeric protein is a zinc finger nuclease chimeric protein. The zinc finger nuclease chimeric protein described herein includes a nuclease domain that generates a 3'-overhang double-strand break in DNA upon dimerization (i.e., the nuclease activity is "dimerization-dependent"); an optional amino acid linker; and a zinc finger domain that includes one or more carboxyl-terminal or amino-terminal zinc fingers. The monomeric form of the zinc finger nuclease chimeric protein that includes one or more carboxyl-terminal or amino-terminal zinc fingers can be linked together after or before binding to the target site to form a dimer, thereby activating nuclease cleavage. The zinc finger nuclease chimeric protein described herein can be used to generate insertion / deletion mutations (insertions and deletions (indels)) at high frequencies via repair of nuclease-induced DNA breaks by non-homologous end joining.

[0381] Zinc finger nuclease chimeric proteins can also be used to replicate, incorporate or insert an exogenous nucleic acid sequence of interest into a target site of a genomic locus of a cell. In some embodiments, the methods provided herein include providing to the nucleus of a cell an exogenous nucleic acid "donor template" sequence and a zinc finger nuclease chimeric protein or another nucleic acid sequence encoding the zinc finger nuclease chimeric protein itself. In some cases, both the exogenous nucleic acid "donor template" sequence and the zinc finger nuclease chimeric protein are delivered by lipid-containing particles provided herein. The exogenous nucleic acid donor template sequence contains terminal sequences homologous to sequences within the target site of the genomic locus. The zinc fingers can be designed to specifically recognize and bind to the genomic target site. After binding to the target site, the dimerized nuclease domain of the chimeric protein can generate a 3'-overhang double-strand break within the target site to induce homology-directed repair between the sequences flanking the break and the exogenous nucleic acid sequence, thereby replicating, incorporating and / or inserting the exogenous nucleic acid sequence into the target site of the genomic locus of the cell.

[0382] A zinc finger nuclease chimeric protein can comprise any nuclease domain capable of generating a 3'-overhang double-strand break in DNA upon dimerization.

[0383] The nuclease domain can be, for example, a type IIS restriction enzyme nuclease domain, including the Acul, Alol, Bpml, Bael or Mmel nuclease domains. In some cases, the Acul nuclease domain can have an amino acid sequence.

[0384] Exemplary nucleotide and amino acid sequences encoding Acul are known in the art and can be located, for example, at GenBank accession number HQ327692.1.

[0385] In some embodiments, the type IIS restriction enzyme nuclease domain includes an isoschizomer of Acul, such as Eco57I. The nucleotide and amino acid sequences encoding Eco57I can be located, for example, at UniProt database reference number P25239.

[0386] Exemplary nucleotide and amino acid sequences encoding Alol are known in the art and can be located, for example, at GenBank accession number AJ312389.1.

[0387] Exemplary nucleotide and amino acid sequences encoding Bpml are known in the art and can be located, for example, at GenBank accession number ADK30556.1. Exemplary nucleotide and amino acid sequences encoding Bael are known in the art and can be located, for example, at GenBank accession number ABS74060.1.

[0388] Exemplary nucleotide and amino acid sequences encoding Mmel are known in the art and can be found, for example, at GenBank accession number EU616582.1.

[0389] Any type IIS restriction enzyme nuclease domain having dimerization-dependent nuclease activity can be fused to a zinc finger domain and used to practice the methods described herein. In some embodiments, the nuclease domain is attached to the C-terminus of the zinc finger domain. In other embodiments, the nuclease domain is attached to the N-terminus of the zinc finger domain.

[0390] The dimerization-dependent nuclease domain and the zinc finger domain of the zinc finger nuclease chimeric protein can be joined together by an amino acid linker. The terms linked, joined, and fused are used interchangeably herein to refer to the manner in which the two domains of the chimeric protein are joined. The amino acid linker can comprise any sequence of at least one amino acid and up to 10 amino acids. In certain embodiments, the linker can comprise leucine, arginine, glycine, and serine (LRGS (SEQ ID NO:2)); glycine, glycine, glycine, glycine, and serine (GGGGS (SEQ ID NO:3)); or the non-standard amino acids, threonine, glutamate, and asparagine (XTEN), as described in Shellenberger et al., Nat Biotechnol. December 2009; 27(12):1186-90.

[0391] In some embodiments, the dimerization-dependent nuclease domain, zinc finger domain, TALE, and / or dCas9 domain can have an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the exemplary amino acid sequences of the dimerization-dependent nuclease domain, zinc finger domain, TALE, and / or dCas9 described herein.

[0392] After binding to the target site and forming a dimer complex, the nuclease domain of the zinc finger nuclease chimeric protein can generate a 3'-overhang double-strand break within the target site to induce homologous directed repair, thereby resulting in the replication, incorporation, and / or integration of an exogenous nucleic acid sequence or a portion thereof within the target site. In the presence of nucleotide sequence homology, a donor template oligonucleotide sequence (single-stranded or double-stranded) can serve as a template for repairing the target DNA sequence that has undergone a double-strand break, resulting in the transfer of genetic information from the donor to the target. Such transfer can involve mismatch correction of the heteroduplex DNA formed between the broken target and the donor, and / or synthesis-dependent strand annealing (where the donor is used to resynthesize the genetic information that will become part of the target), and / or related processes. Homologous directed repair typically results in an alteration of the sequence of the target nucleotides such that some or all of the sequence of the donor nucleotide sequence is replicated and / or incorporated into the target nucleotides.

[0393] The zinc finger nuclease chimeric protein can generate a double-strand break at a predetermined site within the target sequence, and an exogenous nucleic acid sequence serving as a donor template that is homologous to the nucleotide sequence in the broken region can be replicated, incorporated, and / or introduced into the genomic locus. The presence of the double-strand break has been shown to greatly enhance the efficiency of these different repair outcomes. The donor sequence can be physically integrated, or alternatively, the donor nucleotides are used as a template for repairing the break via homologous recombination, resulting in the introduction of all or part of the nucleotide sequence as in the donor into the genomic locus. Thus, the sequence in the genomic locus can be altered and, in certain embodiments, can be converted into the sequence present in the donor nucleotides.

