Compositions and methods for promoting payload delivery in cells

Abstract

Disclosed herein, in some aspects, are compositions of lipid delivery particles used to deliver a payload to a target cell and methods of producing the like. Lipid delivery particles can comprise ectosomes, exosomes, nanoparticles, or other classes of extracellular vesicles suitable for transporting a payload. Lipid delivery particles can further comprise a chimeric protein comprising a plasma recruitment element and one, two, or all of: (i) a particle budding motif; (ii) a membrane penetrating peptide; (iii) a post-translational modification motif; and / or (iv) a multimerization motif. Additionally, disclosed herein, in some aspects, are nucleic acid molecules encoding the same.

Description

COMPOSITIONS AND METHODS FOR PROMOTING PAYLOAD DELIVERY INCELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 506,776, filed on June 7, 2023, the entirety of which is incorporated herein by reference.BACKGROUND

[0002] Vehicles for intercellular delivery, such as lipid delivery particles, enhance in vivo intercellular communication. These lipid delivery particles can be used to deliver a payload to a target cell. Developed gene editing agents and other therapeutic agents have broad therapeutic applications in the treatment of diseases and disorders. The delivery of these payloads to the cytosol of target cells however has posed significant challenges and there is a need for novel delivery particles that demonstrate efficient assembly and payload recruitment.SUMMARY

[0003] In an aspect, provided herein is a lipid delivery particle that comprises: (a) a lipid membrane; and (b) a chimeric protein, wherein the chimeric protein comprises a plasma membrane recruitment element and a payload; wherein the chimeric protein further comprises: (i) a particle budding motif; (ii) a membrane penetrating peptide; (iii) a post -translational modification motif that promotes assembly of the lipid delivery particle and / or promotes loading of the payload into the lipid delivery particle; or (iv) a multimerization motif.

[0004] In an aspect, provided herein is a lipid delivery particle that comprises: (a) a lipid membrane; and (b) a chimeric protein, wherein the chimeric protein comprises a plasma membrane recruitment element and a payload; wherein the chimeric protein further comprises: (i) a particle budding motif; (ii) a membrane penetrating peptide; (iii) a post -translational modification motif selected from the group consisting of: myristoylation motif, acetylation motif, isoprenylation motif, palmitoylation motif, and farnesylation motif; or (iv) a multimerization motif.

[0005] In some embodiments, the lipid delivery particle is an ectosome, an exosome, or a viral - like particle.

[0006] In some embodiments, the lipid delivery particle is an ectosome, an exosome, or a viral - like particle.

[0007] In some emb odiments, the plasma membrane recruitment element is a pleckstrin homology (PH) domain.

[0008] In some embodiments, the PH domain is from a protein selected from a group consisting of human phospholipase C51, human Aktl, human Aktl with E17K substitution, human 3 - phosphoinositide-dependent protein kinase 1, human Daapl, mouse Grpl, human Grpl, human OSBP, human Btkl, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, and human MAPKAP1.

[0009] In some embodiments, the plasma membrane recruitment element is linked to a retroviral gag protein. In some embodiments, the plasma membrane recruitment element is linked to an N- terminus of the retroviral gag protein. In some embodiments, the plasma membrane recruitment element is linked to a gag-payload fusion construct. In some embodiments, the plasma membrane recruitment element is linked via its C-terminus to an N-terminus of the payload. In some embodiments, the plasma membrane recruitment element is linked via its N-terminus to a C- terminus of the payload.

[0010] In some embodiments, the payload comprises a therapeutic agent selected from the group consisting of an antibody, a cytokine, a therapeutic protein, a small molecule therapeutic, a therapeutic DNA, and a therapeutic RNA. In some embodiments, the payload comprises a ribonucleoprotein, a zinc-finger, a transcription activator-like effector (TALE), or a CRISPR- based genome editing or modulating protein.

[0011] In some embodiments, the chimeric protein comprises the particle budding motif.

[0012] In some embodiments, the particle budding motif is PTAP, PPXY, LPYX, PPEE, LYPL, LYPSL, PPPY, PPEY, PSAP, LYPAL, PPAP, PPPE, YMYL, YRKL, YQCL, YCYL, LYRTL, YPXnL, or any combination thereof, and n is an integer from 1 to 5.

[0013] In some embodiments, the particle budding motif is linked to the payload or the plasma membrane recruitment element. In some embodiments, is linked between the payload and the plasma membrane recruitment element. In some embodiments, the particle buddingmotif is linked to the chimeric protein based on a construct arrangement listed in Table 12 or Table 15.

