Compositions and Methods for Efficient in vivo Delivery

Abstract

In this specification, in an aspect, compositions, methods, kits and systems are disclosed relating to the efficient delivery of a freight (e.g., a therapeutic freight) to cells, for example, 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-localized protein, and (c) a freight.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Application No. 63 / 342,773, filed May 17, 2022, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Background Retroviruses can be an attractive scaffold for virus - like particles (VLPs). Retroviral capsids generally lack the strict symmetry requirements of many icosahedral viruses that do not have an envelope (Zhang et al., 2015), which suggests an increase in structural freedom for incorporating non - native protein freight. In addition, retroviral tropism can be modulated by pseudotyping virions with different envelope glycoproteins, which may enable targeting VLPs to specific cell types (Cronin et al., 2005). Previous studies have demonstrated that packaging of the desired protein freight can be sufficiently directed into retroviral particles by fusing the freight protein to the C - terminus of the retroviral gag polyprotein (Kaczmarczyk et al., 2011; Voelkel et al., 2010). More recently, a similar strategy has been applied to package Cas9 ribonucleoproteins (RNPs) into retroviral particles (Hamilton et al., 2021; Mangeot et al., 2019). However, VLPs with therapeutic - level in vivo delivery efficiency are still needed.

Prior Art Documents

Non - Patent Literature

[0003]

Non - Patent Literature 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

Means for Solving the Problems

[0004] Abstract In some aspects herein, lipid-containing particles are disclosed that include a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule; a combinatorial protein that includes a plasma membrane-localized protein coupled to a nuclear export sequence (NES); and a therapeutic freight. 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 mobilizing protein. In some cases, the therapeutic freight 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 freight. In some cases, the combinatorial protein comprises, in order from the N-terminus to the C-terminus, a plasma membrane-localized protein, a NES, and a therapeutic freight. In some cases, the combinatorial protein further comprises a cleavable linker, optionally located between the plasma membrane-localized protein and the therapeutic freight, optionally located between the NES and the therapeutic freight, and optionally the combinatorial protein further comprises a nuclear localization sequence (NLS) C-terminal of the cleavable linker. In some cases, the lipid-containing particle comprises a cell; a virus-like particle (VLP); a proteolipid 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 freight that is a binding partner of the therapeutic freight.

[0005] In some aspects, this specification discloses 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 nuclear export sequence (NES) and a plasma membrane-localized protein coupled to a moiety, wherein the moiety comprises a therapeutic moiety or a binding partner of a therapeutic moiety.

[0006] In some cases, the plasma membrane-localized protein includes a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; a humanized structural protein; a pleckstrin homology (PH) domain; or a non-immunogenic plasma membrane mobilizing protein. In some cases, the non-immunogenic plasma membrane mobilizing protein includes Arc, human Arc, an endogenous retrovirus gag protein, or a human endogenous retrovirus gag protein. In some cases, the therapeutic freight includes 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 includes, in order from the N-terminus to the C-terminus, a plasma membrane-localized protein, a NES, and a therapeutic freight. In some cases, the combinatorial protein further includes a cleavable linker, optionally, the cleavable linker is located between the plasma membrane-localized protein and the therapeutic freight, optionally, the cleavable linker is located between the NES and the therapeutic freight, and optionally, the combinatorial protein includes a nuclear localization sequence (NLS) at the C-terminus of the cleavable linker. In some cases, the composition is a lipid-containing particle, optionally, the lipid-containing particle includes a cell; a virus-like particle (VLP); a proteolipid vehicle (PLV); a liposome, optionally a lipid nanoparticle; or an extracellular vesicle, optionally an exosome or an ectosome.

[0007] As used herein, in some aspects, there is disclosed a lipid-containing particle comprising 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-localized protein coupled to a cleavable linker; and a therapeutic freight.

[0008] In some cases, the plasma membrane-localized protein includes 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 mobilizing protein. In some cases, the therapeutic freight includes 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 includes a therapeutic freight. In some cases, the combinatorial protein includes a plasma membrane-localized protein, a cleavable linker, and a therapeutic freight arranged in order from the N-terminus to the C-terminus, and optionally, the combinatorial protein further includes a nuclear localization sequence (NLS) at the C-terminus of the cleavable linker. In some cases, the lipid-containing particle includes a cell; a virus-like particle (VLP); a proteolipid 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 includes a freight, and the freight is a binding partner of the therapeutic freight.

[0009] As used herein, in some embodiments, 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 cleavable linker and a freight, wherein the freight comprises a therapeutic freight or a binding partner of the therapeutic freight, are disclosed.

[0010] In some cases, the plasma membrane-localized protein includes 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 mobilizing protein. In some cases, the non-immunogenic plasma membrane mobilizing protein includes Arc, human Arc, an endogenous retroviral gag protein, or a human endogenous retroviral gag protein.

[0011] In some cases, the plasma membrane-localized protein contains a PH domain or a variant thereof derived from phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), disk and actin-binding protein 1 (Daap1), general receptor for phosphoinositides 1 (Grp1), oxysterol-binding protein 1-Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), phosphatidylinositol 4-phosphate adapter protein 1 (FAPP1), ceramide transport 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). In some cases, the plasma membrane-localized protein contains a PH domain derived from a human protein. In some cases, the plasma membrane-localized protein contains a PH domain or a variant thereof 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. In some cases, the plasma membrane-localized protein contains a membrane protein selected from the group consisting of CD9, CD47, CD63, and CD81 and their transmembrane domains. In some cases, the plasma membrane-localized protein contains a membrane protein selected from the group consisting of human CD9, human CD47, human CD63, and human CD81 and their transmembrane domains. In some cases, the therapeutic agent contains a nuclease, base editor, prime editor, epigenetic editor, restriction endonuclease (type IIS restriction enzyme if necessary), recombinase, transcription factor, antibody, chimeric antigen receptor, T cell receptor, organelle, nucleic acid molecule, DNA, RNA, retrotransposon, reverse transcriptase, oligonucleotide, aptazyme, aptamer, ribozyme, or small molecule compound, or any combination thereof.In some cases, the combinatorial protein comprises a plasma membrane-localized protein, a cleavable linker, and a therapeutic moiety arranged in order from the N-terminus to the C-terminus, and optionally, 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, and optionally, the lipid-containing particle comprises a cell; a virus-like particle (VLP); a proteolipid vehicle (PLV); a liposome, optionally a lipid nanoparticle; or an extracellular vesicle, optionally an exosome or ectosome.

[0012] Disclosed herein are lipid-containing particles comprising a combinatorial protein comprising, in some embodiments, 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 mobilizing protein, and ii) a nuclear export sequence (NES).

[0013] In some cases, the lipid-containing particle further comprises a fret, which is a therapeutic fret or a binding partner of a therapeutic fret. Optionally, the therapeutic fret 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 fret. 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 mobilizing protein, ii) a NES, and iii) a therapeutic fret. In some cases, the combinatorial protein further comprises a cleavable linker, which is optionally located 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 mobilizing protein and ii) the therapeutic fret, and optionally, the cleavable linker is between iii) the NES and iv) the therapeutic fret. Optionally, the combinatorial protein further comprises a nuclear localization sequence (NLS) at the C-terminus of the cleavable linker.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), disk and actin-binding protein 1 (Daap1), general receptor for phosphoinositides 1 (Grp1), oxysterol-binding protein 1-Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), four-phosphate adaptor protein 1 (FAPP1), ceramide transport 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 variant 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 or a variant thereof 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. In some cases, the plasma membrane-localized protein comprises a membrane protein selected from the group consisting of CD9, CD47, CD63, and CD81 and their transmembrane domains. In some cases, the plasma membrane-localized protein comprises a membrane protein selected from the group consisting of 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, endogenous retroviral gag protein, or human endogenous retroviral gag protein. In some cases, the lipid-containing particle comprises a cell; a virus-like particle (VLP); a proteolipid vehicle (PLV); a liposome, optionally a lipid nanoparticle; or an extracellular vesicle, optionally an exosome or ectosome.

[0014] 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 mobilizing protein, ii) a nuclear export sequence (NES), and iii) a freight, wherein the freight is a therapeutic freight or a binding partner of a therapeutic freight.

[0015] In some cases, the therapeutic freight includes 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.

[0016] 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 mobilizing protein, ii) a NES, and iii) a therapeutic freight. In some cases, the combinatorial protein further comprises a cleavable linker, and optionally, the cleavable linker is located 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 mobilizing protein and ii) the therapeutic freight, and optionally, the cleavable linker is between iii) the NES and iv) the therapeutic freight, and optionally, the combinatorial protein further comprises a nuclear localization sequence (NLS) at the C-terminus of the cleavable linker. In some cases, the plasma membrane-localized protein comprises a PH domain or a variant thereof derived from phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), disk and actin-binding protein 1 (Daap1), general receptor for phosphoinositides 1 (Grp1), oxysterol-binding protein 1-Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), four-phosphate adaptor protein 1 (FAPP1), ceramide transport 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). In some cases, the plasma membrane-localized protein comprises a membrane protein selected from the group consisting of CD9, CD47, CD63 and CD81 and their transmembrane domains. 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 pleckstrin homology (PH) domain or a variant thereof 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. In some cases, the plasma membrane-localized protein comprises a membrane protein selected from the group consisting of 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 mobilizing protein, including Arc, human Arc, endogenous retroviral gag protein, or human endogenous retroviral gag protein. In some cases, the composition is a lipid-containing particle, and optionally, the lipid-containing particle is a cell; a virus-like particle (VLP); a proteolipid vehicle (PLV); a liposome, optionally a lipid nanoparticle; or an extracellular vesicle, optionally an exosome or an ectosome.

[0017] As used herein, in some embodiments, there is provided a lipid-containing particle comprising a combinatorial protein comprising i) a humanized retroviral structural protein; an endogenous retroviral (HERV) structural protein, optionally HERV gag; a pleckstrin homology (PH) domain; or a non-immunogenic plasma membrane mobilizing protein, ii) a cleavable linker, and iii) a freight, wherein the freight is a therapeutic freight or a binding partner of a therapeutic freight.

[0018] In some cases, the therapeutic payload comprises a nuclease, base editor, prime editor, epigenetic editor, restriction endonuclease (optionally, a type IIS restriction enzyme), recombinase, transcription factor, antibody, chimeric antigen receptor, T cell receptor, organelle, nucleic acid molecule, DNA, RNA, retrotransposon, reverse transcriptase, oligonucleotide, aptazyme, aptamer, ribozyme, or 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 mobilizing protein, and ii) the therapeutic payload.

[0019] In some cases, the combinatorial protein further comprises a NES, and optionally, the NES is between 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 mobilizing protein, and ii) a therapeutic freight, and optionally, the NES is between 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 mobilizing protein, and ii) a cleavable linker, and optionally, the combinatorial protein further comprises a nuclear localization sequence (NLS) at the C-terminus of the cleavable linker. In some cases, the plasma membrane-localized protein comprises a PH domain or a variant thereof derived from phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), disk and actin-binding protein 1 (Daap1), general receptor for phosphoinositides 1 (Grp1), oxysterol-binding protein 1-Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), phosphatidylinositol phosphate adapter protein 1 (FAPP1), ceramide transport 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). 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 or a variant thereof 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. In some cases, the plasma membrane-localized protein comprises a membrane protein selected from the group consisting of CD9, CD47, CD63, and CD81 and their transmembrane domains.In some cases, the plasma membrane-localized protein includes a membrane protein selected from the group consisting of 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 mobilizing protein, including Arc, human Arc, endogenous retroviral gag protein, or human endogenous retroviral gag protein. In some cases, the lipid-containing particle includes a cell; a virus-like particle (VLP); a proteolipid vehicle (PLV); a liposome, optionally a lipid nanoparticle; or an extracellular vesicle, optionally an exosome or ectosome.

[0020] As used herein, in some embodiments, a composition comprising a nucleic acid molecule encoding a combinatorial protein comprising: i) a humanized retroviral structural protein; an endogenous retroviral (HERV) structural protein, optionally HERV gag; a plekstrin homology (PH) domain; or a non-immunogenic plasma membrane mobilizing protein; ii) a cleavable linker; and iii) a freight, wherein the freight is a therapeutic freight or a binding partner of a therapeutic freight, is disclosed.

[0021] In some cases, the therapeutic freight includes a nuclease, base editor, prime editor, epigenetic editor, restriction endonuclease (optionally, a type IIS restriction enzyme), recombinase, transcription factor, antibody, chimeric antigen receptor, T cell receptor, organelle, nucleic acid molecule, DNA, RNA, retrotransposon, reverse transcriptase, oligonucleotide, aptazyme, aptamer, ribozyme, or 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 mobilizing protein, and ii) the therapeutic freight. In some cases, the combinatorial protein further includes an NES, and optionally, 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 mobilizing protein, and ii) the therapeutic freight, and optionally, 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 mobilizing protein, and ii) the cleavable linker, and optionally, the combinatorial protein further includes a nuclear localization sequence (NLS) at the C-terminus of the cleavable linker.

[0022] In some cases, the plasma membrane-localized protein comprises a PH domain or a variant thereof from phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), disk and actin-binding protein 1 (Daap1), general receptor for phosphoinositides 1 (Grp1), oxysterol-binding protein 1-Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), four-phosphate adaptor protein 1 (FAPP1), ceramide transport 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). In some cases, the plasma membrane-localized protein comprises a PH domain from a human protein. In some cases, the plasma membrane-localized protein comprises a PH domain or a variant thereof 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. In some cases, the plasma membrane-localized protein comprises a membrane protein selected from the group consisting of CD9, CD47, CD63, and CD81 and their transmembrane domains. In some cases, the plasma membrane-localized protein comprises a membrane protein selected from the group consisting of 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 mobilizing protein, including Arc, human Arc, endogenous retrovirus gag protein, or human endogenous retrovirus gag protein.

[0023] In some cases, the composition is a lipid-containing particle, and optionally, the lipid-containing particle comprises a cell; a virus-like particle (VLP); a proteolipid vehicle (PLV); a liposome, optionally a lipid nanoparticle; or an extracellular vesicle, optionally an exosome or an ectosome.

[0024] In some cases, the lipid-containing particles further comprise 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 relative to the total of the second and third nucleic acid molecules in the composition 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 combinatorial protein relative to the total of the nucleic acid molecule encoding the combinatorial 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%.

[0025] In some cases, the lipid-containing particles do not contain non-human gag protein or humanized gag protein. In some cases, the lipid-containing particles further comprise an outer lipid layer and one or more immunomodulatory substances in the outer lipid layer. In some cases, the one or more immunomodulatory substances are immunosuppressive molecules. In some cases, the immunosuppressive molecules include 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 include 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, B7-1 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.

[0026] In some cases, the composition does not contain a nucleic acid molecule encoding a non-human gag protein or a humanized gag protein.

[0027] Disclosed herein are methods, in some embodiments, comprising contacting a cell with a lipid-containing particle disclosed herein.

[0028] Disclosed herein are methods, in some embodiments, comprising administering a lipid-containing particle disclosed herein to a subject in need thereof.

[0029] In some aspects, provided herein is a method for producing lipid-containing particles, the method comprising expressing a system that includes a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule; a combinatorial protein that includes a plasma membrane-localized protein coupled to a nuclear export sequence (NES); and a freight, to produce lipid-containing particles; and, optionally, recovering and purifying the lipid-containing particles.

[0030] In some aspects, provided herein is a method for producing lipid-containing particles, the method comprising expressing a system that includes a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule; a combinatorial protein that includes a plasma membrane-localized protein coupled to a cleavable linker; and a freight, to produce lipid-containing particles; and, optionally, recovering and purifying the lipid-containing particles.

[0031] In some aspects, provided herein is a method for producing lipid-containing particles, the method comprising expressing a system that includes 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 mobilizing protein; and ii) a nuclear export sequence (NES), to produce lipid-containing particles; and, optionally, recovering and purifying the lipid-containing particles.

[0032] In some aspects, this specification discloses a method for producing lipid-containing particles, which includes: i) expressing a combinatorial protein comprising 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 mobilizing protein, ii) a cleavable linker, and iii) a freight, to provide a system for generating lipid-containing particles; and optionally, recovering and purifying the lipid-containing particles. In some cases, the system further expresses a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; or a humanized structural protein. In some cases, the system includes a production cell, a cell-free extract, or a cell lysate.

[0033] Incorporation by reference All published documents, patents and patent applications referred to in this specification are incorporated herein by reference to the same extent as if each individual published document, patent or patent application was specifically and individually indicated to be incorporated by reference.

[0034] A better understanding of the features and advantages of the present disclosure will be obtained by referring to the following detailed description, which illustrates embodiments useful in explaining the principles of the present disclosure, and the accompanying drawings.

Brief Description of the Drawings

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Figure 9C

[0081]

Figure 10A

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Figure 10B

Figure 10C

[0083]

Figure 10D

[0084]

Figure 10E

[0085]

Figure 10F

[0086]

Figure 10G

[0087]

Figure 11A

[0088]

Figure 11B

[0089]

Figure 11C

[0090]

Figure 11D

[0091]

Figure 12A

Figure 12B

[0092]

Figure 13A

[0093]

Figure 13B-C

[0094]

Figure 14A

Figure 14B

Figure 14C

[0095]

Figure 15

[0096]

Figure 16

[0097]

Figure 17

Mode for Carrying Out the Invention

[0098] Detailed Description The implementation of some of the methods disclosed in this specification uses conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the scope of skills in the art unless otherwise indicated. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0099] Definitions

[0100] 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 "a chimeric transmembrane receptor polypeptide" includes multiple chimeric transmembrane receptor polypeptides.

[0101] The terms "about" or "approximately" mean within an acceptable error range for a particular value as determined by a person skilled in the art, and this acceptable error range will depend in part on the method by which the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, in accordance with the practice in the art. Alternatively, "about" can mean within a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or biological processes, the term can mean within one order of magnitude, preferably within five-fold, more preferably within two-fold of the value. When a particular value is recited in the present application, unless otherwise stated, the term "about" is assumed to mean within the acceptable error range of the particular value.

[0102] As used herein, the term "cell" generally can refer to a living cell. A cell can be the basic structure, function, and / or biological unit of a living organism. A 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 eukaryotes, protozoan cells, cells from plants (e.g., plant crops, fruits, vegetables, grains, soybeans, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkins, forage, potatoes, cotton, hemp, tobacco, flowering plants, conifers, gymnosperms, ferns, club mosses, spike mosses, quillworts, mosses), algal cells (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, and 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.). Sometimes, a cell is not derived from a natural organism (e.g., a cell can be synthetically made and sometimes is called an artificial cell).

[0103] 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 antigen molecule that can be bound by a selective binding agent such as an immunological protein (e.g., an antibody). An antigen sometimes refers to a molecule or a fragment thereof that can be used in an animal to produce an antibody that can bind to that antigen.

[0104] As used herein, the term "antibody" refers to a proteinaceous binding molecule having immunoglobulin-like function. The term antibody includes, of course, 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 (e.g., IgG1, IgG2, etc.). Derivatives, variants or fragments thereof can refer to functional derivatives or fragments that retain the binding specificity (e.g., complete and / or partial) 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 antibodies ("sdAb" or "nanobody" or "camelid"). The term antibody includes optimized, engineered or chemically conjugated antibodies and antigen-binding fragments of antibodies. Examples of optimized antibodies include affinity matured antibodies. Examples of engineered antibodies include Fc-optimized antibodies (e.g., antibodies in which the fragment crystallizable region is optimized) and multispecific antibodies (e.g., bispecific antibodies).

[0105] 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 the monomeric units of nucleic acid sequences (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), which are ribonucleoside triphosphates, 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, and nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. The term nucleotide, as used herein, can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Examples useful in the description 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 using quantum dots. Detectable labels can include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. Examples of 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), Cascade Blue, Oregon Green, Texas Red, cyanine, and 5-(2’-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Specific examples of fluorescently labeled nucleotides 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, tetramethyl-rhodamine-6-dUTP, IR770-9-dATP, fluorescein-12-ddUTP, fluorescein-12-UTP, and fluorescein-15-2'-dATP, available from Boehringer Mannheim, Indianapolis, Ind.; and chromosomal 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 marked by chemical modification.The chemically modified single nucleotide can be biotin-dNTP. Some examples of biotinylated dNTPs 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).

[0106] The terms "polynucleotide", "oligonucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably to refer to nucleotides, deoxyribonucleotides, or ribonucleotides in polymeric form, in any length, in either single-stranded, double-stranded, or multi-stranded form, or analogs thereof. Polynucleotides can be exogenous or endogenous to a cell. Polynucleotides can exist 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 function, known or unknown. Polynucleotides can contain one or more analogs (e.g., modifications to the backbone, sugar, or nucleobase). When present, modifications to the nucleotide structure can be imparted before or after polymerization of the polymer. Some examples of analogs include 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. Examples of polynucleotides include the coding or non-coding regions of a gene or gene fragment, locus (loci) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, 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. There may also be non-nucleotide constituents interrupting the nucleotide sequence.