[0394] This article also describes dCas9 nuclease chimeric proteins and methods for using them to increase the frequency of homologous directed repair at the site of nuclease-induced double-strand breaks. The dCas9 nuclease chimeric protein comprises a catalytically inactivated Cas9 carboxy-terminal or amino-terminal domain linked to a dimerization-dependent nuclease domain that generates 3'-overhang double-strand breaks in DNA. The catalytically inactivated Cas9 domain contains mutations that result in the loss of native endonuclease activity (e.g., D10A and / or H841A) (Qi et al., Cell (2013)). In contrast, the endonuclease activity is provided by the linked dimerization-dependent nuclease domain fused thereto. The dCas9 nuclease chimeric protein in monomeric form is linked together before or after binding to the dCas9 target site to form a dimer, thereby activating nuclease cleavage. Clustered regularly interspaced short palindromic repeats (CRISPR) and associated Cas proteins constitute the CRISPR-Cas system. The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner (Gasiunas, G. et al., Proc Natl Acad Sci USA 109, E2579-E2586 (2012); Jinek, M. et al., Science 337, 816-821 (2012); Sternberg, S.H. et al., Nature 507, 62 (2014); Deltcheva, E. et al., Nature 471, 602-607 (2011)), and has been widely used for programmable genome editing in a variety of organisms and model systems (Cong, L. et al., Science 339, 819-823 (2013); Jiang, W. et al., Nat. Biotechnol 31, 233-239 (2013); Sander, J.D. & Joung, J.K., Nature Biotechnol. 32, 347-355. (2014)). Cas9 requires a guide RNA consisting of two RNAs that are joined or covalently linked together to make the guide RNA; CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA). If the nucleotide sequence of the genomic locus of interest is complementary to the guide RNA, Cas9 recognizes and cleaves that site.The ternary complex of Cas9 with crRNA and tracrRNA or the binary complex of Cas9 with guide RNA can bind to the dsDNA protospacer sequence and cleave the dsDNA protospacer sequence, which is complementary to the crRNA spacer and is also adjacent to the short protospacer adjacent motif. dCas9 can still associate with the crRNA / tracrRNA complex or guide RNA and then recognize and bind to the target site even if its native catalytic activity is inactivated. The nucleotide and amino acid sequences encoding Cas9 are known in the art and can be located, for example, at GenBank accession number NC_002737.2.

[0395] The dCas9 nuclease chimeric proteins described herein can be used to induce homologous directed repair events at target sites in genomic loci of cells. The method includes providing to the nucleus of the cell an exogenous nucleic acid sequence, a nucleic acid sequence encoding a dCas9 nuclease chimeric protein, and one or more (e.g., at least two) guide RNAs. The exogenous nucleic acid sequence contains terminal sequences homologous to sequences within the target site of the genomic locus. The guide RNAs are designed to direct two dCas9 nuclease chimeras to a predetermined target site, where each dCas9 / gRNA complex binds to one of two "half-sites". The dCas9 domain will specifically recognize and bind to its target site complementary to the guide RNA and the adjacent PAM sequence. After binding to the target site, the linked nuclease domain of the chimeric protein acts as a dimer to generate a 3'-overhang double-strand break within the target site to induce homologous directed repair between the sequences surrounding the break and the exogenous nucleic acid sequence, thereby replicating, incorporating, and / or inserting the exogenous nucleic acid sequence into the target site of the genomic locus of the cell. The nucleotide and amino acid sequences encoding dCas9 are known in the art and can be located, for example, at GenBank accession number KR011748.1. dCas9 is also described by Zetsche et al., Nature Biotechnology 33, 139-142 (2015).

[0396] The dCas9 nuclease chimeric protein can comprise any nuclease domain capable of generating a 3'-overhang double-strand break in DNA upon dimerization. The nuclease domain can be, for example, a type IIS restriction enzyme nuclease domain, including Acul, Alol, Bpml, Bael or Mmel nuclease domains. The dimerization-dependent nuclease domain of the dCas9 nuclease chimeric protein and the dCas9 domain are linked together by an optional amino acid linker. The amino acid linker can comprise any sequence of at least one amino acid and a sequence of up to 10 amino acids. In certain embodiments, the amino acid linker can comprise, for example, glycine, glycine, glycine, glycine and serine (GGGGS (SEQ ID NO:3)) or non-standard amino acids, threonine, glutamic acid and asparagine (XTEN).

[0397] In any of the methods and compositions described herein, the exogenous nucleotide sequence serving as a donor can comprise a sequence that is homologous but not identical to the genomic sequence at the target site, thereby stimulating homologous directed repair to replicate, incorporate and / or insert a non-identical sequence within the target site. Thus, in certain embodiments, the portion of the donor sequence that is homologous to the sequence in the region of interest exhibits sequence identity with the genomic sequence being replaced of between about 80 to 99% (or any integer therebetween). In other embodiments, for example, if there is only 1 nucleotide difference between the donor sequence and the genomic sequence over more than 100 contiguous base pairs, the homology between the donor sequence and the genomic sequence is greater than 99%. In some cases, the non-homologous portion of the donor sequence can comprise a sequence not present at the target site, such that a new sequence is introduced into the region of interest. In these instances, the non-homologous sequence is typically flanked by a sequence of 50-1,000 base pairs (or any integer value therebetween) or any number of base pairs greater than 1,000 that are homologous or identical to the sequence at the target site.

[0398] In some embodiments, the entire donor template sequence or a portion of the donor template sequence is integrated at the target site. Any of the methods described herein can be used to partially or completely inactivate one or more genomic loci in a cell by targeting the integration of a donor sequence that disrupts the expression of a gene of interest. Any of the methods described herein can be used to replace a mutated sequence within the target site, thereby correcting a mutated gene or inducing the expression of a previously inactivated gene. The nature of the exogenous nucleic acid sequence to be incorporated will depend on the therapeutic goal to be achieved and can range from inducing or suppressing gene transcription, to replacing the mutated sequence of a defective gene or adding or deleting sequences within a gene.