[0014] In some embodiments, the chimeric protein comprises the membrane penetrating peptide.

[0015] In some embodiments, the membrane penetrating peptide is linked to the payload or the plasma membrane recruitment element. In some embodiments, the membrane penetrating peptide is linked between the payload and the plasma membrane recruitment element. In some embodiments, the membrane penetrating peptide is linked to the chimeric protein based on a construct arrangement listed in Table 13 .

[0016] In some embodiments, the chimeric protein comprises the post-translational modification motif.

[0017] In some embodiments, the chimeric protein comprises the post-translational modification motif to one or more amino acids of the payload or one or more amino acids of the plasmamembrane recruitment element. In some embodiments, the chimeric protein comprises the post- translational modification motif to one or more amino acids of a region in the chimeric protein outside of the payload and the plasma membrane recruitment element. In some embodiments, the chimeric protein comprises the post-translational modification motif to one or more amino acids of a region in the chimeric protein between the payload and the plasma membrane recruitment element. In some embodiments, the post translational modification motif is linked to the chimeric protein based on a construct arrangement listed in Table 14 or Table 15.

[0018] In some embodiments, the chimeric protein comprises the multimerization motif.

[0019] In some embodiments, the multimerization motif comprises a leucine zipper oligomerization motif. In some embodiments, the multimerization motif is linked to the pay load or the plasma membrane recruitment element. In some embodiments, the multimerization motif is linked between the payload and the plasma membrane recruitment element. In some embodiments, the multimerization motif is linked to the chimeric protein based on a construct arrangement listed in Table 16.

[0020] In some embodiments, the chimeric protein comprises the particle budding motif and the membrane penetrating peptide.

[0021] In some embodiments, the chimeric protein comprises the particle budding motif and the post-translational modification motif.

[0022] In some embodiments, the chimeric protein comprises the particle budding motif and the multimerization motif.

[0023] In some embodiments, the chimeric protein comprises the membrane penetrating peptide and the post-translational modification motif.

[0024] In some embodiments, the chimeric protein comprises the membrane penetrating peptide and the multimerization motif.

[0025] In some embodiments, the chimeric protein comprises the post-translational modification motif and the multimerization motif.

[0026] In some embodiments, the chimeric protein comprises the particle budding motif, the membrane penetrating peptide, and the post -translational modification motif.

[0027] In some embodiments, the chimeric protein comprises the particle budding motif, the membrane penetrating peptide, and the multimerization motif.

[0028] In some embodiments, the chimeric protein comprises the particle buddingmotif, the post- translational modification motif, and the multimerization motif.

[0029] In some embodiments, the chimeric protein comprises the post-translational modification motif, the membrane penetrating peptide, and the multimerization motif.

[0030] In some embodiments, the chimeric protein comprises the particle budding motif, the membrane penetrating peptide, the post-translational modification motif, and the multimerization motif.

[0031] In some embodiments, the chimeric protein comprises a tandem repeat of the particle budding motif, the membrane penetrating peptide, or the post -translational modification motif. In some embodiments, the tandem repeat of the particle budding motif further comprises a linker sequence.

[0032] In some embodiments, the chimeric protein further comprises a nuclear localization signal (NLS) or a nuclear export signal (NES).

[0033] In some embodiments, the nuclear localization signal is linked to the payload or the plasma membrane recruitment element.

[0034] In some embodiments, the nuclear localization signal comprises a sequence having atleast about 80% sequence identity to any one of SEQ ID NOs: 268-280. In some embodiments, the nuclear localization signal comprises a sequence having at least one but not more than 8 modifications of an amino acid sequence as setforth in any one of SEQ ID NOs: 268 -280. In some embodiments, the nuclear localization signal comprises a sequence as set forth in any one of SEQ ID NOs: 268-280.

[0035] In some embodiments, the lipid delivery particle comprises 2, 3, 4, 5, or more NLS sequences.

[0036] In some embodiments, the nuclear export signal is linked to the payload or the plasma membrane recruitment element. In some embodiments, the nuclear export signal is linked between the payload and the plasma membrane recruitment element. In some embodiments, the nuclear export signal is linked to the particle budding motif, the membrane penetrating particle, and / or the post-translational modification motif. In some embodiments, the nuclear export signal is linked to a cleavage site.