[0107] As used herein, the term "gene" refers to nucleic acids (e.g., DNA, such as genomic DNA and cDNA) involved in encoding RNA transcripts and their corresponding nucleotide sequences. When used herein with respect to genomic DNA, the term includes intervening, non-coding regions, as well as regulatory regions, and may include 5' and 3' termini. In some uses, the term encompasses transcriptional sequences that include 5' and 3' untranslated regions (5'-UTR and 3'-UTR), exons, and introns. In some genes, the transcriptional region will contain an "open reading frame" that encodes a polypeptide. In some uses of the term, "gene" includes only the coding sequences necessary to encode a polypeptide (e.g., an "open reading frame" or "coding region"). In some cases, genes, such as ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes, do not encode polypeptides. In some cases, the term "gene" includes not only transcriptional sequences, but also non-transcriptional regions, including upstream and downstream regulatory regions, enhancers, and promoters. A gene can refer to an "endogenous gene" or native gene in its natural position in an organism's genome. A gene can refer to a "foreign gene" or non-native gene. A non-native gene can refer to a gene that is not normally found in the host organism but is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene that is not in its natural position in an organism's genome. A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that includes mutations, insertions, and / or deletions (e.g., non-native sequences).

[0108] The terms "target polynucleotide", "target nucleic acid", and "target sequence", as used herein, refer to a nucleic acid or polynucleotide targeted by the freight of the present disclosure. The target polynucleotide can be DNA (e.g., endogenous or exogenous). DNA can refer to a template for generating an mRNA transcript and / or various regulatory regions that regulate the transcription of mRNA from a DNA template. The target polynucleotide can be a portion 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 that can function as a template for encoding a protein. The target polynucleotide containing RNA can include various regulatory regions that regulate the translation of a protein from an mRNA template. The target polynucleotide can contain a regulatory sequence that encodes a gene product (e.g., DNA encoding an RNA transcript, or RNA encoding a protein product) or regulates the expression of a gene product. Generally, the term "target sequence" refers to a nucleic acid sequence on a single strand of a target nucleic acid. The target sequence can be a part of a gene, a regulatory sequence, genomic DNA, cell-free nucleic acid including cfDNA and / or cfRNA, cDNA, chimeric gene, and RNA including mRNA, miRNA, rRNA, etc. When the target polynucleotide is targeted by the freight, the gene expression and / or activity can be changed as a result. When the target polynucleotide is targeted by the freight, an edited nucleic acid sequence can be brought about as a result. 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 from the 5' end of the target nucleic acid.In some embodiments, the replacement does not occur within 5, 10, 15, 20, 25, 30, or 35 nucleotides from the 3' end of the target nucleic acid.

[0109] The term "expression" refers to one or more processes by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcripts), and / or the transcribed mRNA is then translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may together be referred to as a "gene product". When the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells. With respect to expression, "upregulated" generally refers to an increase in the level of expression of a polynucleotide (e.g., RNA, e.g., mRNA) and / or polypeptide sequence compared to its level of expression in the wild-type state, while "downregulated" generally refers to a decrease in the level of expression of a polynucleotide (e.g., RNA, e.g., mRNA) and / or polypeptide sequence compared to its expression in the wild-type state.

[0110] As used herein, the terms "complementary sequence(singular)", "complementary sequence(plural)", "complementary", and "complementarity" generally refer to a sequence that is completely complementary to and capable of hybridizing to a given sequence. In some cases, a sequence that hybridizes to a given nucleic acid is called the "complementary sequence" or "reverse complementary sequence" of the given molecule if its base sequence can bind complementarily over a given region such that A-T, A-U, G-C, and G-U base pairs are formed with the sequence of its binding partner. Generally, a first sequence that is capable of hybridizing to a second sequence is specifically or selectively capable of hybridizing to the second sequence, and thus, during a hybridization reaction, hybridization to the second sequence or a set 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, 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, over all or a portion of their respective lengths.Array identity, e.g., percent complementarity, for purposes of evaluating array identity can be measured by any suitable alignment algorithm, including the Needleman-Wunsch algorithm (e.g., see the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html with default settings as needed), the BLAST algorithm (e.g., see the BLAST alignment tool available at blast.ncbi.nlm.nih.gov / Blast.cgi with default settings as needed), or the Smith-Waterman algorithm (e.g., see the EMBOSS Water aligner available at www.ebi.ac.uk / Tools / psa / emboss_water / nucleotide.html with default settings as needed). The optimal alignment can be evaluated using any suitable parameters of the selected algorithm, including default parameters.

[0111] Complementarity can be perfect or substantial / adequate. Perfect complementarity between two nucleic acids can mean that the two nucleic acids can form a duplex in which all bases in the duplex are paired to complementary bases by Watson-Crick pairing. Substantial or adequate complementarity can mean that the sequence in one strand is not completely and / or perfectly complementary to the sequence in the opposite strand, but there is sufficient bonding between bases on the two strands to form a stable hybrid 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 for predicting the Tm of the hybridized strands, or by the empirical determination of Tm using conventional methods.

[0112] With respect to expression or activity, the term "modulating" as used herein refers to changing the level of expression or activity. Modulation can occur at the transcriptional, post-transcriptional, translational, and / or post-translational levels.

[0113] The terms "peptide", "polypeptide", and "protein" are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by peptide bonds. The term is not meant to imply any specific length of the polymer, nor is it intended to imply or distinguish whether the peptide was produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The term applies to naturally occurring amino acid polymers as well as amino acid polymers that include at least one modified amino acid. In some cases, non-amino acids may interrupt the polymer. The term includes amino acid chains of any length, including full-length proteins, as well as proteins that do or do not have secondary and / or tertiary structures (e.g., domains). The term also includes amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation, such as conjugation with a labeling component. The terms "amino acid(s)" and "amino acid(s)" as used herein generally refer to natural or 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 the amino acid. 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 may lack an N-terminal methionine. For example, SEQ ID NOs: 1-5, 7-11, 15-17, and 22-77 may lack an N-terminal methionine.

[0114] As used herein with respect to a polypeptide, the term "variant" refers to a polypeptide that is related to a wild-type polypeptide but is not identical thereto by virtue of, for example, any of amino acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity), and / or function. A variant includes a polypeptide that contains one or more amino acid differences (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof as compared to the wild-type polypeptide. A variant also includes derivatives and fragments of the wild-type polypeptide.

[0115] As used herein, the term "percent identity (%)" refers to 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, if necessary, to achieve the maximum percent identity (i.e., gaps may be introduced into one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences may be ignored for comparison purposes). The alignment can be achieved using various methods within the skill in the art for determining percent identity, e.g., using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. The percent identity between two sequences can be calculated by aligning the test sequence with the 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.

[0116] The Cas protein referred to in this specification 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 inactive, partially active, constitutively active, fully active, inducibly active, and / or more active (e.g., 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 inactive, and / or conditionally enzymatically inactive site-directed polypeptide) can bind to a target nucleic acid. The Cas protein (e.g., variant, mutated, enzymatically inactive, and / or conditionally enzymatically inactive endoribonuclease) can bind to a target RNA or DNA.

[0117] 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 an exemplary wild-type crRNA (e.g., a crRNA from S. pyogenes). A 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 an exemplary wild-type crRNA (e.g., a crRNA from S. pyogenes, S. aureus, etc.). A crRNA can refer to a modified form of crRNA that can include nucleotide changes, such as deletions, insertions or substitutions, variants, mutations, or chimeras. A crRNA can be a nucleic acid having at least about 60% sequence identity to an exemplary wild-type crRNA sequence (e.g., a crRNA from S. pyogenes, S. aureus, etc.) over a stretch of at least 6 contiguous nucleotides. For example, a 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 an exemplary wild-type crRNA sequence (e.g., a crRNA from S. pyogenes, S. aureus, etc.) over a stretch of at least 6 contiguous nucleotides.

[0118] 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 an exemplary wild-type tracrRNA sequence (e.g., tracrRNA from S. pyogenes). A 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 an exemplary wild-type tracrRNA sequence (e.g., tracrRNA from S. pyogenes, S. aureus, etc.). A tracrRNA can refer to a modified form of tracrRNA that can include nucleotide changes, such as deletions, insertions or substitutions, variants, mutations, or chimeras. A tracrRNA can refer to a nucleic acid that can be at least about 60% identical to an exemplary wild-type tracrRNA (e.g., tracrRNA from S. pyogenes, S. aureus, etc.) sequence over a stretch of at least 6 consecutive nucleotides. For example, a 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 an exemplary wild-type tracrRNA (e.g., tracrRNA from S. pyogenes, S. aureus, etc.) sequence over a stretch of at least 6 consecutive nucleotides.

[0119] As used herein, "guide nucleic acid" can refer to a nucleic acid capable of hybridizing 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 the sequence of a nucleic acid. The target nucleic acid, 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 the double-stranded target polynucleotide that is complementary to the guide nucleic acid and hybridizes to the guide nucleic acid can be called the complementary strand. The strand of the double-stranded target polynucleotide that is complementary to the complementary strand and thus may not be complementary to the guide nucleic acid can be called the non-complementary strand. The guide nucleic acid can contain a polynucleotide strand and can be called 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 strands and can be called a "double guide nucleic acid". The double guide nucleic acid can contain crRNA and tracrRNA. Unless otherwise specified, the term "guide nucleic acid" is inclusive and can refer to both single guide nucleic acids and double guide nucleic acids.

[0120] The guide nucleic acid can contain a segment that can be called a "nucleic acid targeting segment" or "nucleic acid targeting sequence". The nucleic acid targeting segment can contain a sub-segment that can be called a "protein binding segment" or "protein binding sequence" or "Cas protein binding segment".

[0121] As used herein, the term "targeting array" refers to a nucleotide sequence and corresponding amino acid sequence that encodes a targeting polypeptide that mediates the localization (or retention) of a protein to an intracellular location, such as the plasma membrane, or the membrane of a given organelle, nucleus, cytosol, mitochondria, endoplasmic reticulum (ER), Golgi, chloroplast, apoplast, peroxisome or another organelle. For example, a targeting array can direct a protein (e.g., a receptor polypeptide or an adaptor polypeptide) to the nucleus using a nuclear localization signal (NLS); outside the nucleus of the cell, e.g., into the cytoplasm, using a nuclear export signal (NES); to the mitochondria using a mitochondrial targeting signal; to the endoplasmic reticulum (ER) using an ER retention signal; to the peroxisome using a peroxisome targeting signal; to the plasma membrane using a membrane localization signal; or combinations thereof.

[0122] As used herein, "nuclear localization domain" can refer to a nuclear localization signal or other sequence or domain that crosses the nuclear membrane and thereby allows entry into the nucleus. A nuclear localization domain may be fused in-frame with a polypeptide, in which case the nuclear localization domain may be referred to as a "heterologous nuclear localization domain".

[0123] As used herein, "nuclear export domain" can refer to a nuclear export signal or other sequence or domain that is present in a protein and can target the protein for transport from the cell nucleus to the cytoplasm via the nuclear pore complex using nuclear import. A nuclear export domain may be fused in-frame with a polypeptide, in which case the nuclear export domain may be referred to as a "heterologous nuclear export domain".

[0124] As used herein, "fusion" or "chimera" can refer to a protein and / or nucleic acid that includes one or more non-native sequences (e.g., moieties). A chimera or fusion can include one or more of the same non-native sequence. A chimera or fusion can include one or more of different non-native sequences. A chimera or fusion can be chimeric. A chimera or fusion can include a nucleic acid affinity tag. A chimera or fusion can include a barcode. A fusion can include a peptide affinity tag. A chimera or fusion can effect intracellular localization of a site-directed polypeptide (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a mitochondrial localization signal for targeting to mitochondria, a chloroplast localization signal for targeting to chloroplasts, and an endoplasmic reticulum (ER) retention signal, etc.). A chimera or fusion can provide a non-native sequence (e.g., an affinity tag) that can be used to track or purify it.

[0125] A fusion or chimera can refer to any protein that has a functional effect. For example, a chimeric protein can include 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.

[0126] As used herein, "non-native" can refer to a nucleic acid or polypeptide sequence that is not found in native nucleic acids or proteins. Non-native can refer to an affinity tag. Non-native can refer to a chimera or fusion, such as a chimeric protein or chimeric nucleic acid. Non-native can refer to a naturally occurring nucleic acid or polypeptide sequence that includes mutations, insertions, and / or deletions. A non-native sequence can exhibit and / or encode an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitination activity, etc.) that is also exhibited by the nucleic acid and / or polypeptide sequence to which the non-native sequence is fused. A non-native nucleic acid or polypeptide sequence can be ligated to a naturally occurring nucleic acid or polypeptide sequence (or variant thereof) by genetically engineering the non-native nucleic acid or polypeptide sequence to generate a chimeric nucleic acid and / or polypeptide sequence that encodes the chimeric nucleic acid and / or polypeptide.

[0127] 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, livestock, sport animals, and pets. Also included are tissues, cells, and progeny thereof of biological entities obtained in vivo or cultured in vitro.

[0128] As used herein, the terms "treatment" and "treating" refer to an approach for obtaining a beneficial or desired result, including a therapeutic benefit and / or a prophylactic benefit. For example, treatment may include administering a system or cell population disclosed herein. A therapeutic benefit means any therapeutically significant improvement in, or any therapeutically significant effect on, one or more diseases, conditions or symptoms being treated. For prophylactic benefit, a composition may 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.

[0129] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition, such as a composition comprising immune cells, such as lymphocytes (e.g., T lymphocytes and / or NK cells) including the systems of the present disclosure, that is sufficient to provide the desired activity upon administration to a subject in need thereof. In the context of the present disclosure, the term "therapeutically effective" refers to an amount of a composition that is sufficient to delay the manifestation of, arrest the progression of, or alleviate or mitigate at least one symptom of, a disorder being treated by the methods of the present disclosure. Lipid-containing particles

[0130] In some embodiments, the present disclosure relates to delivery vehicles for the delivery of therapeutic freight and / or other molecules to cells in vitro, ex vivo, or in vivo. In some cases, the delivery vehicles of the present disclosure have high in vivo delivery efficiency of therapeutic freight and / or other molecules to target cells. In some cases, the delivery vehicles of the present disclosure include lipid-containing particles such as virus-like particles, exosomes, lipid nanoparticles, proteolipid vehicles, extracellular vesicle mimics, and membrane vesicles. The delivery vehicles (e.g., lipid-containing particles) disclosed herein may have high in vivo delivery efficiency of freight upon administration to a subject. For example, a high percentage of the freight loaded in the lipid-containing particles is delivered to the cells of the subject and to the desired intracellular location (e.g., the cell nucleus or cytoplasm) of the cells of the subject. In some cases, the lipid-containing particles are used to deliver a genome editing system to target cells and may have high in vivo gene editing efficiency performed by the genome editing system. In some cases, the lipid-containing particles are used to deliver an expression construct encoding a therapeutic protein (e.g., an antibody, a transcription factor, or a chimeric antigen receptor (CAR)) to target cells and may have high expression efficiency of the therapeutic protein in the subject.

[0131] In some cases, the lipid-containing particles provided herein include a lipid-based outer layer that encloses a lumen (e.g., a protein core). The freight can be loaded inside the protein core in the lipid-containing particles. In some cases, the freight is loaded into the lipid-containing particles by attaching to the outer lipid-based layer. The outer lipid-based layer can be a single lipid layer or a lipid bilayer made 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 outside of the outer lipid layer. The membrane fusion proteins can assist in the fusion of the lipid-containing particles with the membrane of the target cells and thus in the delivery of the freight loaded in the lipid-containing vesicles to the target cells.

[0132] The size (e.g., diameter) of the lipid-containing particles is 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 include virus-like particles, lipid nanoparticles, or proteolipid vehicles 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, from about 10 nm to about 30 nm, from about 10 nm to about 40 nm, from about 10 nm to about 50 nm, from about 10 nm to about 60 nm, from about 10 nm to about 80 nm, from about 20 nm to about 30 nm, from about 20 nm to about 40 nm, from about 20 nm to about 50 nm, from about 20 nm to about 60 nm, from about 20 nm to about 80 nm, from about 40 nm to about 50 nm, from about 40 nm to about 60 nm, or from about 40 nm to about 80 nm.In some cases, the lipid-containing particles include exosomes and have a size of about 50 nm to about 200 nm, such as about 50 nm to about 80 nm, about 50 nm to about 100 nm, about 50 nm to about 120 nm, about 50 nm to about 150 nm, about 50 nm to about 160 nm, about 50 to about 180 nm, about 60 nm to about 80 nm, about 60 nm to about 100 nm, about 60 nm to about 120 nm, about 60 nm to about 160 nm, about 60 nm to about 160 nm, about 60 nm to about 180 nm, about 80 nm to about 100 nm, about 80 nm to about 120 nm, about 80 nm to about 160 nm, about 80 nm to about 180 nm, about 80 nm to about 180 nm, about 100 nm to about 120 nm, about 100 nm to about 150 nm, about 100 nm to about 180 nm, about 120 nm to about 150 nm, about 120 nm to about 180 nm, about 150 nm to about 180 nm, or about 150 nm to about 200 nm.

[0133] In some embodiments, provided herein are lipid-containing particles that include a cell fusion molecule or membrane fusogen (e.g., a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule); a combinatorial protein that includes a plasma membrane-localized protein (e.g., coupled to a nuclear export sequence (NES)); and a freight (e.g., a therapeutic freight or a binding partner of a therapeutic freight).

[0134] In some embodiments, provided herein are lipid-containing particles that include a membrane fusion molecule (e.g., a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule); a combinatorial protein that includes a plasma membrane-localized protein (e.g., coupled to a cleavable linker); and a freight (e.g., a therapeutic freight or a binding partner of a therapeutic freight).

[0135] In some embodiments, lipid-containing particles are provided herein that include a plasma membrane-localized molecule (e.g., a humanized retroviral structural protein or a human endogenous retrovirus (HERV) structural protein, such as HERV gag, a pleckstrin homology (PH) domain, or a non-immunogenic plasma membrane mobilizing protein) and a nuclear export sequence (NES).

[0136] In some embodiments, lipid-containing particles are provided herein that include a combinatorial protein comprising: i) a plasma membrane-localized molecule (e.g., a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, such as HERV gag; a pleckstrin homology (PH) domain; or a non-immunogenic plasma membrane mobilizing protein), ii) a cleavable linker, and iii) a freight (e.g., a therapeutic freight or a binding partner of a therapeutic freight).

[0137] In some embodiments, lipid-containing particles are provided herein that include: i) a plasma membrane-localized molecule (e.g., a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, such as HERV gag; a pleckstrin homology (PH) domain; or a non-immunogenic plasma membrane mobilizing protein) and ii) a freight (e.g., a therapeutic freight or a binding partner of a therapeutic freight).

[0138] In some embodiments, lipid-containing particles are provided herein that include a combinatorial protein comprising: i) a membrane fusogen (e.g., a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule), ii) a plasma membrane-localized molecule (e.g., a humanized retroviral structural protein; a human endogenous retrovirus (HERV) structural protein, such as HERV gag; a pleckstrin homology (PH) domain; or a non-immunogenic plasma membrane mobilizing protein), and iii) a freight (e.g., a therapeutic freight or a binding partner of a therapeutic freight).

[0139] In one aspect, provided herein are lipid-containing particles 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-localized protein, wherein the plasma membrane-localized protein is selected from the group consisting of 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 (c) a flotillin. In some cases, the combinatorial protein comprises a plasma membrane-localized protein coupled to a flotillin. In some cases, the combinatorial protein comprises a plasma membrane-localized protein coupled to a nuclear export sequence (NES). In some cases, the combinatorial protein comprises a plasma membrane-localized protein, an NES, and a flotillin arranged in order from the N-terminus to the C-terminus of the combinatorial protein. In some cases, the combinatorial protein comprises a plasma membrane-localized protein, an NES, a flotillin, and a second NES arranged in order from the N-terminus to the C-terminus of the combinatorial protein. 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 located between the plasma membrane-localized protein and the flotillin. In some cases, the cleavable linker is located between the NES and the flotillin. In some cases, the combinatorial protein further comprises a nuclear localization sequence (NLS) at the C-terminus of the cleavable linker. In some cases, the lipid-containing particles comprise 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, the human endogenous retrovirus (HERV) envelope protein, humanized envelope protein, or non-immunogenic membrane fusion molecule is attached to the lipid-containing membrane.