[0399] In other embodiments, the DBD (e.g., zinc finger or dCas9) nuclease chimeric protein introduces variable length insertion or deletion mutations that overlap in part or in whole with the nuclease target site of a genomic locus of a cell by non-homologous end joining or microhomology-mediated end joining. In these embodiments, no exogenous donor sequence is provided. Instead, a nucleic acid sequence encoding the zinc finger nuclease chimeric protein or an isolated zinc finger nuclease chimeric protein is provided to the nucleus of the cell, and the zinc finger nuclease chimeric protein binds to the nuclease target site to generate a 3'-overhang double-strand break within the nuclease target site, which is subsequently repaired by non-homologous end joining or microhomology-mediated end joining. Either non-homologous end joining or microhomology-mediated end joining can generate insertions or deletions that interfere with or inhibit gene transcription at the nuclease target site.

[0400] Examples of targetable 3'-overhang nucleases (e.g., type IIS restriction enzymes, e.g., DBD nuclease chimeric proteins), sequences encoding nucleases, compositions, methods of use, and systems are included in those described in International Publication No. WO2020160481, which is incorporated herein by reference in its entirety.

[0401] Base editing

[0402] In some cases, the cargo to be delivered by the lipid-containing particles of the present disclosure comprises one or more components of a base editor (also referred to as a "base editor") or a base editing (also referred to as "base editing") complex.

[0403] As used herein, the term "base editor (BE)" or "nuclear base editor (NBE)" can refer to an agent that comprises a polypeptide capable of modifying a base (e.g., A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor is capable of deaminating a base within a nucleic acid. In some embodiments, the base editor is capable of deaminating a base within a DNA molecule. In some embodiments, the base editor is capable of deaminating adenosine (A) in DNA. In some embodiments, the base editor is capable of deaminating cytosine (C) in DNA.

[0404] In some cases, the base editors disclosed herein comprise a deaminase or a functional domain thereof that catalyzes the deamination reaction ("deaminase domain").

[0405] As used herein, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is adenosine deaminase, which catalyzes the deamination of adenosine to convert it to inosine. In some embodiments, the deaminase or deaminase domain is cytidine deaminase, which catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase domain that catalyzes the hydrolytic deamination of cytosine to uracil. In some embodiments, the deaminase or deaminase domain is a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism that does not exist in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring deaminase from an organism.

[0406] As used herein, "adenosine deaminase" is an enzyme that catalyzes the deamination of adenosine to convert it to inosine. Under standard Watson-Crick hydrogen bonding, the adenosine base forms hydrogen bonds with the thymine base (or uracil in the case of RNA). When adenine is converted to inosine, inosine undergoes hydrogen bonding with cytosine. Thus, during cell repair and / or replication processes, the conversion of "A" to inosine by adenosine deaminase will result in the insertion of "C" rather than "T". Since cytosine "C" pairs with guanine "G", adenosine deaminase in concert with DNA replication results in the conversion of A·T pairing to C·G pairing in a double-stranded DNA molecule.

[0407] In some embodiments, the base editor is a chimeric protein that comprises a nucleic acid programmable R / DNA binding protein (napR / DNAbp) fused to a deaminase domain (e.g., a cytidine deaminase or an adenosine deaminase). The term “nucleic acid programmable D / RNA binding protein (napR / DNAbp)” refers to any protein that can associate (e.g., form a complex) with one or more nucleic acid molecules (i.e., it can be broadly referred to as a “napR / DNAbp programmable nucleic acid molecule” and includes, for example, guide RNAs in the case of a Cas system), which nucleic acid molecules direct or otherwise program the protein to localize to a specific target nucleotide sequence (e.g., a genomic locus of a gene, or an RNA molecule), the specific target nucleotide sequence being complementary to one or more nucleic acid molecules (or a portion or region thereof) associated with the protein, such that the protein binds to the nucleotide sequence at the specific target site. The term napR / DNAbp includes CRISPR Cas9 proteins, as well as Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or recombinant), and can include Cas 9 equivalents from any type of CRISPR system (e.g., type II, type V, type VI), including Cpf1 (type V CRISPR-Cas system), C2c1 (type V CRISPR-Cas system), C2c2 (type VI CRISPR-Cas system), and C2c3 (type V CRISPR-Cas system). Further Cas equivalents are described in Makarova et al., “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector,” Science 2016; 353(6299) (the content of which is incorporated herein by reference). However, the nucleic acid programmable R / DNA binding proteins (napR / DNAbp) that can be used in conjunction with the present disclosure are not limited to CRISPR-Cas systems. The present disclosure encompasses any such programmable proteins, such as the Argonaute protein from Natronobacterium gregoryi (NgAgo), which can also be used for DNA-guided genome editing. The NgAgo-guided DNA system does not require a PAM sequence or guide RNA molecules, meaning that genome editing can be simply performed by expressing the general NgAgo protein and introducing synthetic oligonucleotides on any genomic sequence.See Gao F, Shen XZ, Jiang F, Wu Y, Han C. DNA-guided genome editing using the Natronobacterium gregoryi Argonaute. Nat Biotechnol 2016;34(7):768-73, which is incorporated herein by reference.

[0408] In some cases, the napR / DNAbp is derived from nucleases disclosed herein, such as Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cas14, Cpfl, C2cl, C2c2, C2c3, Argonaute proteins or variants thereof. In some embodiments, the base editor comprises Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpfl, C2cl, C2c2, C2c3 or Argonaute protein fused to a deaminase (e.g., a cytidine deaminase or an adenosine deaminase). In some embodiments, the base editor comprises Cas9 nickase (nCas9) fused to a deaminase (e.g., a cytidine deaminase or an adenosine deaminase). In some embodiments, the base editor comprises a CasX protein fused to a deaminase (e.g., a cytidine deaminase or an adenosine deaminase). In some embodiments, the base editor comprises nuclease-inactivated Cas9 (dCas9) fused to a deaminase (e.g., a cytidine deaminase or an adenosine deaminase). In some embodiments, the base editing comprises a CasY protein fused to a deaminase (e.g., a cytidine deaminase or an adenosine deaminase). In some embodiments, the base editor comprises a Cas14 protein fused to a deaminase (e.g., a cytidine deaminase or an adenosine deaminase). In some embodiments, the base editor comprises a Cpfl protein fused to a deaminase (e.g., a cytidine deaminase or an adenosine deaminase). In some embodiments, the base editor comprises a C2cl protein fused to a deaminase (e.g., a cytidine deaminase or an adenosine deaminase). In some embodiments, the base editor comprises a C2c2 protein fused to a deaminase (e.g., a cytidine deaminase or an adenosine deaminase). In some embodiments, the base editor comprises a C2c3 protein fused to a deaminase (e.g., a cytidine deaminase or an adenosine deaminase). In some embodiments, the base editor comprises an Argonaute protein fused to a deaminase (e.g., a cytidine deaminase or an adenosine deaminase).

[0409] In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenosine. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenosine in the deoxyadenosine residues of DNA. The adenosine deaminase can be derived from any suitable organism (e.g., Escherichia coli). In some embodiments, the adenosine deaminase is a naturally occurring adenosine deaminase that includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). Those skilled in the art will be able to identify corresponding residues in any homologous protein and the corresponding coding nucleic acid by methods well known in the art, such as by sequence alignment and determination of homologous residues. Thus, those skilled in the art will be able to generate mutations in any naturally occurring adenosine deaminase (e.g., homologous to ecTadA) that correspond to any of the mutations described herein, e.g., any of the mutations identified in ecTadA. In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefacien, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from Escherichia coli.

[0410] In some cases, the deaminase domain of the base editors disclosed herein is derived from a cytidine deaminase. In some cases, the cytidine deaminase domain is derived from the apolipoprotein B mRNA editing complex (APOBEC) family of deaminases, such as the APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, or APOBEC3H deaminase.

[0411] In some embodiments, the base editor is fused to a base excision repair inhibitor or further includes a base excision repair inhibitor as part of a chimeric protein, such as a uracil glycosylase inhibitor (UGI) domain.

[0412] In some cases, the base editors disclosed herein are chimeric proteins that include structures such as NH2-[deaminase domain]-[napR / DNAbp]-[UGI domain]-COOH; NH2-[deaminase domain]-[napR / DNAbp]-[UGI]-[UGI]-COOH; NH2-[deaminase domain]-[napR / DNAbp]-[UGI]-COOH; NH2-[UGI]-[deaminase domain]-[napR / DNAbp]-COOH; NH2-[deaminase domain]-[UGI]-[napR / DNAbp]-COOH; NH2-[napR / DNAbp]-[UGI]-[deaminase domain]-COOH; or NH2-[napR / DNAbp]-[deaminase domain]-[UGI]-COOH; wherein each instance of "-" includes an optional linker.

[0413] In some cases, the base editor is fused to a uracil-binding protein (UBP), or further includes a uracil-binding protein (UBP) as part of the chimeric protein. As used herein, the term "uracil-binding protein" or "UBP" refers to a protein capable of binding to uracil. In some embodiments, the uracil-binding protein is a uracil-modifying enzyme. In some embodiments, the uracil-binding protein is a uracil base excision enzyme. In some embodiments, the uracil-binding protein is a uracil DNA glycosylase (UDG). In some embodiments, the uracil-binding protein binds to uracil with an affinity that is at least 1%, 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or at least 95% of the affinity with which wild-type UDG (e.g., human UDG) binds to uracil. As used herein, the term "base excision enzyme" or "BEE" refers to a protein capable of removing a base (e.g., A, T, C, G or U) from a nucleic acid molecule (e.g., DNA or RNA). In some embodiments, the BEE is capable of removing cytosine from DNA. In some embodiments, the BEE is capable of removing thymine from DNA. Exemplary BEEs include, but are not limited to, UDG Tyr147Ala and UDG Asn204Asp, as described in Sang et al., "A Unique Uracil-DNA binding protein of the uracil DNA glycosylase superfamily," Nucleic Acids Research, Vol. 43, No. 17 2015 (the entire content of which is hereby incorporated by reference).

[0414] In some embodiments, the UBP is a uracil-modifying enzyme. In some embodiments, the UBP is a uracil base excision enzyme. In some embodiments, the UBP is a uracil DNA glycosylase. In some embodiments, the UBP is any of the uracil-binding proteins provided herein. For example, the UBP can be UDG, UdgX, UdgX*, UdgX_On, or SMUG1. In some embodiments, the UBP comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a uracil-binding protein, a uracil base excision enzyme, or a uracil DNA glycosylase (UDG).

[0415] In some cases, the base editor is fused to a nucleic acid polymerase domain (NAP), or comprises a nucleic acid polymerase domain (NAP) as part of a chimeric protein. For example, the nucleic acid polymerase domain is a eukaryotic nucleic acid polymerase domain. In some cases, the nucleic acid polymerase domain is a DNA polymerase domain. In some cases, the nucleic acid polymerase domain has translesion polymerase activity. In some cases, the nucleic acid polyme...

Claims

1. A lipid-containing particle, comprising: (a) a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule; (b) a combined protein comprising a plasma membrane targeting protein selected from the pleckstrin homology (PH) domain of human Daap1, the PH domain of murine Grp1, the PH domain of human Grp1, the PH domain of human OSBP, the PH domain of human Btk, the PH domain of human FAPP1, the PH domain of human CERT, the PH domain of human PKD, the PH domain of human PHLPP1, the PH domain of human SWAP70, and the PH domain of human MAPKAP1; and (c) a cargo.

2. The lipid-containing particle according to claim 1, wherein the combined protein comprises the plasma membrane targeting protein conjugated to the cargo.

3. The lipid-containing particle according to claim 1 or 2, wherein the combined protein comprises the plasma membrane targeting protein conjugated to a nuclear export sequence (NES).