[0140] In some embodiments, provided herein are lipid-containing particles comprising: (a) a glycoprotein derived from a virus selected from the group consisting of RD114, Fug-E, FuG-E (P440E), and MLV 10A1; (b) a combinatorial protein comprising a plasma membrane-localized protein coupled to a nuclear export sequence (NES); and (c) a fret. In some cases, the combinatorial protein further comprises a fret. 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 fret. In some cases, the combinatorial protein further comprises a cleavable linker. In some cases, the cleavable linker is located between the plasma membrane-localized protein and the fret. In some cases, the cleavable linker is located between the NES and the fret. In some cases, the combinatorial protein comprises a nuclear localization sequence (NLS) at the C-terminus of the cleavable linker. In some cases, the plasma membrane-localized protein comprises: (a) a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; (b) a humanized structural protein; (c) a plekstrin homology (PH) domain; or (d) a non-immunogenic plasma membrane mobilizing protein. In some cases, the non-immunogenic plasma membrane mobilizing protein comprises Arc, human Arc, an endogenous retrovirus gag protein, or a human endogenous retrovirus gag protein.

[0141] In some embodiments, provided herein are lipid-containing particles comprising: (a) a glycoprotein derived from a virus selected from the group consisting of RD114, Fug-E, FuG-E (P440E), and MLV 10A1; (b) a combinatorial protein comprising a plasma membrane-localized protein coupled to a cleavable linker; and (c) a fret. In some cases, the combinatorial protein further comprises a fret. 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, a cleavable linker, and a fret. In some cases, the combinatorial protein further comprises a nuclear localization sequence (NLS) at the C-terminus of the cleavable linker. In some cases, the plasma membrane-localized protein comprises (a) a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; (b) a humanized structural protein; (c) a plekstrin homology (PH) domain; or (d) a non-immunogenic plasma membrane mobilizing 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.

[0142] In any one of the above or related embodiments, the plasma membrane-localized protein may contain a PH domain. In some cases, the PH domain is the PH domain of phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), disk and actin-binding protein 1 (Daap1), general receptor for phosphoinositides 1 (Grp1), oxysterol-binding protein 1-Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), phosphatidylinositol 4-phosphate adapter protein 1 (FAPP1), ceramide transport 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 variant thereof. In some cases, the PH domain contains the PH domain of a human protein. In some cases, the PH domain contains the PH domain of 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 variant thereof. In some cases, the PH domain is selected from the group consisting of the 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. In some cases, the PH domain contains 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.

[0143] In some cases, the plasma membrane-localized protein includes a membrane protein selected from the group consisting of CD9, CD47, CD63, and CD81 and their transmembrane domains. In some cases, the plasma membrane-localized protein includes a membrane protein selected from the group consisting of human CD9, human CD47, human CD63, and human CD81 and their transmembrane domains. In some cases, the membrane protein includes 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 plasma membrane-localized protein includes a non-immunogenic plasma membrane mobilizing protein, including Arc, human Arc, endogenous retroviral gag protein, or human endogenous retroviral gag protein. In some cases, the non-immunogenic plasma membrane mobilizing protein includes hArc or hGAGK con including an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the sequences.

[0144] In any one of the above or related embodiments, the phage may further include a therapeutic phage or a binding partner of a therapeutic phage.

[0145] In any one of the above or related embodiments, the combinatorial protein may include an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequences selected from SEQ ID NOs: 50, 52-55, and 67-77.

[0146] In some embodiments, provided herein are lipid-containing particles comprising a combinatorial protein 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. In some cases, the lipid-containing particles comprise: (a) a human endogenous retrovirus (HERV) envelope protein, optionally 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, a human endogenous retrovirus (HERV) envelope protein comprising 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 selected from the group consisting of BaEVTR, BaEVTRless, FuG-E, FuG-E(P440E), MVL ENV (amphotropic), MVL ENV (ecotropic), MLV 10A1, VSVG, GP64, gp160, and RD114 ENV, and optionally comprising 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 fret, or a combination thereof. In some cases, the lipid-containing particles comprise 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, humanized envelope protein, non-immunogenic membrane fusion molecule, or virus-derived glycoprotein is attached to the lipid-containing membrane.

[0147] In one aspect, a combinatorial protein comprising a plasma membrane-localized protein and a heterologous sequence is disclosed, wherein the plasma membrane-localized protein is selected from the group consisting of 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. 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. As used herein, in some aspects, lipid-containing particles comprising the combinatorial proteins described herein are also disclosed. In some cases, the lipid-containing particles comprise 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 particles further comprise 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 viral glycoprotein is selected from the group consisting of BaEVTR, BaEVTRless, FuG-E, FuG-E(P440E), MVL ENV (amphotropic), MVL ENV (ecotropic), MLV 10A1, VSVG, GP64, gp160, and RD114 ENV; and optionally, the viral 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.

[0148] In any one of the above or related aspects, the protein core may include a structural protein that includes a second plasma membrane-localized protein. In some cases, the structural protein further includes a retroviral protease (pro) protein. In some cases, the second plasma membrane-localized protein is (a) a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; (b) a humanized structural protein; (c) a pleckstrin homology (PH) domain, optionally where the PH domain is phosphatidylinositol phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), disk and actin-binding protein 1 (Daap1), general receptor for phosphoinositides 1 (Grp1), oxysterol-binding protein 1-Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), tetraphosphate adapter protein 1 (FAPP1), ceramide transport 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) PH domain or a variant thereof, optionally where the PH domain is from a human, optionally where the PH domain is the PH domain of human phosphatidylinositol 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 PH domain or a variant thereof; or (d) a non-immunogenic plasma membrane mobilizing protein, optionally where the non-immunogenic plasma membrane mobilizing protein includes a membrane protein selected from the group consisting of CD9, CD47, CD63, and CD81 and their transmembrane domains.In some cases, the second plasma membrane-localized protein contains a PH domain, and the PH domain contains 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 contains a membrane protein that contains 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 contains a non-immunogenic plasma membrane mobilizing protein that includes Arc, human Arc, endogenous retroviral gag protein, or human endogenous retroviral gag protein. In some cases, the second plasma membrane-localized protein is hArc or hGAGK listed in Table 3. con Contains a non-immunogenic plasma membrane mobilizing protein that contains an 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 the 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 the amino acid sequences selected from SEQ ID NOs: 50, 52-55, and 67-77. In some cases, the plasma membrane-localized protein of the combinatorial protein forms part of the protein core. In some cases, the lipid-containing membrane contains an immunomodulatory substance. In some cases, the immunomodulatory substance is in the phospholipid bilayer. In some cases, the immunomodulatory substance is an immunosuppressive molecule.

[0149] In any one of the above or related embodiments, the lipid-containing particle includes (a) a cell; (b) a virus-like particle (VLP); (c) a proteolipid vehicle (PLV); (d) a liposome, optionally a lipid nanoparticle; or (e) an extracellular vesicle, optionally an exosome or ectosome. Combinatorial protein

[0150] In some embodiments, combinatorial proteins suitable for the assembly of VLPs and delivery of VLPs to cells are disclosed herein. The combinatorial protein can form at least a portion of the lumen (e.g., the protein core) of the lipid-containing particle. The lipid-containing particle can contain 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 include a plasma membrane-localized 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 VL protein or polypeptide (e.g., a therapeutic VL). In some cases, the combinatorial protein includes a VL that is a binding partner of a therapeutic VL (e.g., the binding partner can bind directly to the therapeutic VL, or e.g., the binding partner can bind to another molecule coupled to or interacting with the therapeutic VL). For example, the combinatorial protein can include a plasma membrane-localized protein (e.g., a retroviral gag protein, a human endogenous retroviral gag protein, or a plekstrin homology domain) coupled to a nucleic acid-binding protein that can bind to a nucleic acid molecule, e.g., an RNA (e.g., mRNA) or DNA. In some cases, the combinatorial protein is suitable for delivery by the lipid-containing particles disclosed herein.

[0151] The plasma membrane-localized proteins disclosed herein can be derived from a virus, human, or any other suitable source. In some cases, the plasma membrane-localized protein is a human endogenous protein.

[0152] In some cases, the combinatorial proteins disclosed herein include a nuclear localization sequence (NLS). In some cases, the NLS facilitates delivery of the combinatorial protein, or a moiety released from the combinatorial protein (e.g., released from the combinatorial protein after cleavage of a cleavable linker), to the nucleus of the target cell. In some cases, the NLS is an endogenous NLS. In some cases, the endogenous NLS naturally exists within a portion of the moiety. In some cases, the NLS is an exogenous NLS. In some cases, the exogenous NLS does not naturally exist within a portion of the moiety. In some cases, the exogenous NLS is engineered to become a part of the moiety.

[0153] In some cases, the combinatorial proteins disclosed herein include at least one NLS sequence, for example, two or more, three or more, four or more, or five or more NLS sequences. In some cases, one or more NLS sequences (two or more, three or more, four or more, or five 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 of the terminus). In some cases, one or more NLS sequences (two or more, three or more, four or more, or five or more NLS sequences) are located at or near the N-terminus of the combinatorial protein (e.g., within 50 amino acids of the terminus). In some cases, one or more NLS sequences (two or more, three or more, four or more, or five or more NLS sequences) are located at or near the C-terminus of the combinatorial protein (e.g., within 50 amino acids of the terminus). In some cases, one or more NLS sequences (three or more, four or more, or five 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 of both). In some cases, an NLS sequence is located at the N-terminus of the combinatorial protein and an NLS sequence is located at the C-terminus.

[0154] In some cases, the freight is a protein and is delivered as part of the combinatorial proteins disclosed herein, for example, operably linked to a structural protein (e.g., a human endogenous retrovirus (HERV) structural protein, or a plasma membrane mobilization domain). In some embodiments, one or more NLS sequences are located at one or both ends of, or near one or both ends of, the freight protein sequence of the combinatorial protein. For example, in some cases, one or more NLS sequences (two or more, three or more, four or more, or five or more NLS sequences) are located at or near the N-terminus and / or C-terminus of the freight protein sequence (e.g., within 50 amino acids of the terminus). In some cases, one or more NLS sequences (two or more, three or more, four or more, or five or more NLS sequences) are located at or near the N-terminus of the freight protein sequence (e.g., within 50 amino acids of the terminus). In some cases, one or more NLS sequences (two or more, three or more, four or more, or five or more NLS sequences) are located at or near the C-terminus of the freight protein sequence (e.g., within 50 amino acids of the terminus). In some cases, one or more NLS sequences (three or more, four or more, or five or more NLS sequences) are located at or near both the N-terminus and C-terminus of the freight protein sequence (e.g., within 50 amino acids of both). In some cases, the NLS sequence is located at the N-terminus of the freight protein sequence and the NLS sequence is located at the C-terminus.

[0155] In some cases, the combinatorial proteins disclosed herein include NLS sequences between 1 and 10 (e.g., NLS sequences between 1 and 9, 1 and 8, 1 and 7, 1 and 6, 1 and 5, 2 and 10, 2 and 9, 2 and 8, 2 and 7, 2 and 6, or 2 and 5). In some cases, the combinatorial protein includes (fused thereto) an NLS sequence between 2 and 5 (e.g., NLS between 2 and 4, or 2 and 3). Examples of NLS sequences include NLS sequences derived from the following: the NLS of SV40 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 of the IBB domain from importin-alpha, RMRIZFKNKGKDT AELRRRRVE V S VELRK AKKDEQILKRRN V (SEQ ID NO: 134); the sequences of the myogenic T protein, VSRKRPRP (SEQ ID NO: 135) and PPKKARED (SEQ ID NO: 136); the sequence of human p53, PQPKKKPL (SEQ ID NO: 137); the sequence of mouse c-abl IV, SALIKKKKKMAP (SEQ ID NO: 138); the sequences of influenza virus NS1, DRLRR and PKQKKRK; the sequence of hepatitis delta antigen, RKLKKKIKKL (SEQ ID NO: 139); the sequence of mouse Mx1 protein, REKKKFLKRR; the sequence of human poly(ADP-ribose) polymerase, KRKGDE VDGVDEV AKKKS KK (SEQ ID NO: 140); and the sequence of the steroid hormone receptor (human) glucocorticoid, RKCLQAGMNLEARKTKK (SEQ ID NO: 141), and sequences having at least 80% identity to the foregoing. In some cases, the NLS includes the amino acid sequence MDSLLMNRRKFLY QFKNVRWAKGRRETYLC (SEQ ID NO: 142).

[0156] 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 la., 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.

[0157] 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 into the cytosol of the target cell as compared to the nucleus.

[0158] In some cases, the combinatorial proteins disclosed herein include at least one NES sequence, for example, two or more, three or more, four or more, or five or more NES sequences. In some cases, one or more NES sequences (two or more, three or more, four or more, or five 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 of the terminus). In some cases, the combinatorial proteins disclosed herein include only one NES sequence. In some cases, the combinatorial proteins disclosed herein include three NES sequences. In some cases, one or more NES sequences (two or more, three or more, four or more, or five or more NES sequences) are located at or near the N-terminus of the combinatorial protein (e.g., within 50 amino acids of the terminus). In some cases, one or more NES sequences (two or more, three or more, four or more, or five or more NES sequences) are located at or near the C-terminus of the combinatorial protein (e.g., within 50 amino acids of the terminus). In some cases, one or more NES sequences (three or more, four or more, or five 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 of both). In some cases, an NES sequence is located at the N-terminus of the combinatorial protein and an NES sequence is located at the C-terminus.

[0159] In some cases, the freight is a protein and is delivered as part of a combinatorial protein disclosed herein, for example, operably linked to a structural protein (e.g., a human endogenous retrovirus structural protein, or a plasma membrane mobilization domain). In some embodiments, one or more NES sequences are located at one or both ends of the freight protein sequence within the combinatorial protein, or near one or both ends. For example, in some cases, one or more NES sequences (two or more, three or more, four or more, or five or more NES sequences) are located at or near the N-terminus and / or C-terminus of the freight protein sequence (e.g., within 50 amino acids of the terminus). In some cases, one or more NES sequences (two or more, three or more, four or more, or five or more NES sequences) are located at or near the N-terminus of the freight protein sequence (e.g., within 50 amino acids of the terminus). In some cases, one or more NES sequences (two or more, three or more, four or more, or five or more NES sequences) are located at or near the C-terminus of the freight protein sequence (e.g., within 50 amino acids of the terminus). In some cases, one or more NES sequences (three or more, four or more, or five or more NES sequences) are located at or near both the N-terminus and C-terminus of the freight protein sequence (e.g., within 50 amino acids of both). In some cases, the NES sequence is located at the N-terminus of the freight protein sequence and the NES sequence is located at the C-terminus. In some cases, the combinatorial protein disclosed herein contains only one NES sequence. In some cases, the combinatorial protein contains only one NES sequence, and the NES sequence is located at or near the N-terminus of the freight protein (e.g., within 50 amino acids of the terminus).

[0160] In some embodiments, the combinatorial protein comprises one NES sequence and two NLS sequences. In some cases of these embodiments, the NES sequence, the NLS sequence, and the fret protein sequence are positioned as follows, in order from the N-terminus to the C-terminus: NES-NLS-fret protein-NLS. In some embodiments, the combinatorial protein comprises two or more NES sequences and two NLS sequences. In some cases of these embodiments, the NES sequence, the NLS sequence, and the fret protein sequence are positioned as follows, in order from the N-terminus to the C-terminus: n×NES (n>=2)-NLS-fret protein-NLS.

[0161] In some cases, the combinatorial proteins disclosed herein comprise NES sequences between 1 and 10 (e.g., NES sequences of 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2-6, or 2-5). In some cases, the combinatorial protein comprises NES sequences between 2 and 5 (e.g., 2-4, or 2-3 NES) (fused thereto).

[0162] In some cases, the NES sequences that can be used in combinatorial proteins include LQLPPLERLTL (SEQ ID NO: 151) derived from the HIV-1 Rev protein, and sequences having at least 80% identity thereto. In some cases, the NSE sequences include LALKLAGLDI (SEQ ID NO: 152) derived from PKIα, and sequences having at least 80% identity thereto. 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. 2012 Sep;23(18):3673-6, each of which is hereby incorporated by reference in its entirety. Any of the NES sequences described in the NES sequence database (NESdb(C); prodata.swmed.edu / LRNes) or (NESbase; services.healthtech.dtu.dk / datasets / NESbase-1.0) can be used in the combinatorial proteins disclosed herein, for example, for the purpose of packaging a freight (e.g., a protein) into a lipid-containing particle, such as a virus-like particle.

[0163] In some cases, a combinatorial protein comprises a cleavable linker between two or more components. For example, a combinatorial protein can comprise a cleavable linker between a frataxin protein sequence and a plasma membrane-localized protein sequence (e.g., a retroviral gag protein sequence). In some cases, the cleavable linker separates the plasma membrane-localized protein sequence from an NLS sequence and / or an NES sequence at its N-terminus or C-terminus. The cleavable linker can separate the frataxin protein sequence from a plasma membrane-localized protein sequence, an NLS sequence, and / or an NES sequence at its N-terminus or C-terminus. The cleavable linker sequences provided herein can be cleavable sequences that are recognized and cleaved by a viral protease, a bacterial protease, or a eukaryotic protease (e.g., a protease derived from a plant, animal, or fungus). 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 a combinatorial protein 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 to the foregoing.

[0164] In some cases, a 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, a 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 without being part of the combinatorial protein.

[0165] In some cases, the combinatorial proteins disclosed herein also include one or more non-cleavable linkers that operably link the components to each other. The non-cleavable linker can be any suitable linker sequence used in combinatorial protein construction, for example, a peptide linker consisting of glycine (Gly) and serine (Ser) residues. In some embodiments, the non-cleavable linker comprises an amino acid sequence selected from the group consisting of (GS)x, (GGS)x, (GGGGS)x, (GGSG)x, and (SGGG)x, where x is an integer from 1 to 50.

[0166] In some cases, the combinatorial protein has the following arrangement of components placed in order from the N-terminus to the C-terminus: [Plasma membrane-localized protein]-[n*NES]-[Cleavable linker]-[m1*NLS]-[Flotillin protein]-[m2*NLS]; [Plasma membrane-localized protein]-[Cleavable linker]-[m1*NLS]-[Flotillin protein]-[m2*NLS]-[n*NES]; [Plasma membrane-localized protein]-[Cleavable linker 1]-[m1*NLS]-[Flotillin protein]-]-[m2*NLS]-[Cleavable linker 2]-[n*NES]; and [Plasma membrane-localized protein]-[Cleavable linker 1]-[m1*NLS]-[Flotillin protein]-[m2*NLS]; [m1*NLS]-[Flotillin protein]-[m2*NLS]-[Cleavable linker]-[n*NES]-[Plasma membrane-localized protein]; [n*NES]-[m1*NLS]-[Flotillin protein]-[m2*NLS]-[Cleavable linker]-[Plasma membrane-localized protein]; [n*NES]-[Cleavable linker 1]-[m1*NLS]-[Flotillin protein]-[m2*NLS]-[Cleavable linker 2]-[Plasma membrane-localized protein]; and [m1*NLS]-[Flotillin protein]-[m2*NLS]-[Cleavable linker]-[Plasma membrane-localized protein] has one of them, where n, m1, and m2 are each integers in the range of 0 to 10, indicating the number of repeats of the respective arrays they refer to. The non-cleavable linker array may or may not be present between any two adjacent components in any of the above arrangements.

[0167] In some cases, the combinatorial protein includes one of the following arrangements in which the components are placed in the order from the N-terminus to the C-terminus, or an arrangement as shown in FIGS. 15-16. In some cases, at least two combinatorial proteins each independently include one of the following arrangements in which the components are placed in the order from the N-terminus to the C-terminus, or an arrangement as shown in FIGS. 15-16. 1. [Plasma membrane-localized protein]-[NES]-[Flate] 2. [Plasma membrane-localized protein]-[NES]-[Flate]-[NES] 3. [Plasma membrane-localized protein]-[Flate]-[NES] 4. [Plasma membrane-localized protein]-[Cleavable linker]-[Flate] 5. [Plasma membrane-localized protein]-[NES]-[Cleavable linker]-[Flate] 6. [Plasma membrane-localized protein]-[NES]-[Cleavable linker]-[Flate]-[NES] 7. [Plasma membrane-localized protein]-[Protease] 8. [PH_AKT]-[NES]-[Cas9 / NLS]-[NES] 9. [PH_AKT]-[NES]-[MMLV cleavage sequence]-[Cas9 / NLS]

Table 3-1-1

Table 3-1-2

Table 3-1-3

[0168] In some embodiments, combinatorial proteins comprising a plasma membrane-localized protein and a heterologous sequence are disclosed herein. In some cases, the plasma membrane-localized protein is selected from the group consisting of 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. 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.

[0169] Plasma membrane-localized protein In some cases, the plasma membrane localization described herein forms the basic structure of the lipid-containing particles disclosed herein. In some cases, the plasma membrane-localized proteins described herein form structural proteins that are at least part of the protein core of the lipid-containing particles. 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 protein can promote the localization and packaging of lipid-containing particles (e.g., virus-like particles) to the plasma membrane by forming a membrane enclosure.