4. The lipid-containing particle according to claim 3, wherein the combined protein comprises the plasma membrane targeting protein, the NES, and the cargo arranged in sequence from the N-terminus to the C-terminus of the combined protein.

5. The lipid-containing particle according to claim 3 or 4, wherein the combined protein comprises the plasma membrane targeting protein, the NES, the cargo, and a second NES arranged in sequence from the N-terminus to the C-terminus of the combined protein.

6. The lipid-containing particle according to claim 4, wherein the combined protein further comprises a cleavable linker.

7. The lipid-containing particle according to claim 6, wherein the cleavable linker is positioned between the plasma membrane targeting protein and the cargo; optionally wherein the cleavable linker is positioned between the NES and the cargo; and optionally wherein the combined protein further comprises a nuclear localization sequence (NLS) C-terminus of the cleavable linker.

8. The lipid-containing particle according to any one of the preceding claims, wherein the lipid-containing particle comprises the human endogenous retrovirus envelope protein; optionally wherein the human endogenous retrovirus envelope protein is from hENVH1, hENVH2, hENVH3, hENVK1, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)1, or hENVKcon; and optionally wherein the human endogenous retrovirus envelope protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the sequences in Table 2-1.

9. A lipid-containing particle according to any one of the preceding claims, wherein the plasma membrane-localized protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from SEQ ID NOs: 11-21 and 60-66.

10. A lipid-containing particle according to any one of the preceding claims, wherein the lipid-containing particle comprises a lipid-containing membrane encapsulating a protein core; optionally wherein the lipid-containing membrane comprises a phospholipid bilayer.

11. The lipid-containing particle according to claim 10, wherein the human endogenous retrovirus (HERV) envelope protein, the humanized envelope protein or the non-immunogenic membrane fusion molecule is attached to the lipid-containing membrane.

12. A lipid-containing particle, comprising: (a) a virus-derived glycoprotein selected from RD114, Fug-E, FuG-E(P440E) and MLV 10A1; (b) a combined protein comprising a plasma membrane-localized protein coupled to a nuclear export sequence (NES); and (c) a cargo.

13. The lipid-containing particle according to claim 12, wherein the combined protein further comprises the cargo.

14. The lipid-containing particle according to claim 13, wherein the combined protein comprises the plasma membrane-localized protein, the NES and the cargo arranged in sequence from the N-terminus to the C-terminus of the combined protein.

15. The lipid-containing particle according to claim 14, wherein the combined protein further comprises a cleavable linker; optionally wherein the cleavable linker is positioned between the plasma membrane-localized protein and the cargo; optionally wherein the cleavable linker is positioned between the NES and the cargo; and optionally wherein the combined protein comprises the nuclear localization sequence (NLS) C-terminus of the cleavable linker.

16. A lipid-containing particle, comprising: (a) a virus-derived glycoprotein selected from RD114, Fug-E, FuG-E(P440E) and MLV 10A1; (b) a combined protein comprising a plasma membrane-localized protein coupled to a cleavable linker; and (c) a cargo.

17. The lipid-containing particle according to claim 16, wherein the combined protein further comprises the cargo.

18. The lipid-containing particle according to claim 17, wherein the combined protein comprises the plasma membrane-localized protein, the cleavable linker and the cargo arranged in sequence from the N-terminus to the C-terminus of the combined protein; optionally wherein the combined protein further comprises the nuclear localization sequence (NLS) C-terminus of the cleavable linker.

19. The lipid-containing particle according to any one of claims 12-18, wherein the plasma membrane-localized protein comprises: (a) a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; (b) a humanized structural protein; (c) a pleckstrin homology (PH) domain; or (d) A non-immunogenic plasma membrane recruitment protein, and optionally wherein the non-immunogenic plasma membrane recruitment protein comprises Arc, human Arc, an endogenous retroviral gag protein or a human endogenous retroviral gag protein.

20. The lipid-containing particle according to claim 19, wherein the plasma membrane-localizing protein comprises the PH domain, and wherein the PH domain comprises the PH domain of phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), discoidin (Disc) and actin-related protein 1 (Daap1), phosphatidylinositol 1 universal receptor (Grp1), oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton's tyrosine kinase (Btk), tetraspanin adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), switching B cell complex subunit SWAP70 or MAPK-related protein 1 (MAPKAP1) or a mutant thereof.

21. The lipid-containing particle according to claim 19, wherein the plasma membrane-localizing protein comprises the PH domain, and wherein the PH domain comprises the PH domain of a human protein; optionally wherein the PH domain comprises the PH domain of human phospholipase Cδ1, human Akt1, human 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), human Daap1, murine Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70 or human MAPKAP1 or a mutant thereof.

22. The lipid-containing particle according to claim 19, wherein the plasma membrane-localizing protein comprises the PH domain, and wherein the PH domain is selected from: the PH domain of human Daap1, the PH domain of murine Grp1, the PH domain of human Grp1, the PH domain of human OSBP, the PH domain of human Btk, the PH domain of human FAPP1, the PH domain of human CERT, the PH domain of human PKD, the PH domain of human PHLPP1, the PH domain of human SWAP70 and the PH domain of human MAPKAP1.

23. The lipid-containing particle according to claim 19, wherein the plasma membrane-localizing protein comprises a membrane protein selected from CD9, CD47, CD63 and CD81 and their transmembrane domains.

24. The lipid-containing particle according to claim 19, wherein the plasma membrane-localizing protein comprises a membrane protein selected from human CD9, human CD47, human CD63 and human CD81 and their transmembrane domains.

25. The lipid-containing particle according to claim 19, wherein the plasma membrane-localizing protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences listed in Table 3.

26. The lipid-containing particle according to any one of claims 1-25, wherein the cargo further comprises a therapeutic cargo or a binding partner of the therapeutic cargo.

27. The lipid-containing particle according to any one of claims 1-26, wherein the combinatorial protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from SEQ ID NOs: 50, 52-55 and 67-77.

28. The lipid-containing particle according to claims 12-26, wherein the lipid-containing particle comprises a lipid-containing membrane encapsulating a protein core; optionally wherein the lipid-containing membrane comprises a phospholipid bilayer.

29. The lipid-containing particle according to claim 28, wherein the virus-derived glycoprotein is attached to the lipid-containing membrane.

30. A lipid-containing particle comprising a combinatorial protein, the combinatorial protein comprising an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from SEQ ID NOs: 50, 52-55 and 67-77.

31. The lipid-containing particle according to claim 30, wherein the lipid-containing particle comprises: (a) a human endogenous retrovirus (HERV) envelope protein; optionally wherein the human endogenous retrovirus envelope protein is from hENVH1, hENVH2, hENVH3, hENVK1, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)1 or hENVKcon; and optionally wherein the human endogenous retrovirus envelope protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences in Table 2-1; (b) a humanized envelope protein; (c) a non-immunogenic membrane fusion molecule; or (d) a virus-derived glycoprotein; optionally wherein the virus-derived glycoprotein is selected from: BaEVTR, BaEVTRless, FuG-E, FuG-E(P440E), MVL ENV (amphotropic), MVL ENV (ecotropic), MLV 10A1, VSVG, GP64, gp160 and RD114 ENV; and optionally wherein the virus-derived glycoprotein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences in Table 1.

32. The lipid-containing particle according to claim 30 or 31, wherein the combinatorial protein further comprises a cleavable linker, a nuclear export sequence (NES), a cargo or a combination thereof.

33. The lipid-containing particle according to any one of claims 30-32, wherein the lipid-containing particle comprises a lipid membrane encapsulating a protein core; optionally wherein the lipid membrane comprises a phospholipid bilayer.

34. The lipid-containing particle according to claim 33, wherein the human endogenous retrovirus (HERV) envelope protein, the humanized envelope protein, the non-immunogenic membrane fusion molecule or the virus-derived glycoprotein is attached to the lipid membrane.

35. The lipid-containing particle according to any one of claims 10-11, 28-29 or 33-34, wherein the protein core comprises a structural protein, and the structural protein comprises a second plasma membrane-localized protein; optionally wherein the structural protein further comprises a retroviral protease (pro) protein.

36. The lipid-containing particle according to claim 35, wherein the second plasma membrane-localized protein comprises: (a) a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; (b) a humanized structural protein; (c) a pleckstrin homology (PH) domain, optionally wherein the PH domain comprises a PH domain of phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), discoidin (Disc) and actin-related protein 1 (Daap1), phosphatidylinositol 1 universal receptor (Grp1), oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton's tyrosine kinase (Btk), phosphatidylinositol tetraphosphate adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), switch B cell complex subunit SWAP70 or MAPK-related protein 1 (MAPKAP1) or a mutant thereof, optionally wherein the PH domain is from human, and optionally wherein the PH domain comprises a PH domain of human phospholipase Cδ1, human Akt1, human 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), human Daap1, murine Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70 or human MAPKAP1 or a mutant thereof; or (d) a non-immunogenic plasma membrane recruitment protein, optionally wherein the non-immunogenic plasma membrane recruitment protein comprises a membrane protein selected from CD9, CD47, CD63 and CD81 and their transmembrane domains, optionally wherein the membrane protein is selected from: human CD9, human CD47, human CD63 and human CD81 and their transmembrane domains, and optionally wherein the non-immunogenic plasma membrane recruitment protein comprises Arc, human Arc, endogenous retrovirus gag protein or human endogenous retrovirus gag protein.

37. The lipid-containing particle according to claim 35, wherein the second plasma membrane-localizing protein comprises a PH domain, and the PH domain comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences listed in Table 3.

38. The lipid-containing particle according to any one of claims 10-11, 28-29 or 33-37, wherein the combined protein forms part of the protein core.

39. The lipid-containing particle according to claim 38, wherein the plasma membrane-localizing protein of the combined protein forms part of the protein core.

40. The lipid-containing particle according to any one of claims 10-11, 28-29 or 33-39, wherein the lipid-containing membrane comprises an immunomodulator; optionally wherein the immunomodulator is in the phospholipid bilayer; and optionally wherein the immunomodulator is an immunosuppressive molecule.

41. The lipid-containing particle according to any one of the preceding claims, wherein the lipid-containing particle comprises: (a) a cell; (b) a virus-like particle (VLP); (c) a protein-lipid vehicle (PLV); (d) a liposome, optionally a lipid nanoparticle; or (e) an extracellular vesicle, optionally an exosome or a nucleosome.

42. A composition comprising: (a) a first nucleic acid molecule encoding a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein or a non-immunogenic membrane fusion molecule; (b) a second nucleic acid molecule encoding a combined protein comprising a plasma membrane-localizing protein, wherein the plasma membrane-localizing protein is selected from: the pleckstrin homology (PH) domain of human Daap1, the PH domain of murine Grp1, the PH domain of human Grp1, the PH domain of human OSBP, the PH domain of human Btk, the PH domain of human FAPP1, the PH domain of human CERT, the PH domain of human PKD, the PH domain of human PHLPP1, the PH domain of human SWAP70 and the PH domain of human MAPKAP1; and (c) a cargo or a third nucleic acid molecule encoding the cargo.

43. The composition according to claim 42, wherein the second nucleic acid molecule encodes a combined protein comprising the plasma membrane-localizing protein conjugated to the cargo, or wherein the second nucleic acid molecule comprises the third nucleic acid molecule.

44. The composition according to claim 42 or 43, wherein the second nucleic acid molecule encodes a combined protein comprising the plasma membrane-localizing protein conjugated to a nuclear export sequence (NES).

45. The composition according to claim 44, wherein the combined protein comprises the plasma membrane-localizing protein, the NES and the cargo arranged in sequence from the N-terminus to the C-terminus of the combined protein.

46. The composition according to any one of claim 45, wherein the combined protein further comprises a cleavable linker.

47. The composition according to claim 46, wherein the cleavable linker is positioned between the plasma membrane targeting protein and the cargo; optionally wherein the cleavable linker is positioned between the NES and the cargo; and optionally wherein the fusion protein further comprises the C-terminus of the nuclear localization sequence (NLS) of the cleavable linker.