[0170] In some cases, the plasma membrane-localized protein is a viral protein, for example, derived from a virus. In some cases, the plasma membrane-localized protein is a mammalian protein, for example, derived from a mammal such as a human. In some cases, the plasma membrane-localized protein is a human endogenous protein.

[0171] 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 includes a retroviral polyprotein containing one or more of a retroviral gag protein, for example, a matrix (MA) polypeptide, an RNA-binding phosphorylated protein 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, a betaretrovirus, a gammaretrovirus, a deltaretrovirus, an epsilonretrovirus, or a spumavirus. In some cases, the retroviral gag polyprotein is a gag polyprotein of human immunodeficiency virus.

[0172] Examples of plasma membrane-localized proteins include the 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, Hendra 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, homologs thereof, fragments thereof, variants thereof, or any combination of these.

[0173] In some cases, the plasma membrane-localized protein sequence includes a human endogenous retrovirus (HERV) gag protein. In some cases, the plasma membrane-localized protein sequence includes a pleckstrin 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes an amino acid sequence having at least about 99% sequence identity to any one of the sequences in Table 3.

[0174] Membrane fusion / envelope protein The membrane fusion proteins disclosed herein are present on the outer membrane of lipid-containing particles (e.g., inserted into, attached to, or tethered to the lipid layer) and can refer to proteins that facilitate the fusion of lipid-containing particles with a membrane, such as a target cell membrane. In some cases, the membrane fusion protein mediates the tropism of lipid-containing particles to one or more specific cell types, e.g., preferential fusion of lipid-containing particles. In some cases, the membrane fusion protein results in mixing of the lipids in the lipid-containing particle and the lipids in the target cell. In some cases, the lipid-containing particle comprises a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule. Examples of HERV envelope proteins can include those described in Table 2 and Table 2-1.

[0175] 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to 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 to the sequence of the HERV envelope protein listed in Table 2-1.

[0176] 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, e.g., 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 native derivative, a synthetic protein, a fragment thereof, a variant thereof, a protein chimera comprising one or more of the membrane fusion protein or fragment, and any combination thereof.

[0177] The non-immunogenic membrane fusion proteins provided herein may have reduced immunogenicity for a human subject as compared to proteins that are heterologous to the human subject. For example, the non-immunogenic membrane fusion proteins can be humanized to reduce their immunogenicity for a human subject. In some embodiments, the membrane fusion proteins can be modified to reduce immunoreactivity. For example, the membrane fusion proteins can be decorated with molecules that reduce immune interactions, such as PEG, such as those described in Croyle MA, et al., J Virol.2004 Jan;78(2):912-21, which is incorporated herein by reference in its entirety. Thus, in some embodiments, the envelope protein comprises PEG, for example, a PEGylated polypeptide. Amino acid residues in the membrane fusion protein targeted by the immune system can be altered so as not to be recognized by the immune system, as described, for example, in Lech PJ, et al., Virology.2014 Apr;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 (humanized) to resemble an amino acid sequence found in humans. In some embodiments, the protein sequence of the membrane fusion protein is changed to a protein sequence that binds to the MHC complex with lower avidity. In some embodiments, the membrane fusion protein is derived from a virus or organism that does not infect humans (and for which the human has not been vaccinated), thereby increasing the likelihood that the patient's immune system will be tolerant to the membrane fusion protein (e.g., humoral or cell-mediated adaptive immune responses to the membrane fusion protein can be ignored) (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 change immune interactions or to reduce immunoreactivity.

[0178] In some cases, the membrane fusion protein comprises a sequence selected from nipah virus protein F, measles virus F protein, tupaia paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, henipavirus F protein, Morbillivirus F protein, respirovirus F protein, Sendai virus F protein, rubulavirus F protein, or avulavirus F protein, or derivatives thereof.

[0179] In some cases, the membrane fusion protein includes mammalian proteins. Examples of mammalian membrane fusion proteins 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) (such as those 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 (such as those 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 proteins or fragments thereof. In some embodiments, the membrane fusion protein includes curvature-generating proteins, such as epsin 1, dynamin, or proteins containing a BAR domain, such as those described in Kozlov et al., CurrOp StrucBio 2015 2015 Aug;33: 61-67; Zimmerberg et al., Nat Rev Mol Cell Biol.2006 Jan;7(1):9-19; Richard et al., Biochem J. 2011 Dec 1;440(Pt 2): 185-193, each of which is incorporated herein by reference in its entirety.

[0180] In some cases, the membrane fusion protein includes non-mammalian proteins, such as viral membrane fusion proteins. 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 Spodoptera exigua MNPV (SeMNPV) F protein and Lymantria dispar MNPV (LdMNPV), influenza HA, parainfluenza F, HIV Env, Ebola 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, the class II viral membrane fusion protein, such as dengue E glycoprotein, has a β-sheet structural signature that forms an elongated ectodomain that refolds into a trimer of hairpins. In an embodiment, the class II viral membrane fusion protein lacks a central coiled coil. Examples of class II viral membrane fusion proteins that can be used in the VLPs disclosed herein include tick bone encephalitis E (TBEV E), Semliki Forest virus E1 / E2, and membrane fusion proteins derived from Sindbis, rubella virus, and dengue virus. In an embodiment, the class III viral membrane fusion protein, such as vesicular stomatitis virus G glycoprotein, has the structural signatures found in classes I and II combined.In embodiments, class III viral membrane fusion proteins include a helix (e.g., folding the protein back by forming a six-helix bundle similar to class I viral membrane fusion proteins) and three sheets having an amphiphilic membrane fusion protein at its end that associates with 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., glycoprotein B of herpes simplex virus 1 (HSV-1)), Epstein-Barr virus glycoprotein B (EBV gB), Severe acute respiratory syndrome coronavirus G, baculovirus gp64 (e.g., Autographa California multiple nuclear polyhedrosis virus (AcMNPV) gp64), and Borna disease virus (BDV) glycoprotein (BDV G). In embodiments, 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 embodiments, 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).

[0181] Examples of other viral membrane fusion proteins that can be used in the VLPs disclosed herein include viral syncytial 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 to LFA-1 to form lymphocyte syncytia, HIV gp41, HIV gp160, or HIV transcriptional transactivator (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; a fusion protein of vesicular stomatitis virus Indiana strain glycoprotein and rabies virus glycoprotein (FuG-E); modified FuG-E (FuG-E(P440E)); gibbon ape leukemia virus glycoprotein (GaLV); G-type glycoproteins in rabies, Mokola, vesicular stomatitis virus and togavirus; mouse 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; measles virus F and H, HN or G genes; 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; human, bovine and orangutan herpesvirus gB; envelope glycoproteins of Friend murine leukemia virus and Mason-Pfizer monkey virus; mumps virus hemagglutinin neuraminidase, as well as glycoproteins F1 and F2; membrane glycoprotein from Venezuelan equine encephalomyelitis; paramyxovirus F protein; SIV gp160 protein; Ebola virus G protein; or Sendai virus membrane fusion protein, or homologs thereof, fragments thereof, variants thereof, or any combination of these are included.In some cases, the viral membrane fusion protein comprises a measles virus hemagglutinin (HA) protein and / or a measles virus membrane fusion glycoprotein, an influenza virus neuraminidase (NA) protein, a measles virus F protein, an influenza virus HA protein, a Moloney virus MLV-A protein (amphotropic), a Moloney virus MLV-E protein (ecotropic), a baboon endogenous retrovirus (BAEV) glycoprotein or a modified baboon endogenous retrovirus glycoprotein (BaEVTRless), an Ebola virus glycoprotein, a foamy virus membrane fusion protein, or a homolog thereof, a fragment thereof, a variant thereof, or any combination thereof.

[0182] 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 A, the E1 and E2 subunits of the E protein of Togaviridae - CHIV (included together and separately in the complex); the S, E or MN proteins from Cornaviridae - 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 from Phenuviridae - Sindbis virus (included together and separately in the complex); the GP protein from Arenavirida - Lassa virus; the Gn and Gc proteins from Hantaviridae - Hantavirus (included together and separately in the complex); the G protein from Bornaviridae - Borna disease virus; the Gn and Gc proteins from Bunyaviridae - Crimean - Congo hemorrhagic fever virus (included together and separately in the complex); 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 - Vaccinia virus; the S, L or M proteins from Hepatitis D; or the glycoprotein from Hepeviridae - Hepatitis E virus, or homologs thereof, fragments thereof, variants thereof, as well as protein chimeras containing one or more proteins or fragments thereof.

[0183] In some embodiments, the membrane fusion protein is derived from a paramyxovirus. In some embodiments, the membrane fusion protein is Nipah virus protein F, measles virus F protein, tupaia paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, henipavirus F protein, morbillivirus F protein, respirovirus F protein, Sendai virus F protein, rubulavirus F protein, or alphavirus F protein.

[0184] 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 Nos. 6,416,997, 7,329,807, US2017 / 0112773, US2009 / 0202622, and US2004 / 0009604, and International Patent Publications WO2006 / 027202 and WO2020102709, each of which is incorporated herein by reference in its entirety.

[0185] In some embodiments, the membrane fusion protein includes EFF-1, AFF-1, gap junction proteins such as connexins (e.g., Cn43, GAP43, CX43) (DOI: 10.1021 / jacs.6b05191), other tumor junction proteins, homologs thereof, fragments thereof, variants thereof, and protein fusions containing one or more of such proteins or fragments.

[0186] The membrane fusion proteins disclosed herein can be retargeted by mutating amino acid residues in the membrane fusion protein (e.g., hemagglutinin protein). In some embodiments, the envelope protein is mutated randomly. In some embodiments, the envelope protein is mutated rationally. In some embodiments, the envelope protein is subjected to directed evolution.

[0187] The membrane fusion proteins disclosed in this specification can be re-targeted by covalently conjugating them to a targeting moiety. For example, the membrane fusion proteins can be covalently conjugated to a targeting moiety by expressing a chimeric protein that includes an envelope protein linked to the targeting moiety. The target of the targeting moiety includes any peptide (e.g., receptor) presented on the target cell. In some examples, the target is expressed at a higher level on the target cell than on non-target cells.

[0188] The targeting moiety can be selected, for example, 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, exosomes are targeted to brain tissue.

[0189] Specific examples of the targeting moiety include a muscle-specific peptide discovered by phage display for targeting skeletal muscle; 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 ducts and pancreatic epithelium. Alternatively, immunoglobulins and their derivatives such as scFv antibody fragments can be expressed as membrane fusion proteins to target specific antigens such as VEGFR for cancer gene therapy. Alternatively, the natural ligand of the receptor, for example, NGF that binds to NGFR and confers neuron-specific targeting, can be expressed as a membrane fusion protein to confer specificity.

[0190] 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, e.g., sdAb (either VL or VH), nanobody, or camelid VHH domain), an antigen-binding fibronectin type III (Fn3) scaffold, e.g., 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 the target cell, redirecting the membrane fusion activity towards the cells presenting the target of the antibody.

[0191] The targeting moiety can include, for example, humanized antibody molecules, intact IgA, IgG, IgE or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies® etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets 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., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™”); single-chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies®; minibodies; BiTE®; ankyrin repeat proteins or DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; microproteins; Fynomers®; Centyrins®; and KALBITOR®.

[0192] In embodiments, the targeting moiety linked to the membrane fusion protein binds to a cell surface marker on the target cell, such as a protein, glycoprotein, receptor, cell surface ligand, agonist, lipid, sugar, class I transmembrane protein, class II transmembrane protein, or class III transmembrane protein.

[0193] In some cases, the lipid-containing particles (e.g., VLPs, exosomes, or lipid nanoparticles) disclosed herein also present a targeting moiety that is not conjugated to a membrane fusion protein or to any other protein, 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 to the target cell. Virus-like particles

[0194] In some embodiments, compositions, methods, and systems related to virus-like particles that can be utilized to deliver freight to cells are disclosed herein.

[0195] The virus-like particles (VLPs) disclosed herein can include one or more virus-derived proteins, such as structural proteins of the VLP, and envelope proteins. In some cases, the virus-derived proteins are present as part of a combinatorial protein that forms the VLP.

[0196] In some cases, the payload capacity of the VLPs disclosed herein has a payload capacity that 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 greater than that of conventional VLPs. Structural proteins of the VLP

[0197] In some cases, the structural proteins described herein form at least a part of the basic structure of the virus-like particle, such as the capsid that encapsulates the protein core of the VLP. The structural proteins of the virus-like particle can include plasma membrane-localized proteins. In some cases, the plasma membrane-localized proteins described herein also promote self-assembly of the VLP and promote plasma membrane localization and packaging of the virus-like particle, for example, by forming a membrane enclosure. In some cases, the structural proteins described herein promote release of the VLP from the production cells in which the VLP is produced.

[0198] In some cases, the structural proteins of the VLP (e.g., plasma membrane-localized proteins) are viral proteins, such as those derived from a virus. In some cases, the structural proteins of the VLP are mammalian proteins, such as those derived from a mammal, e.g., a human. In some cases, the structural proteins of the VLP are human endogenous proteins.

[0199] 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, a fragment thereof, a variant thereof, or any combination thereof. For example, the structural protein of the VLP (e.g., a plasma membrane-localized protein) includes a retroviral polyprotein containing one or more of a retroviral gag protein, such as a matrix (MA) polypeptide, an RNA-binding phosphorylated protein 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, a betaretrovirus, a gammaretrovirus, a deltaretrovirus, an epsilonretrovirus, or a spumavirus. In some cases, the retroviral gag polyprotein is a gag polyprotein of human immunodeficiency virus.

[0200] Examples of VLP structural proteins (e.g., 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, Hendra 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, homologs thereof, fragments thereof, variants thereof, or any combination of these. Envelope protein

[0201] In some cases, the VLPs disclosed herein include an outer lipid-based membrane (“envelope”). In some cases, the envelope comprises a single layer of lipids. In some cases, the envelope comprises a lipid bilayer. In some cases, the envelope further comprises a membrane fusion protein (also referred to as the “envelope protein” of the VLP) that is inserted into, attached to, or tethered to the lipid layer.

[0202] Envelope proteins can facilitate the fusion of VLPs to membranes, such as the cell membrane. In some cases, envelope proteins mediate the tropism of VLPs, such as the preferential fusion of VLPs, to one or more specific cell types. In some cases, envelope proteins result in the mixing of lipids in the VLPs and lipids in the target cells. The envelope protein can be any of the membrane fusion proteins disclosed above. In some cases, the envelope protein can be a chimeric protein containing the targeting moiety disclosed above.

[0203] In some cases, the envelope protein of the VLP is engineered to pseudotype the VLP for a particular property, such as specific tropism for a selected cell population. In some cases, the envelope protein of the VLP is a viral glycoprotein or a variant thereof, such as a pseudotyping viral glycoprotein, such as a hepatitis B virus (HBV) glycoprotein, a hepatitis C virus (HCV) glycoprotein, a Marburg virus glycoprotein, an Ebola virus glycoprotein, a VSV-G glycoprotein, or a variant thereof, and the target cell is a hepatocyte. In some cases, the envelope protein of the VLP is a pseudotyping viral glycoprotein, for example, the viral glycoprotein is selected from influenza virus hemagglutinin, SARS-CoV glycoprotein, respiratory syncytial virus glycoprotein, human parainfluenza virus glycoprotein, and VSV-G, or a variant thereof, and the target cell is a lung cell. In some cases, the envelope protein of the VLP is a pseudotyping viral glycoprotein, for example, the viral glycoprotein is measles virus hemagglutinin and / or measles virus membrane fusion glycoprotein, or a variant thereof, and the target cell is CD34 +It is a cell. In some cases, the envelope protein of the VLP is a pseudotyped virus glycoprotein. For example, the viral glycoprotein is selected from measles virus hemagglutinin and / or measles virus membrane fusion glycoprotein, HTLV-1 glycoprotein, and VSV-G glycoprotein, or variants thereof, and the target cell is CD8 +It is a T cell. In some cases, the envelope protein of the VLP is a pseudotyped virus glycoprotein. For example, the viral glycoprotein is selected from HIV-1 envelope, HTLV-1 glycoprotein, measles virus hemagglutinin, and VSV-G glycoprotein, or variants thereof, and the target cell is a CD4+ T cell. In some cases, the envelope protein of the VLP is a pseudotyped virus glycoprotein, such as Ross River virus glycoprotein or VSV-G glycoprotein, or variants thereof, and the target cell is a skeletal muscle cell. In some cases, the envelope protein of the VLP is a pseudotyped virus glycoprotein. For example, the viral glycoprotein is selected from Ebola virus glycoprotein, Marburg virus glycoprotein, and VSV-G, or variants thereof, and the target cell is an eye cell (e.g., among retinal cells, photoreceptor cells, etc.). In some cases, the envelope protein of the VLP is a pseudotyped virus glycoprotein. For example, the viral glycoprotein is selected from Ebola virus glycoprotein, Marburg virus glycoprotein, and VSV-G, or variants 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 virus glycoprotein. For example, 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 variants thereof, and 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 includes 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 sequence 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 sequence 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 sequence shown in Table 1.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

[0204] Human endogenous VLP and humanized VLP In some embodiments, virus-like particles with reduced or no immunogenicity in a human subject, such as non-viral human endogenous virus-like particles (heVLPs), or humanized VLP comprising humanized structural proteins (e.g., humanized viral structural proteins) or humanized envelope proteins (e.g., humanized viral envelope proteins) are provided herein. In some embodiments, the humanized envelope proteins disclosed herein are derived from viral envelope proteins, for example, 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, for example, by mutating or engineering a viral structural protein, such as a retroviral gag protein, such that the protein is non-immunogenic to humans.

[0205] Unlike virus-like particles according to some embodiments of the present disclosure, the heVLPs or humanized VLPs described herein can package protein freight by integrating all production DNA into the genomic DNA of the production cell line. Once the cell line is generated, protein delivery heVLPs can be produced in a constitutive or inducible manner. The protein freight is packaged into heVLPs by fusing a selected human endogenous GAG protein or other plasma membrane-localized protein (also referred to herein as a "plasma membrane recruitment domain") to the protein-based freight.

[0206] The heVLP or humanized VLP systems described herein may be simpler, more efficient and safer than conventional, artificially induced lipid / gold nanoparticle and virus particle-based delivery systems because the heVLPs or humanized VLPs are composed of human-derived or humanized components. The freight inside the particle may or may not be of human origin, but the heVLPs or humanized VLPs are of human origin or contain human endogenous or synthetic non-immunogenic components.

[0207] The "synthetic" component has been demonstrated to be non-immunostimulatory and can be used to enhance the targeting and cellular uptake of heVLPs. It includes surface scFvs / nanobodies / darpin peptides. This means that the outer surface of the particles lacks components that can be significantly immunostimulatory, and the lack can minimize the immunogenicity and antibody neutralization of these particles.

[0208] In some cases, except for the freight, the heVLPs provided herein do not contain exogenous viral components unique to other VLPs, which represents a significant new advance in the art. Additionally, heVLPs can utilize (but do not require) chemical-based dimerizers, and heVLPs can package and deliver therapeutic or diagnostic agents, including freight molecules that include combinations of biomolecules and chemical compounds, such as special single-stranded and / or double-stranded DNA molecules (e.g., plasmids, minicircles, covalently closed circular DNA, AAV DNA, episomes, bacteriophage DNA, homologous recombination 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 splicing RNA, microRNA, small interfering RNA, small hairpin RNA, piwi-interacting RNA, toehold switch RNA, RNA that can be bound by RNA-binding proteins, bacteriophage RNA, RNA containing internal ribosome entry sites, etc.), proteins, chemical compounds and / or molecules (e.g., small molecules), and the freight (e.g., AAV particles) listed above.

[0209] The heVLPs described herein differ from conventional retroviral particles, virus-like particles (VLPs), exosomes, and other previously described extracellular vesicles that can be loaded with freight in that the heVLPs can be produced, at least, by the strategic overexpression of human-derived components in human cells, that the heVLPs have an enormous diversity of possible freight and loading strategies, that there are no constraints limiting DNA / RNA length in the heVLPs, that the heVLPs lack proteins derived from pol and exogenous gag, and that the heVLPs have a unique cell entry mechanism.

[0210] Compositions and methods for freight delivery are described herein that can be used with a wide variety of proteins and nucleic acid molecules, including genome editing, epigenome modulation, transcriptome editing, and proteome modulation reagents, which are applicable to the treatment of many diseases.

[0211] Provided herein are engineered heVLPs comprising a membrane containing a phospholipid bilayer having on the outside one or more HERV-derived ENV / glycoproteins (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 exogenous envelope proteins; and within the core of the heVLP inside the membrane (e.g., within a protein core sealed by the phospholipid bilayer), a human endogenous GAG protein, other plasma membrane-localized proteins (e.g., as shown in Table 3), and / or a biomolecule / chemical freight.

[0212] In some cases, the lipid-containing particles (e.g., VLPs) provided herein It includes a plasma membrane-localized protein that is a PH domain or a variant thereof derived from phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDPK1), disk and actin-binding protein 1 (Daap1), general receptor for phosphoinositides 1 (Grp1), oxysterol-binding protein 1-Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), phosphatidylinositol 4-phosphate adapter protein 1 (FAPP1), ceramide transport 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). 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 or a variant thereof 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. In some cases, the plasma membrane-localized protein includes a membrane protein selected from the group consisting of CD9, CD47, CD63, and CD81 and their transmembrane domains. In some cases, the plasma membrane-localized protein includes a membrane protein selected from the group consisting of 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 mobilizing protein including Arc, human Arc, endogenous retroviral gag protein, or human endogenous retroviral gag protein. In some cases, the plasma membrane-localized protein includes any one of the sequences in Table 3.

[0213] In one aspect, provided herein are humanized VLPs comprising a membrane comprising a phospholipid bilayer having on the outside 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 exogenous envelope proteins; and viral structural proteins (e.g., retroviral gag proteins) inside the membrane (e.g., in a protein core sealed by the phospholipid bilayer).

[0214] In one aspect, provided herein are humanized VLPs comprising a membrane comprising a phospholipid bilayer having one or more viral envelope proteins disclosed herein; and human endogenous GAG proteins, other plasma membrane-localized proteins, and / or biomolecule / chemical freight disposed inside the core of the heVLP (e.g., in a protein core sealed by the phospholipid bilayer).

[0215] The freight may be fused to human endogenous GAG or other plasma membrane-localized proteins. In some cases, the freight is not fused to either human endogenous GAG or other plasma membrane-localized proteins. In some cases, the heVLP or humanized VLP does not contain 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 by the gag proteins encoded in the human genome or consensus sequences derived from gag proteins found in the human genome. The human-derived GAG or other plasma membrane-localized proteins fused to the freight may be overexpressed from an exogenous source, such as a plasmid or a stably integrated transgene, in heVLP-producing cells.

[0216] Human endogenous GAG proteins and human pleckstrin homology (PH) domains can localize to biological membranes. The PH domain can interact with phosphatidylinositol lipids and proteins within the biological membrane, such as PIP2, PIP3, the βγ subunits of GPCRs, and PKC. However, in addition to localization to the phospholipid bilayer, human endogenous GAG proteins can also drive budding and particle formation. This dual functionality of human endogenous GAGs may enable the packaging and budding / particle formation of virions. One such human endogenous GAG protein used for this purpose is the human Arc protein, which can be fused to protein-based virions to recruit virions to the cytosolic 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 virions via a variable-length polypeptide linker, regardless of the position(s) of one or more nuclear localization sequences (NLSs) within the virion. In some cases, the linker between the protein-based virion and the human endogenous GAG phospholipid bilayer recruitment domain is a polypeptide linker 5-20, such as 8-12, such as 10 amino acids in length, composed mainly of glycine and serine. [Table 2] [Table 2-1-1] [Table 2-1-2] [Table 2-1-3] [Table 2-1-4] [Table 2-1-5] [Table 2-1-6]

Table 3-1

Table 3-2

Table 3-3

[0217] Human endogenous GAG or other phospholipid bilayer trafficking domains can localize flotillin to the phospholipid bilayer, and this protein flotillin is packaged within heVLPs or humanized VLPs that bud from the producing cells into the extracellular space. The use of these human endogenous GAGs and other phospholipid bilayer trafficking domains is novel and unique in that these human endogenous GAGs and other proteins can facilitate the localization of flotillin to the cytoplasmic side surface of the plasma membrane within heVLP or humanized VLP producing cells. The use of these human endogenous GAGs and other phospholipid bilayer trafficking domains can enable flotillin to localize to the nucleus of transduced cells without using exogenous retroviral GAGs nor chemical- and / or light-based dimerization systems.

[0218] heVLPs can also package and deliver a combination of DNA and RNA when the heVLPs are produced by transient transfection of a production cell line. The DNA transfected into the cells will have size-dependent mobility such that a fraction of the transfected DNA will remain in the cytosol while another fraction of the transfected DNA will localize to the nucleus. A fraction of the transfected cells in the nucleus can express the components to make heVLPs, and other fractions in the cytosol / near the plasma membrane will be encapsulated and delivered by the heVLPs.

[0219] Combinations of exogenous DNA, exogenous RNA, and proteins (exogenous and / or endogenous proteins) will be referred to as type 1 frates (T1 heVLPs), exogenous RNA and proteins (exogenous and / or endogenous proteins) will be referred to as type 2 frates (T2 heVLPs), combinations of exogenous DNA and proteins (exogenous and / or endogenous proteins) will be referred to as type 3 frates (T3 heVLPs), and proteins (exogenous and / or endogenous proteins) will be referred to as type 4 frates (T4 heVLPs). Thus, T1 contains DNA, RNA, + / - exogenous proteins, T2 contains RNA + / - exogenous proteins, T3 contains DNA + / - exogenous proteins, and T4 is a particle with or without an exogenous protein frate. Therefore, T4 without exogenous proteins is considered an "empty particle" because it has no "exogenous frate". An "exogenous frate" is a frate that is not endogenous to the producer cell that can be packaged and / or incorporated into the heVLP. In addition, T1-T4 heVLPs can package exogenous chemical molecules in addition to the types of frates present in the T1-T4 heVLPs. For example, RNA in this context can be single-guide RNA (sgRNA) that encodes a frate, clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA), and / or mRNA. As used herein, "small molecule" refers to a small organic or inorganic molecule with a molecular weight of less than about 3,000 Daltons. Generally, small molecules useful in the present 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., about 100 to about 3,000 Da, about 100 to about 2500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or between about 100 and about 250 Da).

[0220] The fret is limited by the diameter of the particles and, for example, in some embodiments, is in the range of 150 nm to 500 nm.

[0221] 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 hereby incorporated by reference in its entirety.

[0222] In some embodiments, for the efficient trafficking of the fret to heVLPs or humanized VLPs, the fret comprises a covalent or non-covalent linker to a human endogenous GAG or other plasma membrane trafficking domain, such as those shown in Table 3. For example, a covalent linker can include a direct protein-protein chimera generated from a single reading frame, an intein capable of forming a peptide bond, and other proteins capable of forming a covalent linker during R-group and / or RNA splicing. For example, non-covalent linkers include DNA / DNA, DNA / RNA, and / or RNA / RNA hybrids (nucleobase pairing with other nucleic acids by hydrogen bond interactions); protein domains that dimerize or multimerize with or without the need for chemical compounds / molecules that induce protein-protein binding (e.g., DmrA / DmrB / DmrC (Takara Bio), FKBP / FRB, dDZF, and leucine zipper); single-chain variable fragments; nanobodies; affibodies; proteins that bind DNA and / or RNA; proteins with quaternary structure interactions; optogenetic protein domains that can dimerize or multimerize in the presence of a specific light wavelength; and / or split proteins that reconstitute spontaneously.

[0223] In some embodiments, the fret comprises a fusion with a dimerization domain or protein-protein binding domain that may or may not require a molecule to induce dimerization or protein-protein binding.

[0224] 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 full or active peptide domain of human CD47 can be incorporated onto the heVLP surface to reduce immunogenicity. Examples of AAV proteins included herein are AAV REP52, REP78, and VP1-3. The capsid site where the protein can be inserted is T138, counted starting from the VP1 amino acid. For example, a dimerization domain can be inserted at this location of the capsid. Examples of dimerization domains included herein that may or may not require small molecule inducers are dDZF1, dDZF2, DmrA (Takara Bio), DmrB (Takara Bio), DmrC (Takara Bio), FKBP, FRB, GCN4 scFv, 10× / 24× GCN4, GFP nanobody and GFP. Examples of split inteins included herein are Npu DnaE, Cfa, Vma, and Ssp DnaE. Examples of other split proteins that together make a covalent bond included herein are Spy Tag and Spy Catcher. 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 multimerize as a result of quaternary structure included herein are E. 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.

[0225] In another embodiment, the T1-T4 heVLPs produced and isolated can be loaded with biomolecules or chemical molecule freight by utilizing nucleofection, electroporation, lipid, polymer or CaCl2 transfection, sonication, freeze-thaw, incubation at various temperatures, and / or heat shock of the purified particles mixed with the freight. These techniques are adapted from those used to load exosomes with freight for therapeutic or research applications. For example, 100 ug of heVLP or humanized VLP can be resuspended in 50 mM trehalose in 200 - 450 ul of PBS, mixed with the freight at the desired concentration, and electroporated at 0.200 kV and 125 uF in a 0.4 cm cuvette (GenePulser II Electroporation System with Capacitance Extender, Bio-Rad, Hercules, CA, USA).

[0226] In some embodiments, heVLPs or humanized VLPs are recovered from the cell culture media supernatant 36 to 48 hours after transfection, or when the heVLPs or humanized VLPs in the media of the production cells reach their maximum concentration (the production cells secrete the particles into the media at several time points, and the particle concentration in the media becomes optimal for particle recovery). 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 polyvinylidene fluoride hydrophilic membrane with a pore size of 0.45 μm (Millipore Millex-HV) or a mixed cellulose ester hydrophilic membrane with a pore size of 0.8 μm (Millipore Millex-AA) to remove particles larger than, for example, 1 μm. After filtration, the supernatant can be further purified and concentrated by using, for example, ultracentrifugation at a speed of 80,000 to 100,000×g at a temperature between 1°C and 5°C for 1 to 2 hours or at a speed of 8,000 to 15,000 g at a temperature between 1°C and 5°C for 10 to 16 hours. After this centrifugation step, the heVLPs or humanized VLPs are concentrated in the form of a centrifugal precipitate (pellet), which can be resuspended to a desired concentration, mixed with a transduction enhancement reagent, subjected to buffer exchange, or used as is. In some embodiments, the heVLP-containing supernatant or humanized VLP-containing supernatant can be filtered, precipitated, centrifuged, and resuspended in 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.

[0227] The purified particles are stable and can be stored at 4°C for up to 1 week or at -80°C for several years without apparent loss of activity.

[0228] Preferably, the heVLP or humanized VLP is resuspended or undergoes buffer exchange to suspend the particles in a suitable carrier. In some embodiments, the buffer exchange can be performed by ultrafiltration (Sartorius Vivaspin 500 MWCO 100,000). Exosome

[0229] In some aspects, the lipid-containing particles disclosed herein are exosomes. In aspects, compositions, methods, and systems related to exosomes that can be utilized to deliver cargo to cells are disclosed herein. The term "exosome" as used herein can refer to small membrane-bound vesicles (30 - 100 nm) of endosomal origin. In some cases, exosomes are released into the extracellular environment after membrane fusion of multivesicular bodies 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.

[0230] Source cells according to the present disclosure can be selected from a wide range of cells, such as mesenchymal stem or stromal cells or fibroblasts (which can be obtained, for example, from bone marrow, adipose tissue, Wharton's jelly, peripartum tissue, dental pulp, umbilical cord blood, skin tissue, etc.), amniotic cells, more specifically amniotic epithelial cells, and myeloid suppressor cells. Generally, both primary cells and cell lines are suitable sources of exosomes. By way of example, some 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, lung, GI tract, urinary tract, etc., dendritic cells (DC) or other cells from the hematopoietic system, such as macrophages, monocytes, B or T cells, NK cells, neutrophils, eosinophils, mast cells or basophils, erythrocytes or erythrocyte progenitor cells, platelets and megakaryocytes, etc., cells of different origins, such as placenta-derived cells (e.g., decidual placenta cells), syncytiotrophoblasts and amniotic epithelial cells, etc., and cells from the CNS and PNS, such as microglia, astrocytes, oligodendrocytes and Schwann cells, ependymal cells and neurons, etc., adipocytes from brown and white adipose, muscle cells of both smooth muscle origin and skeletal muscle origin, and cardiomyocytes. Generally, exosomes can be derived from essentially any cell source, whether a primary cell source or a cell line. Exosome source cells can be any embryonic, fetal and adult somatic stem cell type, including induced pluripotent stem cells (iPSCs) and other stem or progenitor cells obtained by any method. When treating neurological diseases, it may be contemplated to utilize, for example, primary neurons, astrocytes, oligodendrocytes, microglia, and neural progenitor cells as source cells. The source cells can in fact be allogeneic, autologous, or even xenogeneic to the patient to be treated, i.e., the cells can be from the patient themselves or from unrelated compatible or incompatible donors. In certain situations, allogeneic cells may be preferred from a medical perspective as they can provide an immunomodulatory effect that in some cases cannot be obtained from the patient's own cells suffering from a particular indication.

[0231] In some cases, exosomes are produced by many different cell types, including immune cells such as B lymphocytes, T lymphocytes, dendritic cells (DCs), and mast cells. In some cases, exosomes are also produced by, for example, glioma cells, platelets, reticulocytes, neurons, intestinal epithelial cells, and tumor cells. In some cases, exosomes for use 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.

[0232] In some cases, exosomes are derived from immature DCs. In some cases, exosomes produced from immature DCs do not express MHC-II, MHC-I, or CD86. Therefore, such exosomes do not significantly stimulate naive T cells and cannot induce a response in a mixed lymphocyte reaction. Thus, exosomes produced from immature dendritic cells can be an ideal candidate for use in the delivery of freight, such as therapeutic freight.

[0233] In some cases, exosomes are obtained from any autologous patient-derived, allogeneic haplotype-matched, or allogeneic stem cells in order to reduce or avoid the generation of an immune response in the patient to whom the exosomes are delivered. Any cell that produces exosomes can be utilized for this particular purpose.

[0234] 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 the isolation of exosomes from cell culture media or tissue supernatants.

[0235] Exosomes produced from cells can be collected from the culture medium 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., low-speed (<20000g) centrifugation to pellet larger particles, followed by high-speed (>100000g) centrifugation to pellet exosomes; size filtration using a suitable filter (e.g., a 0.22 μm filter); gradient ultracentrifugation (e.g., using a sucrose gradient); or a combination of these methods.

[0236] In some cases, exosomes are loaded with a freight, e.g., a therapeutic freight, e.g., a protein, a nucleic acid molecule, or a small molecule. In some cases, exosomes are prepared and then loaded with the desired therapeutic freight for delivery.

[0237] In some embodiments, the exosomes disclosed herein are engineered to target a desired cell type or tissue. 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 target cell. In some cases, the targeting moiety is a peptide expressed as a chimeric protein with a transmembrane protein that can be expressed on the surface of the exosome.

[0238] In some cases, exosomes are targeted to a specific cell type or tissue by expressing targeting moieties such as peptides on their surface. Suitable peptides bind to cell surface moieties such as receptors found on the cell surface of the target cells or their ligands. 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 peptide targeting moiety can be less than 100 amino acids in length, such as less than 50 amino acids in length, less than 30 amino acids in length, down to a minimum length of 10, 5, or 3 amino acids.

[0239] The targeting moiety can be selected, for example, 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 exosomes are targeted to brain tissue.

[0240] Specific examples of targeting moieties include muscle-specific peptides discovered by phage display for targeting skeletal muscle; 29-amino acid fragments of the rabies virus glycoprotein that bind to the acetylcholine receptor, or fragments of nerve growth factor that target its receptor to target neurons; and secretin peptides that bind to the secretin receptor that can be used to target bile ducts and pancreatic epithelium. Alternatively, immunoglobulins, and derivatives thereof including scFv antibody fragments, can be expressed as membrane fusion proteins to target specific antigens such as VEGFR for cancer gene therapy. Alternatively, the natural ligand of the receptor, for example, NGF that binds to NGFR and confers neuron-specific targeting, can be expressed as a membrane fusion protein to confer specificity.

[0241] By expressing the peptide targeting moiety as a membrane fusion protein with an exosome transmembrane protein, it can be expressed on the surface of exosomes. It is known that several proteins are associated with exosomes, i.e., they are incorporated into exosomes when exosomes are formed. In some cases, the targeting moiety includes or is derived from a transmembrane protein. Examples include Lamp-1, Lamp-2, CD13, CD86, flotillin, syntaxin-3, CD2, CD36, CD40, CD40L, CD41a, CD44, CD45, ICAM-1, integrin alpha4, LiCAM, LFA-1, Mac-1 alpha and beta, Vti-1A and B, CD3 epsilon and zeta, CD9, CD18, CD37, CD53, CD63, CD81, CD82, CXCR4, FcR, GluR2 / 3, HLA-DM (MHC II), immunoglobulin, MHC-I or MHC-II components, TCR beta, and tetraspanin. In a particularly preferred embodiment of the present disclosure, the transmembrane protein is selected from Lamp-1, Lamp-2, CD13, CD86, flotillin, syntaxin-3. In some cases, the targeting moiety includes or is derived from a variant, modified, altered, or derivatized form of the amino acid sequence of the proteins discussed above. It will be understood that such variants, modifications, alterations, or derivatized forms of the polypeptide as described herein are subject to the requirement of retaining any further activity or characteristics such that the polypeptide may be specified in subsequent sections of the present disclosure.

[0242] 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, e.g., sdAb (either VL or VH), nanobody, or camelid VHH domain), an antigen-binding fibronectin type III (Fn3) scaffold, e.g., fibronectin polypeptide minibody, ligand, cytokine, chemokine, or 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., 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 the target cell, redirecting the fusion activity towards the cell presenting the target of the antibody.

[0243] The targeting moiety can include, for example, humanized antibody molecules, intact IgA, IgG, IgE or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies® etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single-chain Fv; polypeptide-Fc chimeras; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); camelized animal antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™”); single-chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies®; minibodies; BiTE®; ankyrin repeat proteins or DARPINs®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; microproteins; Fynomers®; Centyrins®; and KALBITOR®.

[0244] In embodiments, the targeting moiety linked to the membrane protein binds to a cell surface marker on the target cell, such as a protein, glycoprotein, receptor, cell surface ligand, agonist, lipid, sugar, class I transmembrane protein, class II transmembrane protein, or class III transmembrane protein.

[0245] 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 cells used to produce exosomes. Expression of this membrane fusion protein in the cells enables incorporation of the membrane fusion protein into exosomes as the exosomes are produced from the cells.

[0246] In some cases, the targeting moieties disclosed herein that are applicable to exosomes can also be used with other lipid-containing particles disclosed herein, such as virus-like particles, lipid nanoparticles, and proteolipid vehicles.

[0247] For example, a polynucleotide construct expressing a membrane fusion protein, such as a DNA plasmid, is transfected into cells. Any suitable method can be used for introducing the polynucleotide construct into the cells. The polynucleotide construct contains a promoter sequence suitable for expressing the encoded membrane fusion protein in the cells. A signal peptide sequence is also included to incorporate the protein into the membrane of the endoplasmic reticulum when the endoplasmic reticulum is produced. Thus, subsequently, the membrane protein is incorporated into exosomes after being transported to the exosome / lysosome compartment. The signal sequence can include the signal peptide sequence of an exosome transmembrane protein.

[0248] In some cases, exosomes produced from cells can be collected from the culture medium 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., low-speed (<20000g) centrifugation to pellet larger particles, followed by high-speed (>100000g) centrifugation to pellet exosomes; size filtration using a suitable filter (e.g., a 0.22 μm filter); gradient ultracentrifugation (e.g., using a sucrose gradient); or a combination of these methods.

[0249] In some cases, it is not necessary to include a specific targeting moiety in the exosome. For example, the exosome can be administered directly to the site in need of treatment. Alternatively, for example, when the exosome contains genetic material encoding an immunogen, in some cases there is no need to specifically target a particular site, and delivery, for example, by intradermal or intramuscular delivery, can sufficiently generate the desired immune response without targeting the exosome to any specific cell type. In some cases, no targeting moiety is included on the surface of the exosome. However, the exosome is selected such that it is more likely to target a specific tissue type. For example, exosomes derived from different cells can have a natural affinity for specific cell subtypes as required by their physiological functions, for example, the well-established affinity of exosomes derived from mature dendritic cells for T cells. This affinity can be utilized to specifically deliver the above-mentioned freight to the tissue.