48. The composition according to any one of claims 42-47, wherein the first nucleic acid molecule encodes the human endogenous retrovirus envelope protein; optionally wherein the human endogenous retrovirus envelope protein is from hENVH1, hENVH2, hENVH3, hENVK1, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)1 or hENVKcon; and optionally wherein the human endogenous retrovirus envelope protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences in Table 2-1.

49. The composition according to any one of claims 42-48, wherein the plasma membrane targeting protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from SEQ ID NOs: 11-21 and 60-66.

50. A composition comprising: (a) a first nucleic acid molecule encoding a virus-derived glycoprotein selected from RD114, Fug-E, FuG-E(P440E) and MLV10A1; (b) a second nucleic acid molecule encoding a fusion protein comprising a plasma membrane targeting protein coupled to a nuclear export sequence (NES); and (c) a cargo or a third nucleic acid molecule encoding the cargo.

51. The composition according to claim 50, wherein the fusion protein further comprises the cargo, or wherein the second nucleic acid molecule comprises the third nucleic acid molecule.

52. The composition according to claim 51, wherein the fusion protein comprises the plasma membrane targeting protein, the NES and the cargo arranged in sequence from the N-terminus to the C-terminus of the fusion protein.

53. The composition according to claim 52, wherein the fusion protein further comprises a cleavable linker; optionally wherein the cleavable linker is positioned between the plasma membrane targeting protein and the cargo; optionally wherein the cleavable linker is positioned between the NES and the cargo; and optionally wherein the fusion protein comprises the C-terminus of the nuclear localization sequence (NLS) of the cleavable linker.

54. A composition comprising: (a) A first nucleic acid molecule that encodes a virus-derived glycoprotein selected from RD114, Fug-E, FuG-E(P440E), and MLV10A1; (b) A second nucleic acid molecule that encodes a combinatorial protein that comprises a plasma membrane-localized protein conjugated to a cleavable linker; and (c) A cargo or a third nucleic acid molecule that encodes the cargo.

55. The composition according to claim 54, wherein the combinatorial protein further comprises the cargo, or wherein the second nucleic acid molecule comprises the third nucleic acid molecule.

56. The composition according to claim 55, wherein the combinatorial protein comprises, in order from the N-terminus to the C-terminus of the combinatorial protein, the plasma membrane-localized protein, the cleavable linker, and the cargo, optionally wherein the combinatorial protein further comprises a nuclear localization sequence (NLS) C-terminus of the cleavable linker; optionally wherein the combinatorial protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 67-71.

57. The composition according to any one of claims 50-56, wherein the plasma membrane-localized protein comprises: (a) A human endogenous retrovirus (HERV) structural protein, optionally HERV gag; (b) A humanized structural protein; (c) A pleckstrin homology (PH) domain; or (d) A non-immunogenic plasma membrane recruitment protein, and optionally wherein the non-immunogenic plasma membrane recruitment protein comprises Arc, human Arc, an endogenous retrovirus gag protein, or a human endogenous retrovirus gag protein.

58. The composition according to claim 57, wherein the plasma membrane-localized protein comprises a PH domain, and wherein the PH domain comprises a PH domain of phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), Discoidin and actin-related protein 1 (Daap1), phosphatidylinositol 1 universal receptor (Grp1), oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton's tyrosine kinase (Btk), phosphatidylinositol-4-phosphate adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), transforming B cell complex subunit SWAP70, or MAPK-related protein 1 (MAPKAP1), or a mutant thereof.

59. The composition according to claim 57, wherein the plasma membrane-localized protein comprises the PH domain, and wherein the PH domain comprises the PH domain of a human protein; optionally wherein the PH domain comprises the PH domain of human phospholipase Cδ1, human endogenous retrovirus gag protein, human 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), human Daap1, murine Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70 or human MAPKAP1 or a mutant thereof.

60. The composition according to claim 57, wherein the plasma membrane-localized protein comprises a membrane protein selected from CD9, CD47, CD63 and CD81 and their transmembrane domains.

61. The composition according to claim 57, wherein the plasma membrane-localized protein comprises a membrane protein selected from human CD9, human CD47, human CD63 and human CD81 and their transmembrane domains.

62. The composition according to claim 57, wherein the plasma membrane-localized protein comprises the PH domain, and wherein the PH domain is selected from: the PH domain of human Daap1, the PH domain of murine Grp1, the PH domain of human Grp1, the PH domain of human OSBP, the PH domain of human Btk, the PH domain of human FAPP1, the PH domain of human CERT, the PH domain of human PKD, the PH domain of human PHLPP1, the PH domain of human SWAP70 and the PH domain of human MAPKAP1.

63. The composition according to claim 57, wherein the plasma membrane-localized protein comprises the PH domain, and wherein the PH domain comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences listed in Table 3.

64. The composition according to any one of claims 42-63, wherein the cargo comprises a therapeutic cargo or a binding partner of the therapeutic cargo.

65. The composition according to any one of claims 42-64, wherein the combined protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from SEQ ID NO: 50, 52-55 and 67-77.

66. The composition according to any one of claims 42-64, further comprising a fourth nucleic acid molecule encoding a structural protein comprising a second plasma membrane-localized protein; optionally wherein the structural protein further comprises a retroviral protease (pro) protein.

67. A composition comprising a first nucleic acid molecule encoding a combinatorial protein, the combinatorial protein comprising a first plasma membrane localization domain, and wherein the combinatorial protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence selected from SEQ ID NO: 50, 52 - 55 and 67 - 77.

68. The composition according to claim 67, comprising a second nucleic acid molecule encoding: (a) a human endogenous retrovirus (HERV) envelope protein; optionally wherein the human endogenous retrovirus envelope protein is from hENVH1, hENVH2, hENVH3, hENVK1, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)1 or hENVKcon; and optionally wherein the human endogenous retrovirus envelope protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences in Table 2 - 1; (b) a humanized envelope protein; (c) a non - immunogenic membrane fusion molecule; or (d) a virus - derived glycoprotein; optionally wherein the virus - derived glycoprotein is selected from: BaEVTR, BaEVTRless, FuG - E, FuG - E(P440E), MVL ENV (amphotropic), MVL ENV (ecotropic), MLV 10A1, VSVG, GP64, gp160 and RD114 ENV; and optionally wherein the virus - derived glycoprotein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences in Table 1.