[0250] In some cases, exosomes are produced from cells that have been modified to express a chimeric polypeptide receptor, such as a chimeric antigen receptor (CAR). In some cases, exosomes are produced from cells that have been genetically modified to produce a chimeric polypeptide receptor comprising (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 by the intracellular transcription factor of at least one polynucleotide encoding a gene product comprising at least one exosome polypeptide. In some cases, the gene product further comprises a protein of interest, such as an antibody, single-chain antibody or any other antibody derivative, bispecific T cell engager (BiTE), receptor, cytokine, such as interleukin, enzyme, such as caspase, granzyme, Cas, Cas9, checkpoint inhibitor, costimulation inhibitor, RNA-binding protein, membrane transporter, such as NPC-1, splicing factor, protein associated with intracellular organelles, lysosomal enzyme, transcription factor, mitochondrial protein, intracellular protein, antiviral protein, 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 contain an RNA freight molecule selected from the group consisting of 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 (DC), macrophages, monocytes, neutrophils, epithelial cells, endothelial cells, microglial cells, astrocytes, neurons, stem cells, bone marrow-derived mesenchymal stromal cells, Wharton's jelly-derived MSC, or any other cell type.In some cases, the extracellular recognition domain of the chimeric polypeptide receptor is an antibody, antibody derivative, single-chain fragment, single-chain antibody, nanobody, peptide, receptor ligand, adhesion molecule, receptor, interleukin receptor, 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, from the N-terminus to the C-terminus and by covalent linkage, (i) an extracellular recognition domain not naturally present in the Notch receptor polypeptide; (ii) a Notch regulatory region comprising a transmembrane domain containing Lin 12-Notch repeats, an S2 proteolytic cleavage site, and an S3 proteolytic cleavage site; (iii) a chimeric Notch polypeptide comprising an intracellular transcription factor 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 an intracellular transcription factor that activates transcription of the polynucleotide. In some cases, the Notch regulatory region further comprises a heterodimerization domain containing an S2 proteolytic cleavage site. In some cases, the S1 proteolytic cleavage site is a furin-like protease cleavage site comprising 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 responsive to the transcription factor, which is operably linked to the coding sequence. 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.

[0251] In some cases, loading of protein freight onto exosomes disclosed herein is achieved by expressing a three-domain polypeptide construct in the source cell from which the exosomes are produced. 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. Design of the three-domain polypeptide construct can enable efficient loading of the POI onto exosomes and also drive increased production of exosomes from the source cell.

[0252] 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 wide variety of species and can also present relatively different mechanisms of action (e.g., it can be a heterodimerization domain, or it can be a homotrimerization domain, or a homopentamer domain, etc.). In some cases, the multimerization domain is a homomultimerization domain if they can enable a simple design of the membrane fusion protein and support the controlled loading of one single type of membrane fusion polypeptide construct (rather than multiple membrane fusion constructs) onto the exosomes. The multimerization domain can be any of a dimerization domain, a trimerization domain, a tetramerization domain, or essentially any higher-order multimerization domain, provided that the domain can facilitate 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 S. cerevisiae, the retro leucine zipper homodimerization domain of GCN4 from S. cerevisiae, the Fold-on homodimerization domain of fibritin (from T4 bacteriophage), the fragment X heteromerization domain of a phosphorylated protein (from human respiratory syncytial virus A), the human alpha helix coiled coil oligomerization domain of the collagen superfamily, the leucine zipper homodimerization domain of Fos and Jun (human), the transmembrane homopentamer domain of cardiac phospholamban (human), the homodimerization domain of parathyroid hormone (human), the transmembrane homodimer domain of glycophorin A (human), the trimerization domain of Gp41 (from HIV), the C-terminal homodimer domain of oncoprotein E7 (from HPV 45), and the EVH2 homotetramer domain of a vasodilator-stimulated phosphorylated protein (human), the mitochondrial antiviral signaling protein CARD filament, and / or any combination thereof.

[0253] The multimerization domain can be placed at several different positions in the polypeptide construct. For example, the multimerization domain can be placed between the POI sequence and the exosome sorting domain sequence, within or adjacent to the exosome sorting domain sequence, and / or within or adjacent to the POI sequence. Overall, the design of the 3-domain polypeptide construct (with respect to both the selection of the multimerization domain and its position in the construct, and with respect to the selection of the exosome sorting domain and its position in the construct) can play a role in determining the position of the polypeptide within the exosome that it ultimately ends up in after production in the exosome source cell. For example, by selecting a tetraspanin exosome sorting protein (e.g., CD9, CD63 or CD81) or any other exosome membrane protein (e.g., Lamp2b), it is possible to enrich the POI on the exosome surface. Conversely, by selecting an exosome sorting protein that may be present in the exosome protein core, such as ALIX or syntenin, it becomes possible to enrich the polypeptide construct (and thus the POI) essentially inside the exosome. Inevitably, the polypeptide construct can be present simultaneously on the outside and inside of the exosome, as well as within the exosome membrane. Further, in a preferred embodiment, the membrane fusion polypeptide construct can include various types of linkers between 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 containing the amino acids glycine and serine, or any other suitable linker domain of a type that ensures that the activities of different domains are not restricted when the different domains are present in the membrane fusion polypeptide construct.

[0254] The 3-domain membrane fusion polypeptide construct according to the present disclosure can be described schematically as follows (the following notations should not be construed as indicating any C- and / or N-terminal direction, but are for illustrative purposes only):

[0255] POI-Multimerization Domain-Exosome Sorting Domain The exosome sorting domain of the present disclosure can be selected from any one of the following proteins: CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, syntenin-1, syntenin-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, CD3 epsilon, CD3 zeta, 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-1 alpha, Mac-1 beta, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B, and any combination thereof.

[0256] In some cases, the exosomes are loaded using a cell membrane permeable peptide such as those described in US Patent Publication No. US20190388347, which is hereby incorporated by reference in its entirety.

[0257] Examples of exosomes, source cells from which exosomes are produced, freight that can be delivered by exosomes, methods of loading freight onto exosomes, and methods of producing exosomes include those described in U.S. Patent Publications Nos. US20070298118, US20180177727, US20200062813, US20200206360, US20200023012, US20160137716, US20170173113, US20130053426, US20190167810, US20190388347, US20190224331, US20160137716, US20210188903, US20210069254, and US20200407418, each of which is hereby incorporated by reference in its entirety. Lipid nanoparticles or proteolipid vehicles

[0258] In some aspects, compositions, methods, and systems related to lipid nanoparticles that can be utilized to deliver freight to cells are disclosed herein. In some aspects, compositions, methods, and systems related to proteolipid vehicles that can be utilized to deliver freight to cells are disclosed herein.

[0259] Lipid nanoparticles can provide a biocompatible and biodegradable delivery system for the therapeutic phyllosilicates disclosed herein. In some cases, the lipid nanoparticles disclosed herein include nanostructured lipid carriers (NLCs), polymer nanoparticles (PNPs), or lipid-polymer nanoparticles (PLNs). NLCs are modified solid lipid nanoparticles (SLNs) that retain the characteristics 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 drug release control. Lipid-polymer nanoparticles (PLNs), a new type of carrier that combines liposomes and polymers, can also be used. These nanoparticles have the complementary advantages of PNPs and liposomes. PLNs are composed of a core-shell structure, where the polymer core provides a stable structure and the phospholipid shell provides good biocompatibility. Therefore, the two components increase the rate of drug encapsulation efficiency, facilitate surface modification, and prevent the leakage of water-soluble drugs.

[0260] Examples of lipid nanoparticles disclosed herein include those described in JA Zuris et al., Nat Biotechnol. 2014 Oct 30;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. Phyllosilicate

[0261] The freight can include a therapeutic freight and / or a binding partner of the therapeutic freight. As used herein, "freight" can refer to a chemical substance, such as a small molecule compound, a combination of DNA, RNA, and protein, a combination of RNA and protein, a combination of DNA and protein, or, for example, a protein for therapeutic or diagnostic use or for the application of genome editing, epigenome modulation, and / or transcriptome modulation. In addition, endogenous RNA and protein from producer 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 can package and deliver a variety of freights, such as biomolecules including nucleic acids (DNA, RNA) or proteins, chemical compounds including small molecules and / or other molecules, and any combination thereof, to eukaryotic cells. In some cases, the term "freight" is used synonymously with "cargo".

[0262] In some embodiments, the freight contained in and thereby to be delivered by the lipid-containing particles disclosed herein is a polypeptide, such as 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., zinc finger nuclease (ZFN), transcription activator-like nuclease (TALEN), Cas9, or a homolog thereof), a recombinase, 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, or any combination thereof. In some embodiments, the freight contained in the lipid-containing particles disclosed herein includes a protein that targets a protein in a cell for degradation. In some cases, the freight contained in the lipid-containing particles disclosed herein includes a chimeric antigen receptor (CAR), an antibody, a T cell receptor, or a functional fragment thereof, or any combination thereof.

[0263] In some embodiments, the freight contained in and thereby to be delivered by the lipid-containing particles disclosed herein is a polynucleotide, such as a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule. In some cases, the polynucleotide encodes a polypeptide such as those described in the paragraph above. In some cases, the polynucleotide includes the napR / DNAbp programming nucleic acid molecule described below.

[0264] In some embodiments, the freight contained within and thereby 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 freight may comprise an RNP complex formed by a nucleic acid programmable R / DNA (napR / DNAbp) binding protein and a napR / DNAbp programming nucleic acid molecule, such as a Cas protein and a guide RNA, as described below.

[0265] In some embodiments, the freight contained within and thereby delivered by the lipid-containing particles disclosed herein comprises other therapeutic molecules, such as ribozymes, aptamers, aptazymes, peptides, oligonucleotides, antibody mimetics, peptidomimetics, antibody-drug conjugates, antibiotics, carbohydrates, ribosomes, mitochondria, and small molecule compounds.

[0266] In some embodiments, the freight contained in and thereby delivered by the lipid-containing particles disclosed herein is a polypeptide, such as 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., zinc finger nuclease, transcription activator-like nuclease (TALEN), cas9 and homologs thereof), a recombinase, and any combination thereof. In some embodiments, the protein targets a protein in the cell for degradation. In some embodiments, the protein targets a protein in the cell for degradation by localizing 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 or chimeric protein.

[0267] In some cases, the freight contained in and thus to be delivered by the lipid-containing particles disclosed herein is 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 target tissue or organ or cell type; an antibody, an intrabod, 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 alpha-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., their 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 the RVG peptide, the VSV-G peptide, the p-selectin binding peptide, or the e-selectin binding peptide; a cell-penetrating peptide (CPP) (e.g., Tat, penetratin, TP10, CADY); or a tumor suppressor.

[0268] In some cases, the freight contained in and thus to be delivered by the lipid-containing particles disclosed herein includes an immunogenic molecule, such as a vaccine. The vaccine can be a peptide antigen, RNA (e.g., mRNA or circRNA), DNA (e.g., a DNA molecule encoding an antigen). The freight can also include an adjuvant that enhances the immunogenicity of the vaccine composition.

[0269] In some cases, the freight is a protein loaded onto the lipid-containing particles that functions to bind to another freight molecule (e.g., a nucleic acid molecule, a protein, an RNP, etc.) to be delivered by the lipid-containing particles.

[0270] In some embodiments, the freight contained in and thus to be delivered by the lipid-containing particles disclosed herein is a small molecule, such as an ion (e.g., Ca2+ , 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 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 localizing the protein to a proteasome. In some embodiments, the small molecule is a proteolysis-inducing chimeric molecule (PROTAC).

[0271] In some embodiments, the freight contained in and thereby delivered by the lipid-containing particles disclosed herein is a mixture of proteins, nucleic acids, or metabolites, such as multiple polypeptides, multiple nucleic acids, multiple small molecules; a combination 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.

[0272] In some embodiments, the freight contained in and thereby delivered by the lipid-containing particles disclosed herein is one or more organelles, such as condrosomes, mitochondria, lysosomes, nuclei, cell membranes, cytoplasm, endoplasmic reticulum, ribosomes, vacuoles, endosomes, spliceosomes, polymerases, capsids, acrosomes, autophagosomes, centrosomes, glycosomes, glyoxysomes, hydrogenosomes, melanosomes, mitosomes, myofibrils, trichocysts, peroxisomes, proteasomes, vesicles, stress granules, a network of organelles, and any combination thereof.

[0273] In some cases, the freight contained in and thereby delivered by the lipid-containing particles disclosed herein is RNA (viral or heterologous), DNA (single-stranded, double-stranded), green fluorescent protein, nuclease, iron oxide NP (IONP), taxol, Alexa Fluor® 488, porphyrin, doxorubicin, fluorescein, DOTA chelator, RNA (messenger, micro, small interfering), ricin toxin 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, gadopentetic acid polymethacrylate, CRISPR (Cas9 and guide RNA), green fluorescent protein or mCherry, CellB protein, [NiFe] hydrogenase, diketopiperazine, triple enzyme cascade (genetically linked), alcohol dehydrogenase, polystyrene sulfonate, RNA, green or teal fluorescent protein, Pseudozyma antarctica lipase B, horseradish peroxidase, DOTAC10 micelle having Gd(III) or Zn(II), Gd(DOTA), fluorescent probe, doxorubicin, DAPI, acridine orange, propidium iodide, proflavine, iron oxide NP, Gd(III), or Tb(III), including one or more of them. In some cases, the freight 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 hereby incorporated by reference in its entirety.

[0274] In some cases, the freight contained in and thereby delivered by the lipid-containing particles of the present disclosure includes polypeptides having a length of 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 freight contained in the disclosed lipid-containing particles of the present disclosure includes polypeptides having a length of 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.

[0275] In some cases, the freight contained in and thereby delivered by the lipid-containing particles disclosed herein comprises a polynucleotide encoding a polypeptide having a length of 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 freight contained in the lipid-containing particles of the present disclosure comprises a polynucleotide encoding a polypeptide having a length of 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.

[0276] In some cases, the polypeptide contained in and thereby to be delivered by the lipid-containing particles disclosed herein 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 and thereby to be delivered by the lipid-containing particles disclosed herein 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.

[0277] In some cases, the freight contained in the lipid-containing particles disclosed herein and thereby to be delivered is a single-stranded polynucleotide having a length of 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 freight contained in the lipid-containing particles of the present disclosure is a single-stranded polynucleotide encoding a polypeptide having a length of 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.

[0278] In some cases, the freight contained in the lipid-containing particles disclosed herein and thereby to be delivered is a double-stranded polynucleotide having a length of 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 freight contained in the lipid-containing particles of the present disclosure is a double-stranded polynucleotide encoding a polypeptide having a length of 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. Nuclease

[0279] Any suitable nuclease can be delivered by the lipid-containing particles disclosed herein that contain either a nuclease or a polynucleotide encoding a nuclease. Suitable nucleases include CRISPR-associated (Cas) proteins or Cas nucleases, such as 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)); as well as any derivatives thereof; any variants thereof; and any fragments thereof.

[0280] In some embodiments, the freight in the lipid-containing particles disclosed herein comprises or encodes a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) protein or Cas nuclease that functions in a naturally non-existent CRISPR / Cas system. In bacteria, this 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).

[0281] One or more components of a CRISPR / Cas system (e.g., modified and / or unmodified) delivered by the lipid-containing particles disclosed herein can be utilized as a genome engineering tool in a variety of organisms including diverse mammals, animals, plants, and yeast. The CRISPR / Cas system can include a guide nucleic acid such as a guide RNA (gRNA) complexed with a Cas protein for targeted regulation of gene expression and / or activity or nucleic acid editing. The RNA-guided Cas protein (e.g., a Cas nuclease, e.g., Cas9 nuclease) can specifically bind to a target polynucleotide (e.g., DNA) in a sequence-dependent manner.When the Cas protein has nuclease activity, it 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 is widely used for programmable genome editing in various 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).

[0282] In some cases, the Cas protein delivered by the lipid-containing particles of the present disclosure is mutated and / or modified to yield a nuclease-deficient protein or a protein with reduced nuclease activity compared to the wild-type Cas protein. A nuclease-deficient protein may retain the ability to bind to DNA but may lack or have reduced nucleic acid cleavage activity. A protein comprising a Cas nuclease (e.g., retaining wild-type nuclease activity, having reduced nuclease activity, and / or lacking nuclease activity), or a protein encoded by a gRNA molecule, can function in the CRISPR / Cas system to regulate (e.g., decrease, increase, or abolish) the level and / or activity of a target gene or protein. The Cas protein can bind to a target polynucleotide, prevent transcription by a physical barrier, or edit a nucleic acid sequence to yield a non-functional gene product.

[0283] In some embodiments, the gRNA 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 gRNA 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 gRNA in the lipid-containing particles disclosed herein comprises or encodes an RNA-binding protein (RBP) optionally complexed with a guide nucleic acid, such as a guide RNA (e.g., sgRNA), that can form a complex with a Cas protein.

[0284] 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 various nomenclature systems. Exemplary nomenclature 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, type II, type III, type IV, type V, type VI system, or any other suitable CRISPR / Cas system. The CRISPR / Cas system, as used herein, can be a class 1, class 2, or any other suitably 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 generally have a multi-subunit crRNA-effector complex, while class 2 systems generally have a single protein, e.g., Cas9, Cpf1, C2c1, C2c2, C2c3, or a crRNA-effector complex. Class 1 CRISPR / Cas systems can perform regulation using a complex of multiple Cas proteins. 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 perform regulation using a single large Cas protein. 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 lend functional units in trans to facilitate CRISPR locus targeting.

[0285] The freight delivered by the lipid-containing particles of the present disclosure can contain or encode a Class 1 or Class 2 Cas protein. The Cas protein can be a Type I, II, III, IV, V, or VI Cas protein. The Cas protein can contain 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 the guide nucleic acid. The nuclease domain can contain catalytic activity for nucleic acid cleavage. The nuclease domain may lack catalytic activity to prevent nucleic acid cleavage. The Cas protein can be a chimeric Cas protein fused to another protein or polypeptide. The Cas protein can be a chimera of various Cas proteins, including, for example, domains from different Cas proteins.

[0286] Examples of Cas proteins that can be delivered by the lipid-containing particles of the present disclosure include c2c1, Cas13a (former name 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 (former name 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, as well as homologs or modified versions thereof. 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 others described in Chuang CK et al., Int J Mol Sci. 2021 Sep 13;22(18):9872, which is incorporated herein by reference in its entirety.

[0287] Another example of a Cas protein that can be delivered by the lipid-containing particles of the present disclosure is Cas14. The Cas14 protein or polypeptide (also referred to as the "CasZ" protein or polypeptide) can bind to and / or modify a target nucleic acid and / or a polypeptide associated with the target nucleic acid (e.g., cleaving, nicking, methylating, demethylating, etc.) (e.g., methylation or acetylation of histone tails). (For example, in some cases, the CasZ protein includes an active chimeric partner, and in some cases, the CasZ protein provides nuclease activity). In some cases, the Cas14 protein or polypeptide is a naturally occurring (e.g., naturally occurring in prokaryotic cells) protein (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 includes three partial RuvC domains (also referred to herein as subdomains, RuvC-I, RuvC-II, and RuvC-III), which are not contiguous with respect to the primary amino acid sequence of the Cas14 protein but form the RuvC domain when the protein is produced and folded. Naturally occurring Cas14 proteins function 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 the target sequence within the target DNA. Naturally occurring Cas14 guide RNAs are crRNAs, and crRNAs include (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 US Patent Publication Nos. US20200172886 and US20210214697, Harrington LB et al., Science. 2018 Nov 16;362(6416):839-842; Aquino-Jarquin G. Nanomedicine. 2019 Jun;18:428-431, each of which is incorporated herein by reference in its entirety. In some cases, the constructs disclosed herein include a Cas14 polypeptide, or a nucleic acid molecule encoding a Cas14 polypeptide. In some cases, the constructs disclosed herein include a Cas14a polypeptide, or a nucleic acid molecule encoding a Cas14a polypeptide. In some cases, the constructs disclosed herein include a Cas14b polypeptide, or a nucleic acid molecule encoding a Cas14b polypeptide. In some cases, the constructs disclosed herein include a Cas14c polypeptide, or a nucleic acid molecule encoding a Cas14c polypeptide.

[0288] 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 viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus 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 embodiments, the organism is Streptococcus pyogenes (S. pyogenes). In some embodiments, the organism is Staphylococcus aureus (S. aureus). In some embodiments, the organism is Streptococcus thermophilus (S. thermophilus).

[0289] Cas proteins are 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.It can be derived from various bacterial species including 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. 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.

[0290] The Cas protein, as disclosed herein, may be a wild-type or modified form of the Cas protein. The Cas protein can be an active variant, an inactive variant, or a fragment of a wild-type or modified Cas protein. The Cas protein can include amino acid changes, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof, compared to the wild-type version of the Cas protein. The Cas protein can be a polypeptide 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 an exemplary wild-type Cas protein. The Cas protein can be a polypeptide having at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity to an exemplary wild-type Cas protein. A variant or fragment can include 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 or modified Cas protein or a portion thereof. A variant or fragment can be targeted to a nucleic acid locus in a complex with a guide nucleic acid but lacks nucleic acid cleavage activity.

[0291] The Cas protein can include one or more nuclease domains, such as a DNase domain. For example, the Cas9 protein can include a RuvC-like nuclease domain and / or an HNH-like nuclease domain. The RuvC and HNH domains can each cut different strands of double-stranded DNA to produce a double-strand break in the DNA. The Cas protein can also include only one nuclease domain (e.g., Cpf1 includes a RuvC domain but lacks an HNH domain).

[0292] The Cas protein can include 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 the nuclease domain of the wild-type Cas protein (e.g., RuvC domain, HNH domain).

[0293] The Cas protein can be modified to optimize the regulation of gene expression. The Cas protein can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, and / or enzyme activity. The Cas protein can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the 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 the activity of the Cas protein can be optimized (e.g., enhanced or reduced) for the regulation of gene expression.

[0294] In some embodiments, the freight delivered by the lipid-containing particles of the present disclosure includes a nuclease-deficient DNA-binding protein derived from a DNA nuclease capable of inducing transcriptional activation or repression of a target DNA sequence. In some embodiments, the freight includes or encodes a nuclease-deficient RNA-binding protein derived from an RNA nuclease capable of inducing transcriptional activation or repression of a target RNA sequence. For example, the freight can include or encode a Cas protein lacking cleavage activity.

[0295] The Cas protein can be a chimeric protein. For example, the Cas protein may be fused to a heterologous functional domain. The heterologous functional domain may include a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. The Cas protein may also be fused to a heterologous polypeptide that results in an increase or decrease in stability. The fusion domain or heterologous polypeptide may be located at the N-terminus, C-terminus, or internally within the Cas protein.

[0296] Regulation of a gene can be the regulation of any gene of interest. It is contemplated that gene homologs of the genes described herein are covered. For example, a gene may exhibit a certain identity and / or homology to a gene disclosed herein. Thus, it is contemplated that genes showing 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) or genes showing the % homology (at the nucleic acid or protein level) can be modified. Also contemplated is that genes showing 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) or genes showing the % identity (at the nucleic acid or protein level) can be modified.

[0297] The Cas protein can be provided in any form. For example, the Cas protein can be provided in the form of a protein alone or 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.

[0298] 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 protein in a specific cell or organism.

[0299] In some embodiments, the Cas protein is an inactive Cas protein. The inactive Cas protein can be a protein lacking nucleic acid cleavage activity.

[0300] 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 changes (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 S. pyogenes). The modified form of the Cas protein may have substantially no nucleic acid cleavage activity. If the Cas protein is in a modified form that has substantially no nucleic acid cleavage activity, it can be referred to as enzymatically inactive and / or "dead" (abbreviated as "d"). The inactive Cas protein (e.g., dCas, dCas9) can bind to the target polynucleotide but may not cleave the target polynucleotide. In some aspects, the inactive Cas protein is an inactive Cas9 protein.

[0301] The dCas9 polypeptide can associate with a single-guide RNA (sgRNA) to activate or repress the transcription of 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 sgRNA targets 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 nucleic acid targeted can be a gene involved in immune cell regulation. In some embodiments, the nucleic acid is cancer-related. The cancer-related nucleic acid 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-deficient CRISPR protein, variants thereof, derivatives thereof, or fragments thereof.

[0302] Enzymatically inactive can refer to a polypeptide that can bind sequence-specifically to a nucleic acid sequence in a polynucleotide but has the potential not to cleave the target polynucleotide. The enzymatically inactive site-directed polypeptide can include an enzymatically inactive domain (e.g., a nuclease domain). Enzymatically inactive can refer to having no activity. Enzymatically inactive can refer to having substantially no activity. Enzymatically inactive can refer to having essentially no activity. Enzymatically inactive can refer to having 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% activity compared to an exemplary activity of the wild type (e.g., nucleic acid cleavage activity, wild-type Cas9 activity).

[0303] One or more nuclease domains of a Cas protein (e.g., RuvC, HNH) can be deleted or mutated such that they are no longer functional or have reduced nuclease activity (inactivated or inactive 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, known as a nickase, can introduce a single-strand break at the CRISPR RNA (crRNA) recognition sequence within double-stranded DNA, but cannot introduce a double-strand break. Such a nickase can cleave either the complementary or non-complementary strand, but may not cleave both. When all of the 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 may or may not have a reduced ability to cleave both strands of double-stranded DNA. Examples of mutations that can convert a Cas9 protein into a nickase are the D10A (from aspartic acid to alanine at position 10 of Cas9) mutation in the RuvC domain of Cas9 from S. pyogenes. H939A (from histidine to alanine at amino acid position 839) or H840A (from histidine to alanine at amino acid position 840) in the HNH domain of Cas9 from S. pyogenes can convert Cas9 into a nickase. Examples of mutations that can convert a Cas9 protein into an inactive Cas9 are the D10A (from aspartic acid to alanine at position 10 of Cas9) mutation in the RuvC domain of Cas9 from S. pyogenes and H939A (from histidine to alanine at amino acid position 839) or H840A (from histidine to alanine at amino acid position 840) in the HNH domain.

[0304] The inactive Cas protein may contain one or more mutations compared to the wild-type version of the protein. As a result of the mutation, 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 in one or more of the multiple nucleic acid cleavage domains of the wild-type Cas protein may result. As a result of the mutation, one or more of the multiple nucleic acid cleavage domains may retain the ability to cleave the complementary strand of the target nucleic acid but reduce its ability to cleave the non-complementary strand of the target nucleic acid. As a result of the mutation, one or more of the multiple nucleic acid cleavage domains may retain the ability to cleave the non-complementary strand of the target nucleic acid but reduce its ability to cleave the complementary strand of the target nucleic acid. As a result of the mutation, one or more of the multiple nucleic acid cleavage domains may lack the ability to cleave both the complementary and non-complementary strands of the target nucleic acid. The residues to be mutated in the nuclease domain may correspond to one or more catalytic residues of the nuclease. For example, residues in the exemplary wild-type S. pyogenes Cas9 polypeptide, such as Asp10, His840, Asn854, and Asn856, may 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 may correspond to residues Asp10, His840, Asn854, and Asn856 in the wild-type S. pyogenes Cas9 polypeptide, determined by, for example, sequence and / or structural alignment.

[0305] As an example, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 (or corresponding mutations of any of the Cas proteins) 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 may also be preferred.

[0306] The D10A mutation can be combined with one or more of the H840A, N854A, or N856A mutations to produce a Cas9 protein (e.g., an inactive 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.

[0307] In some embodiments, the Cas protein is a class 2 Cas protein. In some embodiments, the Cas protein is a type II Cas protein. In some embodiments, the Cas protein is Cas9 protein, a modified version of Cas9 protein, or is derived from Cas9 protein. For example, a Cas9 protein lacking cleavage activity. In some embodiments, the Cas9 protein is the Cas9 protein from S. pyogenes (e.g., SwissProt accession number Q99ZW2). In some embodiments, the Cas9 protein is the Cas9 from S. aureus (e.g., SwissProt accession number J7RUA5). In some embodiments, the Cas9 protein is a modified version of the Cas9 protein from S. pyogenes or S. aureus. In some embodiments, the Cas9 protein is derived from the Cas9 protein from S. pyogenes or S. aureus. For example, a S. pyogenes or S. aureus Cas9 protein lacking cleavage activity.

[0308] 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 an exemplary wild-type Cas9 polypeptide (e.g., Cas9 from S. 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 an exemplary wild-type Cas9 polypeptide (e.g., from S. pyogenes). Cas9 can refer to a wild-type or modified form of the Cas9 protein that may include amino acid changes such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0309] In some embodiments, the freight includes or encodes a "zinc finger nuclease" or "ZFN". A ZFN refers to a chimera of a cleavage domain, such as the cleavage domain of FokI, and at least one zinc finger motif (e.g., at least two, three, four, or five zinc finger motifs) that can bind to polynucleotides, such as DNA and RNA. Heterodimerization of two individual ZFNs at a particular position in the polynucleotide at a particular orientation and spacing can lead to cleavage of the polynucleotide. For example, ZFN binding to DNA can induce double-strand breaks in the DNA. Two individual ZFNs can bind to opposite strands of DNA that are separated by a particular distance such that their C termini are separated to allow the two cleavage domains to dimerize and cleave the DNA. In some cases, the linker sequence between the zinc finger domain and the cleavage domain may require that the 5' edge of each binding site 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 are 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, as well as U.S. Patent Application Publications Nos. 2003 / 0232410 and 2009 / 0203140.

[0310] In some embodiments, a protein comprising a ZFN, or a protein encoded by a fret protein or fret nucleic acid molecule, can generate a double-strand break in a target polynucleotide such as DNA. The double-strand break in the DNA can result in DNA cleavage repair that allows for the introduction of genetic modification (e.g., nucleic acid editing). DNA cleavage repair can be carried out by non-homologous end joining (NHEJ) or homologous recombination repair (HDR). In HDR, a donor DNA repair template containing homologous arm flanking sites of the target DNA can be provided. In some embodiments, the ZFN is a zinc finger nickase that induces site-specific single-strand DNA cleavage or nicks, and thus results 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.

[0311] In some embodiments, the cleavage domain of a protein comprising a ZFN, or a protein encoded by a fret protein or fret nucleic acid molecule, comprises a modified form of the wild-type domain. The modified form of the cleavage domain can include amino acid changes (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 may have substantially no nucleic acid cleavage activity. In some embodiments, the cleavage domain is enzymatically inactive.

[0312] In some embodiments, the protein is a TALEN or a protein encoded by a TAL nucleic acid molecule, where a TALEN is an engineered transcriptional activator-like effector nuclease that generally contains a central domain of DNA-binding tandem repeats and a cleavage domain. A TALEN can be produced by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. In some cases, the DNA-binding tandem repeats contain two hypervariable amino acid residues that are 33-35 amino acids in length and can recognize at least one specific DNA base pair at positions 12 and 13. A transcriptional activator-like effector (TALE) protein can be fused to a nuclease, such as a wild-type or mutated FokI endonuclease, or the catalytic domain of FokI. For use in TALENs, several mutations have been introduced into FokI, which improves, for example, cleavage specificity or activity. Such TALENs can be engineered to bind to any desired DNA sequence. TALENs can be used to create double-strand breaks in a target DNA sequence, such that the target DNA sequence undergoes NHEJ or HDR, resulting in gene modification (e.g., nucleic acid sequence editing). In some cases, a single-stranded donor DNA repair template is provided to facilitate HDR. Detailed descriptions of TALENs and their use for 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.

[0313] 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 may include amino acid changes (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 may 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 may have substantially no nucleic acid cleavage activity. In some embodiments, the nuclease domain is enzymatically inactive.

[0314] In some embodiments, the transcription activator-like effector (TALE) protein can modulate transcription and is fused to a nuclease-free domain. In some embodiments, the transcription activator-like effector (TALE) protein is designed to function as a transcription activator. In some embodiments, the transcription activator-like effector (TALE) protein is designed to function as a transcription repressor. For example, the DNA binding domain of the transcription activator-like effector (TALE) protein can be fused (e.g., linked) to one or more transcription activation domains or to one or more transcription repression domains. Examples of transcription activation domains include the herpes simplex VP16 activation domain and the tetrameric repeat of the VP16 activation domain, such as the VP64 activation domain. Other examples include VP16, VP32, VP64, VPR, p65, RTA, KRAB, or P65HSF1. An example of a transcription repression domain includes the Kruppel-associated box domain.

[0315] In some embodiments, the freight protein, or a protein encoded by a freight nucleic acid molecule, comprises a meganuclease. Meganucleases generally refer to rare-cut endonucleases or homing endonucleases that can be highly specific. Meganucleases can recognize DNA target sites ranging from at least 12 base pairs in length, for example, 12-40 base pairs, 12-50 base pairs, or 12-60 base pairs in length. Meganucleases can be modular DNA-binding nucleases, such as any chimeric protein, that includes at least one catalytic domain of an endonuclease and at least one DNA-binding domain or protein that defines a nucleic acid target sequence. The DNA-binding domain can contain at least one motif that recognizes single-stranded or double-stranded DNA. Meganucleases can be monomers or dimers. 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. Detailed descriptions of useful meganucleases and their applications in gene editing can be found, for example, in Silva et al., Curr Gene Ther, 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.

[0316] 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 may include amino acid changes (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 may 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 may have substantially no nucleic acid cleavage activity. In some embodiments, the nuclease domain is enzymatically inactive. In some embodiments, the meganuclease can bind to DNA but cannot cleave DNA. Targetable 3'-Overhang Nuclease

[0317] In some cases, the freight to be delivered by the lipid-containing particles of the present disclosure includes a nuclease that produces 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. The term "type IIS restriction enzyme" as used herein refers to a restriction enzyme that recognizes an asymmetric DNA sequence and cleaves outside of those recognition sequences. In one embodiment, the restriction enzyme is Acul.

[0318] In some cases, the freight includes a targetable nuclease chimeric protein comprising 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 produces 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.

[0319] In some cases, the DBD is a protein or protein domain that binds sequence-dependently to its target nucleic acid. In some cases, the DBDs disclosed herein are either a zinc finger array or dCas9.

[0320] In some cases, the nuclease chimeric protein is a zinc finger nuclease chimeric protein. The zinc finger nuclease chimeric proteins described herein include a nuclease domain that, when dimerized, produces a 3' overhang double-strand break in DNA (i.e., the nuclease activity is "dimerization-dependent"), an optional amino acid linker, and a zinc finger domain that includes one or more carboxy-terminal or amino-terminal zinc fingers. The monomeric form of the zinc finger nuclease chimeric protein that includes one or more carboxy-terminal or amino-terminal zinc fingers can dimerize either upon or prior to binding to the target site, thereby activating nuclease cleavage. The zinc finger nuclease chimeric proteins described herein can be used to create insertion / deletion mutations (indels) at high frequency by repair of nuclease-induced DNA cleavage by non-homologous end joining.

[0321] A zinc finger nuclease chimeric protein can also be used to copy, incorporate or insert a foreign nucleic acid sequence of interest into a target site of a genomic locus of a cell. In some embodiments, the methods provided herein involve providing to the nucleus of a cell a foreign 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 foreign nucleic acid “donor template” sequence and the zinc finger nuclease chimeric protein are delivered by the lipid-containing particles provided herein. The foreign nucleic acid donor template sequence includes terminal sequences homologous to sequences within the target site of the genomic locus. Zinc fingers can be designed to specifically recognize and bind to the genomic target site. Upon binding to the target site, the dimerized nuclease domain of the chimeric protein creates a 3′ overhang double-strand break within the target site, inducing homologous recombination repair between the sequences surrounding the break and the foreign nucleic acid sequence, thereby allowing the foreign nucleic acid sequence to be copied, incorporated, and / or inserted into the target site of the genomic locus of the cell.

[0322] The zinc finger nuclease chimeric protein can include any nuclease domain that can create a 3′ overhang double-strand break in DNA when dimerized.

[0323] The nuclease domain can be, for example, a type IIS restriction enzyme nuclease domain including, but not limited to, the Acul, Alol, Bpml, Bael or Mmel nuclease domain. In some cases, the Acul nuclease domain can have an amino acid sequence.

[0324] Exemplary nucleotide and amino acid sequences encoding Acul are known in the art and can be found, for example, under GenBank accession number HQ327692.1.

[0325] In some embodiments, the IIS-type restriction enzyme nuclease domain includes an isoschizomer of Acul, such as Eco57I. The nucleotide and amino acid sequences encoding Eco57I can be retrieved, for example, with the UniProt database reference number P25239.

[0326] Exemplary nucleotide and amino acid sequences encoding Alol are known in the art and can be retrieved, for example, with GenBank accession number AJ312389.1.

[0327] Exemplary nucleotide and amino acid sequences encoding Bpml are known in the art and can be retrieved, for example, with GenBank accession number ADK30556.1. Exemplary nucleotide and amino acid sequences encoding Bael are known in the art and can be retrieved, for example, with GenBank accession number ABS74060.1.

[0328] Exemplary nucleotide and amino acid sequences encoding Mmel are known in the art and can be retrieved, for example, with GenBank accession number EU616582.1.

[0329] Any IIS-type restriction enzyme nuclease domain having dimerization-dependent nuclease activity can be fused to the zinc finger domain and used to perform 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.

[0330] The dimerization-dependent nuclease domain and zinc finger domain of a 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 means by which the two domains of the chimeric protein are joined. The amino acid linker can be any sequence of at least 1 amino acid and can include sequences of up to 10 amino acids. In specific embodiments, the linker is leucine, arginine, glycine, and serine (LRGS (SEQ ID NO: 2)); glycine, glycine, glycine, glycine, and serine (GGGGS (SEQ ID NO: 3)); or a non-standard amino acid, threonine, glutamic acid, and asparagine (XTEN) as described by Shellenberger, et al. Nat Biotechnol. 2009 Dec; 27(12): 1186-90.

[0331] 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 amino acid sequence of the exemplary amino acid sequences of the dimerization-dependent nuclease domain, zinc finger domain, TALE, and / or dCas9 described herein.

[0332] When bound 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 recombination repair, resulting in the copying, incorporation, and / or integration of an exogenous nucleic acid sequence or a portion thereof into the target site. When nucleotide sequence homology exists, the donor template oligonucleotide sequence (either single-stranded or double-stranded) can serve as a template for repairing the target DNA sequence that has experienced a double-strand break, leading to the transfer of genetic information from the donor to the target. Such transfer can involve mismatch repair of the heteroduplex DNA formed between the cleaved target and the donor, and / or synthesis-dependent strand annealing to resynthesize genetic information that becomes part of the target using the donor, and / or related processes. Homologous recombination repair often results in a change in the sequence of the target nucleotides such that some or all of the sequence for the donor nucleotide is copied and / or incorporated into the target nucleotides.

[0333] The zinc finger nuclease chimeric protein can create a double-strand break at a predetermined site in the target sequence and copy, incorporate, and / or introduce into the genomic locus an exogenous nucleic acid sequence that serves as a donor template and has homology to the nucleotide sequence within the cleavage region. The presence of the double-strand break has been shown to significantly enhance the efficiency of these different repair outcomes. The donor sequence can be physically incorporated, or alternatively, the donor nucleotides are used as a template for repair of the cleavage by homologous recombination, resulting in the introduction of all or part of the nucleotide sequence into the genomic locus as in the case of the donor. Thus, the sequence at the genomic locus can be altered and, in certain embodiments, can be converted to the sequence present in the donor nucleotides.

[0334] Also described herein are dCas9 nuclease chimeric proteins and methods of using them to enhance the frequency of homologous recombination repair at nuclease-induced double-strand break sites. The dCas9 nuclease chimeric protein comprises a catalytically inactive Cas9 carboxy-terminal or amino-terminal domain linked to a dimerization-dependent nuclease domain that produces 3' overhang double-strand breaks in DNA. The catalytically inactive Cas9 domain contains mutations (e.g., D10A and / or H841A) that result in the loss of native endonuclease activity (Qi et al., Cell (2013)). Instead, the endonuclease activity is provided by the linked dimerization-dependent nuclease domain to which it is fused. The dCas9 nuclease chimeric protein in monomeric form dimerizes together either before or upon binding to the dCas9 target site, 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 is widely used for programmable genome editing in various 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 composed of two RNAs that associate or are covalently linked together to form the guide RNA; CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA). When the nucleotide sequence of the target genomic locus 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 the guide RNA, can bind to and cleave a dsDNA protospacer sequence that is complementary to the crRNA spacer and is also flanked by a short protospacer adjacent motif. dCas9 can still associate with the crRNA / tracrRNA complex or with the guide RNA, despite its native catalytic activity being inactivated, and as a result, recognize and bind to the target site. The nucleotide and amino acid sequences encoding Cas9 are known in the art and can be retrieved, for example, with GenBank accession number NC_002737.2.

[0335] Using the dCas9 nuclease chimeric proteins described herein, homologous recombination repair events can be induced at target sites in the genomic loci of cells. This method involves 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 those target sites that are complementary to the guide RNA and the adjacent PAM sequence. Upon binding to the target site, the linked nuclease domain of the chimeric protein functions as a dimer to generate a 3'-overhang double-strand break within the target site and induce homologous recombination repair between the sequence surrounding the break and the exogenous nucleic acid sequence, thereby copying, 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 retrieved, for example, with GenBank accession number KR011748.1. dCas9 is also described by Zetsche et al, Nature Biotechnology 33, 139-142 (2015).

[0336] The dCas9 nuclease chimeric protein can include any nuclease domain that can cause a 3' overhang double-strand break in DNA when dimerized. The nuclease domain can be, for example, a type IIS restriction enzyme nuclease domain including, but not limited to, the Acul, Alol, Bpml, Bael, or Mmel nuclease domain. The dimerization-dependent nuclease domain of the dCas9 nuclease chimeric protein and the dCas9 domain are joined together by an amino acid linker as needed. The amino acid linker can be any sequence of at least 1 amino acid and can include a sequence of up to 10 amino acids. In a specific embodiment, the amino acid linker can include, for example, glycine, glycine, glycine, glycine, and serine (GGGGS (SEQ ID NO: 3)); or a non-standard amino acid, threonine, glutamic acid, and asparagine (XTEN).