69. The composition according to claim 66 or 67, wherein the combinatorial protein further comprises a cleavable linker, a nuclear export sequence (NES), a cargo, or a combination thereof.

70. The composition according to any one of claims 66 - 69, further comprising a third nucleic acid molecule encoding a structural protein comprising a second plasma membrane - localized protein; optionally wherein the structural protein further comprises a retroviral protease (pro) protein.

71. The composition according to claim 66 or 70, wherein the second plasma membrane - localized protein comprises: (a) a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; (b) a humanized structural protein; (c) Pleckstrin homology (PH) domain, optionally wherein the Pleckstrin homology (PH) domain comprises phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), Discoidin and actin-associated protein 1 (Daap1), phosphatidylinositol 1 general receptor (Grp1), oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton's tyrosine kinase (Btk), tetraspanin adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), switch B cell complex subunit SWAP70 or MAPK-related protein 1 (MAPKAP1) or a PH domain of a mutant thereof, optionally wherein the PH domain is from a human; and optionally wherein the PH domain comprises a PH domain of human phospholipase Cδ1, human Akt1, human 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), human Daap1, murine Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70 or human MAPKAP1 or a mutant thereof; or (d) Non-immunogenic plasma membrane recruitment protein, optionally wherein the non-immunogenic plasma membrane recruitment protein comprises a membrane protein selected from CD9, CD47, CD63 and CD81 and their transmembrane domains, optionally wherein the membrane protein is selected from: human CD9, human CD47, human CD63 and human CD81 and their transmembrane domains, and optionally wherein the non-immunogenic plasma membrane recruitment protein comprises Arc, human Arc, endogenous retroviral gag protein or human endogenous retroviral gag protein.

72. The composition according to claim 66 or 70, wherein the second plasma membrane localization protein comprises a Pleckstrin homology (PH) domain, wherein the PH domain comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of the sequences listed in Table 3.

73. The composition according to any one of claims 66 - 72, wherein the percentage of the second nucleic acid molecule in the composition relative to the sum of the second nucleic acid molecule and the fourth nucleic acid molecule is about, at least or at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.

74. The composition according to any one of claims 70-72, wherein the percentage of the first nucleic acid molecule in the composition relative to the sum of the first nucleic acid molecule and the third nucleic acid molecule is about, at least, or at most 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

75. The lipid-containing particle or composition according to any one of claims 1-29, 32-66, or 69-74, wherein the cargo comprises a nuclease, a base editor, a prime editor, an epigenetic editor, a restriction endonuclease (optionally a type IIS restriction enzyme), a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, DNA, RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, a ribozyme, or a small molecule compound, or any combination thereof.

76. A combinatorial protein comprising a plasma membrane-localized protein and a heterologous sequence, wherein the plasma membrane-localized protein is selected from: the pleckstrin homology (PH) domain of human Daap1, the PH domain of mouse Grp1, the PH domain of human Grp1, the PH domain of human OSBP, the PH domain of human Btk, the PH domain of human FAPP1, the PH domain of human CERT, the PH domain of human PKD, the PH domain of human PHLPP1, the PH domain of human SWAP70, and the PH domain of human MAPKAP1, and wherein the heterologous sequence is an NES, a cleavable linker, or a combination thereof.

77. The combinatorial protein according to claim 76, wherein the plasma membrane-localized protein comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from SEQ ID NOs: 11-21 and 60-66.

78. A lipid-containing particle comprising the combinatorial protein according to claim 76 or 77; optionally wherein the lipid-containing particle comprises a lipid membrane encapsulating a protein core; optionally wherein the lipid membrane comprises a phospholipid bilayer.

79. The lipid-containing particle according to claim 78, which further comprises a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule; optionally wherein the human endogenous retrovirus (HERV) envelope protein, the humanized envelope protein, or the non-immunogenic membrane fusion molecule is attached to the lipid membrane.

80. A composition comprising a nucleic acid molecule encoding the combinatorial protein according to any one of claims 76-77.

81. A cell comprising the lipid-containing particle, composition, or combinatorial protein according to any one of claims 1-80.

82. A system comprising a lipid-containing particle or composition according to any one of claims 1-75 or 78-79, or a cell according to claim 81, optionally wherein the system comprises a production cell, a cell-free extract or a cell lysate.

83. A pharmaceutical composition comprising: (a) (i) a lipid-containing particle according to any one of claims 1-41, 75 or 78-79; or (ii) a system according to claim 82; and (b) a pharmaceutically acceptable excipient.

84. A kit comprising: (a) (i) a lipid-containing particle according to any one of claims 1-41, 75 or 78-79; (ii) a system according to claim 82; or (iii) a pharmaceutical composition according to claim 83; and (b) information material comprising instructions for administering a dose of the lipid-containing particle, the cell or the system or a dosage form of the pharmaceutical composition to a subject.

85. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject (a) a lipid-containing particle according to any one of claims 1-41, 75 or 78-79; (b) a system according to claim 82; or (c) a pharmaceutical composition according to claim 83.

86. A method comprising contacting a cell with a lipid-containing particle according to any one of claims 1-41, 75 or 78-79.

87. A method comprising contacting a cell with a system according to claim 82.

88. A method of producing a lipid-containing particle according to any one of claims 1-41, 75 or 78-79.

89. The method according to claim 88, comprising: (a) contacting a production cell with a composition according to any one of claims 42-75 or 80, and wherein the production cell produces the lipid-containing particle; or (b) providing a system according to claim 82, wherein the system expresses a composition according to any one of claims 42-75 or 80, and wherein the system produces the lipid-containing particle; and optionally wherein the method further comprises harvesting and purifying the lipid-containing particle.

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