[0337] In any of the methods and compositions described herein, the exogenous nucleotide sequence serving as a donor contains a sequence that is homologous but not identical to the genomic sequence at the target site, thereby stimulating homologous recombination repair to copy, incorporate, and / or insert the non-identical sequence into 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 a sequence identity of between about 80% and 99% (or any integer between these values) with respect to the genomic sequence to be replaced. In other embodiments, the homology between the donor and the genomic sequence is higher than 99%, for example, if there is only 1 nucleotide difference over 100 contiguous base pairs between the donor sequence and the genomic sequence. In certain cases, the non-homologous portion of the donor sequence can contain a sequence that is not present at the target site, and thus, a new sequence is introduced into the region of interest. In these cases, the non-homologous sequence is generally flanked by a sequence of 50 to 1,000 base pairs (or any integer value between these) or any number of base pairs greater than 1,000 that is homologous or identical to the sequence at the target site.

[0338] In some embodiments, the entire donor template array or a portion of the donor template array is integrated into the target site. Any of the methods described herein can be used for the partial or complete inactivation of one or more genomic loci in a cell by targeted integration of a donor sequence that interferes with the expression of a gene of interest. Any of the methods described herein can be used to replace a mutated sequence within a target site, thereby correcting a mutated gene or inducing gene expression that was previously inactive. The nature of the exogenous nucleic acid sequence to be incorporated will depend on the therapeutic goal to be achieved and can vary from induction or inhibition of gene transcription to replacement of a mutated sequence of a defective gene or addition or deletion of a sequence within a gene.

[0339] In other embodiments, a DBD (e.g., zinc finger or dCas9) nuclease chimeric protein introduces variable length insertion or deletion mutations that partially or completely overlap 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. More precisely, a nucleic acid sequence encoding a zinc finger nuclease chimeric protein or an isolated zinc finger nuclease chimeric protein is provided to the nucleus of the cell, 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, followed by repair of the break by non-homologous end joining or microhomology-mediated end joining. Both non-homologous end joining and microhomology-mediated end joining can result in insertions or deletions that interfere with or inhibit gene transcription at the nuclease target site.

[0340] 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 include those described in International Publication No. WO2020160481, which is hereby incorporated by reference in its entirety.

[0341] Base editing In some cases, the freight to be delivered by the lipid-containing particles of the present disclosure includes one or more components of a nucleic acid base editor (also referred to as a "base editor") or a nucleic acid base editing (also referred to as "base editing") complex.

[0342] The term "base editor (BE)" or "nucleic acid base editor (NBE)" as used herein can refer to an agent that includes 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 can deaminate a base within a nucleic acid. In some embodiments, the base editor can deaminate a base within a DNA molecule. In some embodiments, the base editor can deaminate adenosine (A) in DNA. In some embodiments, the base editor can deaminate cytosine (C) in DNA.

[0343] In some cases, the base editors disclosed herein include a deaminase or a functional domain thereof (a "deaminase domain") that catalyzes a deamination reaction.

[0344] 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 an adenosine deaminase that catalyzes the deamination of adenosine to convert it to nucleoside hypoxanthine. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase that 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.

[0345] As used herein, "adenosine deaminase" is an enzyme that catalyzes the deamination of adenosine, converting adenosine to nucleoside hypoxanthine. According to standard Watson-Crick hydrogen bonding pair formation, the adenosine base hydrogen bonds to a thymine base (or uracil in the case of RNA). When adenine is converted to hypoxanthine, hypoxanthine undergoes hydrogen bonding pair formation with cytosine. Thus, due to the conversion of "A" to hypoxanthine by adenosine deaminase, "C" will be inserted instead of "T" during the cell repair and / or replication process. 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.

[0346] In some embodiments, the base editor is a chimeric protein that includes a nucleic acid programmable R / DNA-binding protein (napR / DNAbp) fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase) domain. 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., which can be broadly referred to as "napR / DNAbp programming nucleic acid molecules" and which, for example, in the case of the Cas system, includes guide RNAs), and direct or otherwise program the protein to localize to a specific target nucleotide sequence (e.g., a genomic locus, or an RNA molecule) that is complementary to one or more nucleic acid molecules (e.g., a portion or region thereof) associated with the protein, thereby binding the protein to the nucleotide sequence at a specific target site. This term napR / DNAbp encompasses, of course, the CRISPR Cas9 protein as well as Cas9 equivalents, homologs, orthologs or paralogs, whether naturally occurring or not (e.g., engineered or recombinant), and can include Cas9 equivalents from any type of CRISPR system (e.g., types II, V, 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 contents of which are incorporated herein by reference. However, the nucleic acid programmable R / DNA-binding protein (napR / DNAbp) that can be used in connection with the present disclosure is not limited to the CRISPR-Cas system.The present disclosure encompasses any such programmable protein, such as the Argonaute protein from Natronobacterium gregoryi (NgAgo), which can also be used for DNA-guided genome editing. The DNA system guided by NgAgo requires neither a PAM sequence nor a guide RNA molecule, which means that genome editing can be performed for any genomic sequence simply by expression of the general NgAgo protein and introduction of a synthetic oligonucleotide. See Gao F, Shen X Z, 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.

[0347] In some cases, napR / DNAbp is derived from a nuclease disclosed herein, such as Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cas14, Cpfl, C2c1, C2c2, C2c3, an Argonaute protein, or a variant thereof. In some embodiments, the base editor comprises Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpfl, C2c1, C2c2, C2c3, or an Argonaute protein fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises Cas9 nickase (nCas9) fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a CasX protein fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises nuclease-inactive Cas9 (dCas9) fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a CasY protein fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a Cas14 protein fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a Cpf1 protein fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a C2c1 protein fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a C2c2 protein fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a C2c3 protein fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase).In some embodiments, the base editor comprises an Argonaute protein fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase).

[0348] In some embodiments, the adenosine deaminase provided herein is capable of deaminating adenosine. In some embodiments, the adenosine deaminase provided herein is capable of deaminating adenosine in the deoxyadenosine residues of DNA. The adenosine deaminase can be derived from any suitable organism (e.g., E. coli). In some embodiments, the adenosine deaminase is a naturally occurring adenosine deaminase that comprises one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). One of ordinary skill in the art will be able to identify corresponding residues in any homologous protein and in the respective coding nucleic acids by methods well known in the art, e.g., by sequence alignment and determination of homologous residues. Thus, one of ordinary skill in the art will be able to introduce mutations in any naturally occurring adenosine deaminase (e.g., having homology to ecTadA) corresponding 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 p...

Claims

1. (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-localized protein, wherein the plasma membrane-localized protein is selected from the group consisting of 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 PHPP1, the PH domain of human SWAP70, and the PH domain of human MAPKAP1; a combinatorial protein; (c) a fret and a lipid-containing particle comprising.

2. The lipid-containing particle according to claim 1, wherein the combinatorial protein comprises the plasma membrane-localized protein coupled to the fret.

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

4. The lipid-containing particle according to claim 3, wherein the combinatorial protein comprises, in order from the N-terminus to the C-terminus of the combinatorial protein, a plasma membrane-localized protein, the NES, and the fret.

5. The lipid-containing particle according to claim 3 or 4, wherein the combinatorial protein comprises, in order from the N-terminus to the C-terminus of the combinatorial protein, a plasma membrane-localized protein, the NES, the fret, and a second NES.

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

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

8. The lipid-containing particles contain the human endogenous retrovirus envelope protein; optionally, the human endogenous retrovirus envelope protein is from hENVH1, hENVH2, hENVH3, hENVK1, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENV T, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)1, or hENVKcon; and optionally, the human endogenous retrovirus envelope protein contains 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. The lipid-containing particles according to any one of the preceding claims.

9. The lipid-containing particles according to any one of the preceding claims, wherein the plasma membrane-localized 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 NOs: 11-21 and 60-66.

10. The lipid-containing particles according to any one of the preceding claims, wherein the lipid-containing particles contain a lipid-containing membrane encapsulating a protein core; optionally, the lipid-containing membrane contains a phospholipid bilayer.

11. The lipid-containing particles 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) A glycoprotein derived from a virus selected from the group consisting of RD114, Fug-E, FuG-E(P440E), and MLV 10A1; (b) A combinatorial protein containing a plasma membrane-localized protein coupled to a nuclear export sequence (NES); (c) A fret and A lipid-containing particle comprising.

13. The lipid-containing particles according to claim 12, wherein the combinatorial protein further contains the fret.

14. The lipid-containing particles according to claim 13, wherein the combinatorial protein contains the plasma membrane-localized protein, the NES, and the fret arranged in order from the N-terminus to the C-terminus of the combinatorial protein.

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

16. (a)a glycoprotein derived from a virus selected from the group consisting of RD114, Fug-E, Fug-E (P440E), and MLV 10A1; (b)a combinatorial protein comprising a plasma membrane-localized protein coupled to a cleavable linker; (c)a fret and a lipid-containing particle.

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

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

19. The plasma membrane-localized protein is (a)a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; (b)a humanized structural protein; (c)a plekstrin homology (PH) domain; or (d)a non-immunogenic plasma membrane mobilizing protein, optionally comprising Arc, human Arc, an endogenous retrovirus gag protein, or a human endogenous retrovirus gag protein The lipid-containing particle according to any one of claims 12 to 18.

20. The lipid-containing particle according to claim 19, wherein the plasma membrane-localized protein contains the PH domain, and the PH domain contains a PH domain of phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDK1), disk and actin-binding protein 1 (Dap1), general receptor for phosphoinositides 1 (Grp1), oxysterol-binding protein 1-Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), phosphatidylinositol tetra-phosphate adapter protein 1 (FAPP1), ceramide transport protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), switching B cell complex subunit SWAP70, or a PH domain of MAPK-related protein 1 (MAPKAP1) or a variant thereof.

21. The lipid-containing particle according to claim 19, wherein the plasma membrane-localized protein contains the PH domain, and the PH domain contains a PH domain of a human protein; optionally, the PH domain contains a PH domain of human phospholipase Cδ1, human Akt1, human 3-phosphoinositide-dependent protein kinase 1 (hPDK1), human Dap1, mouse Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, or a PH domain of human MAPKAP1 or a variant thereof.

22. The lipid-containing particle according to claim 19, wherein the plasma membrane-localized protein contains the PH domain, and the PH domain is selected from the group consisting of a PH domain of human Dap1, a PH domain of mouse Grp1, a PH domain of human Grp1, a PH domain of human OSBP, a PH domain of human Btk, a PH domain of human FAPP1, a PH domain of human CERT, a PH domain of human PKD, a PH domain of human PHLPP1, a PH domain of human SWAP70, and a PH domain of human MAPKAP1.

23. The lipid-containing particle according to claim 19, wherein the plasma membrane-localized protein contains a membrane protein selected from the group consisting of CD9, CD47, CD63, and CD81 and transmembrane domains thereof.

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

25. The lipid-containing particle according to claim 19, 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 any of the sequences listed in Table 3.

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

27. The lipid-containing particle according to any one of claims 1 to 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 to 55, and 67 to 77.

28. The lipid-containing particle according to claims 12 to 26, wherein the lipid-containing particle comprises a lipid-containing membrane encapsulating a protein core; optionally, 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 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 to 55, and 67 to 77.

31. The lipid-containing particle is (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, hENV T, 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, a human endogenous retrovirus (HERV) envelope protein; (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 the group consisting of 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, a virus-derived glycoprotein The lipid-containing particle according to claim 30, comprising.

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 floret, or a combination thereof.

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

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

36. The second plasma membrane-localized protein is (a) a human endogenous retrovirus (HERV) structural protein, optionally HERV gag; (b) a humanized structural protein; (c) a pleckstrin homology (PH) domain, optionally, the PH domain is phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDK1), disk and actin-binding protein 1 (Daap1), general receptor for phosphoinositides 1 (Grp1), oxysterol-binding protein 1-Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), tetraphosphate adapter protein 1 (FAPP1), ceramide transport protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), switching B cell complex subunit SWAP70, or the PH domain of MAPK-related protein 1 (MAPKAP1) or a variant thereof, optionally, the PH domain is from a human, optionally, the PH domain is the PH domain of human phospholipase Cδ1, human Akt1, human 3-phosphoinositide-dependent protein kinase 1 (hPDK1), 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 variant thereof, a PH domain; or (d) A non-immunogenic plasma membrane mobilizing protein, optionally wherein the non-immunogenic plasma membrane mobilizing protein comprises a membrane protein selected from the group consisting of CD9, CD47, CD63 and CD81 and transmembrane domains thereof, optionally wherein the membrane protein is selected from the group consisting of human CD9, human CD47, human CD63 and human CD81 and transmembrane domains thereof, and optionally wherein the non-immunogenic plasma membrane mobilizing protein comprises Arc, human Arc, endogenous retroviral gag protein, or human endogenous retroviral gag protein, a non-immunogenic plasma membrane mobilizing protein The lipid-containing particle according to claim 35, comprising

37. The lipid-containing particle according to claim 35, wherein the second plasma membrane-localized protein comprises the 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 of the sequences listed in Table 3.

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

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

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

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

42. (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 combinatorial protein comprising a plasma membrane-localized protein, wherein the plasma membrane-localized protein is selected from the group consisting of 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 PHPP1, the PH domain of human SWAP70, and the PH domain of human MAPKAP1; (c) A flate, or a third nucleic acid molecule encoding the flate A composition comprising.

43. The composition according to claim 42, wherein the second nucleic acid molecule encodes a combinatorial protein comprising the plasma membrane-localized protein coupled to the flate, 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 combinatorial protein comprising the plasma membrane-localized protein coupled to a nuclear export sequence (NES).

45. The composition according to claim 44, wherein the combinatorial protein comprises the plasma membrane-localized protein, the NES, and the flate arranged in order from the N-terminus to the C-terminus of the combinatorial protein.

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

47. The composition according to claim 46, wherein the cleavable linker is located between the plasma membrane-localized protein and the flate; optionally, the cleavable linker is located between the NES and the flate; and optionally, the combinatorial protein further comprises a nuclear localization sequence (NLS) at the C-terminus of the cleavable linker.

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

49. The composition according to any one of claims 42 to 48, 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 to 21 and 60 to 66.

50. (a) a first nucleic acid molecule encoding a viral-derived glycoprotein selected from the group consisting of RD114, Fug-E, FuG-E(P440E), and MLV 10A1; (b) a second nucleic acid molecule encoding a combinatorial protein comprising a plasma membrane-localized protein coupled to a nuclear export sequence (NES); (c) a frate, or a third nucleic acid molecule encoding the frate A composition comprising.

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

52. The composition according to claim 51, wherein the combinatorial protein comprises the plasma membrane-localized protein, the NES, and the frate arranged in order from the N-terminus to the C-terminus of the combinatorial protein.

53. The combinatorial protein further comprises a cleavable linker; optionally, the cleavable linker is located between the plasma membrane-localized protein and the fret; optionally, the cleavable linker is located between the NES and the fret; and optionally, the combinatorial protein comprises a nuclear localization sequence (NLS) at the C-terminus of the cleavable linker, The composition according to claim 52.

54. (a) a first nucleic acid molecule encoding a glycoprotein derived from a virus selected from the group consisting of RD114, Fug-E, Fug-E (P440E), and MLV 10A1; (b) a second nucleic acid molecule encoding a combinatorial protein comprising a plasma membrane-localized protein coupled to a cleavable linker; (c) a fret, or a third nucleic acid molecule encoding the fret A composition comprising

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

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

57. The plasma membrane-localized protein is (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 mobilizing protein, optionally, the non-immunogenic plasma membrane mobilizing protein comprises Arc, human Arc, an endogenous retrovirus gag protein, or a human endogenous retrovirus gag protein, a non-immunogenic plasma membrane mobilizing protein The composition according to any one of claims 50 to 56.

58. The composition according to claim 57, wherein the plasma membrane-localized protein contains a PH domain, and the PH domain contains a PH domain of phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDK1), disk and actin-binding protein 1 (Dap1), general receptor for phosphoinositides 1 (Grp1), oxysterol-binding protein 1-Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), phosphatidylinositol phosphate adapter protein 1 (FAPP1), ceramide transport protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), switching B cell complex subunit SWAP70, or a PH domain of MAPK-related protein 1 (MAPKAP1) or a variant thereof.

59. The composition according to claim 57, wherein the plasma membrane-localized protein contains the PH domain, and the PH domain contains a PH domain of a human protein; optionally, the PH domain contains a PH domain of human phospholipase Cδ1, human endogenous retrovirus gag protein, human 3-phosphoinositide-dependent protein kinase 1 (hPDK1), human Dap1, mouse Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, or a PH domain of human MAPKAP1 or a variant thereof.

60. The composition according to claim 57, wherein the plasma membrane-localized protein contains a membrane protein selected from the group consisting of CD9, CD47, CD63, and CD81 and transmembrane domains thereof.

61. The composition according to claim 57, wherein the plasma membrane-localized protein contains a membrane protein selected from the group consisting of human CD9, human CD47, human CD63, and human CD81 and transmembrane domains thereof.

62. The composition according to claim 57, wherein the plasma membrane-localized protein contains the PH domain, and the PH domain is selected from the group consisting of the PH domain of human Dap1, 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 PHLPPI, 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 contains the PH domain, and the PH domain contains an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of the sequences listed in Table 3.

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

65. The composition according to any one of claims 42 to 64, wherein the combinatorial protein contains an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequences selected from SEQ ID NOs: 50, 52-55, and 67-77.

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

67. A composition comprising a first nucleic acid molecule encoding a combinatorial protein containing a first plasma membrane-localized domain, wherein the combinatorial protein contains an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequences selected from SEQ ID NOs: 50, 52-55, and 67-77.

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

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

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

71. The second plasma membrane-localized protein is (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 pleckstrin homology (PH) domain is phospholipase Cδ1 (PLCδ1), Akt1, 3-phosphoinositide-dependent protein kinase 1 (hPDK1), Disc and actin binding protein 1 (Dap1), general receptor for phosphoinositides 1 (Grp1), oxysterol binding protein 1-Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), tetraphosphate adapter protein 1 (FAPP1), ceramide transport protein (CERT), protein kinase D (PKD), PH domain leucine rich repeat protein phosphatase 1 (PHLPP1), switching B cell complex subunit SWAP70, or a PH domain of MAPK-related protein 1 (MAPKAP1) or a variant thereof, optionally, the PH domain is from a human, optionally, the PH domain is a PH domain of human phospholipase Cδ1, human Akt1, human 3-phosphoinositide-dependent protein kinase 1 (hPDK1), human Dap1, mouse Grp1, human Grp1, human OSBP, human Btk1, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, or human MAPKAP1 or a variant thereof, a PH domain; or (d) A non-immunogenic plasma membrane mobilizing protein, optionally, wherein the non-immunogenic plasma membrane mobilizing protein comprises a membrane protein selected from the group consisting of CD9, CD47, CD63 and CD81 and their transmembrane domains, optionally, the membrane protein is selected from the group consisting of human CD9, human CD47, human CD63 and human CD81 and their transmembrane domains, optionally, the non-immunogenic plasma membrane mobilizing protein comprises Arc, human Arc, endogenous retroviral gag protein, or human endogenous retroviral gag protein, a non-immunogenic plasma membrane mobilizing protein The composition according to claim 66 or 70, comprising [

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

73. The percentage of the second nucleic acid molecule in the composition relative to the total 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%. The composition according to any one of claims 66 to 72.

74. The percentage of the first nucleic acid molecule in the composition relative to the total 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%. The composition according to any one of claims 70 to 72.

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

76. A combinatorial protein comprising a plasma membrane-localized protein and a heterologous sequence, wherein the plasma membrane-localized protein is selected from the group consisting of 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 PHLPPI, the PH domain of human SWAP70, and the PH domain of human MAPKAP1, and the heterologous sequence is 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 the amino acid sequences 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-containing membrane encapsulating a protein core; and optionally wherein the lipid-containing membrane comprises a phospholipid bilayer.

79. The lipid-containing particle according to claim 78, further comprising a human endogenous retrovirus (HERV) envelope protein, a humanized envelope protein, or a non-immunogenic membrane fusion molecule; and 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-containing membrane.

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

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

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

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

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

85. A method of treating a disease or condition in a subject in need of treating the disease or condition, comprising administering to the subject (a) a lipid-containing particle according to any one of claims 1 to 41, 75, or 78 to 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 to 41, 75, or 78 to 79.

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

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

89. (a) Contacting a producing cell with a composition according to any one of claims 42 to 75 or 80, wherein the producing 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 to 75 or 80, and wherein the system produces the lipid-containing particle Comprising; and Optionally, the method further comprises recovering and purifying the lipid-containing particle. The method according to claim 88.

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