[0037] In some emb odiments, the nuclear export signal comprises a sequence having at least about 80% sequence identity to any one of SEQ ID NOs: 87-267. In some embodiments, the nuclear export signal comprises a sequence having at least one but not more than 8 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 87-267. In some embodiments, the nuclear export signal comprises a sequence as set forth in any one of SEQ ID NOs: 87 -267.

[0038] In some embodiments, the lipid delivery particle comprises 2, 3, 4, 5, or more NES sequences.

[0039] In some embodiments, the chimeric protein comprises a linker sequence. In some embodiments, the linker sequence comprises a cleavable linker.

[0040] In some embodiments, the particle budding motif comprises a sequence having at least about 80% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 1 -32. In some embodiments, the particle budding motif comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 1- 32. In some embodiments, the particle budding motif comprises a sequence as set forth in any one of SEQ ID NOs: 1-32.

[0041] In some embodiments, the membrane penetrating peptide comprises a sequence having at least about 80% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 33 -56. In some embodiments, the membrane penetrating peptide comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 33-56. In some embodiments, the membrane penetrating peptide comprises a sequence as set forth in any one of SEQ ID NOs: 33 -56.

[0042] In some embodiments, the post-translational modification motif comprises a sequence having at least about 80% sequence identity to SEQ ID NOs: 57-86. In some embodiments, the post-translational modification motif comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 57-86. In some embodiments, the post-translational modification motif comprises a sequence as set forth in SEQ ID NOs: 57-86.

[0043] In some embodiments, the multimerization motif comprises a sequence having at least about 80% sequence identity to SEQ ID NOs: 281 -285. In some embodiments, the multimerization motif comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as setforth in any one of SEQ ID NOs: 281 -285. In some embodiments, the multimerization motif comprises a sequence as setforth in SEQ ID NOs: 281- 285.

[0044] In some embodiments, the particle budding motif interacts with an endosomal sorting complex required for transport (ESCRT).

[0045] In some embodiments, the lipid delivery particle further comprises an envelope protein.

[0046] In some embodiments, the envelope protein comprises a VSV-G glycoprotein, a human immunodeficiency virus (HIV) GP160 glycoprotein, a Baboon Endogenous Retrovirus (BaEVTR) glycoprotein, a fusion protein of Vesicular stomatitis Indiana virus and Rabies virus glycoprotein (FuG-E), an ecotropic Murine Leukemia Virus envelope protein (MLV ENV ecotropic), a human T-cell lymphotropic virus type 1 (HTLV-1) glycoprotein, an amphotrophic Murine Leukemia Virus envelope protein (MLV ENV amphotropic), a moloney murine leukemia virus 10A1 strain glycoprotein (MLV 10A1), a Baculovirus envelope glycoprotein (GP64), a pantropic MLV envelope protein, a xenotropic MLV envelope protein, a xenotropic murineleukemia virus (XMLV) envelope protein, a Moloney murine leukemia virus (MMLV) envelope protein, a Moloney murine sarcoma virus (MoMSVg) envelope protein, a simian endogenous type D retrovirus protein (RD-114), a gibbon ape leukemia virus (GALV) envelope protein, a feline leukemia virus (FLV) envelope protein, a mouse mammary tumor virus (MMTV) envelope protein, an avian leukosis virus envelope protein, a Rous Sarcoma virus envelope protein, or an endogenous feline virus envelope protein (RD114 ENV), or a mutant thereof.

[0047] In some embodiments, the envelope protein comprises a HIV-1 envelope protein, a respiratory syncytial virus glycoprotein, a SARS-CoV glycoprotein, a rabies glycoprotein, a Mokola virus glycoprotein, a Semliki Forest virus glycoprotein, a Venezuelan equine encephalitis virus glycoprotein, an Ebola virus glycoprotein, a Marburg virus glycoprotein, or a mutant thereof.

[0048] In some embodiments, the envelope protein comprises a glycoprotein of a mammalian endogenous retrovirus or mutant thereof. In some embodiments, the glycoprotein of the mammalian endogenous retrovirus is a glycoprotein of a human endogenous retrovirus (hERV). In some embodiments, the glycoprotein of a hERV comprises a hENVHl, a hENVH2, ahENVH3, a hENVKl, a hENVK2, a hENVK3, a hENVK4, a hENVK5, a hENVK6, a hENVT, a hENVW, a hENVFRD, a hENVR, a hENVR(b), a hENVR(c)l, a hENVR(c)2 or a hENVKcon, or a mutant thereof.

[0049] In some embodiments, the envelope protein is fused to a targeting moiety, optionally a scFv.

[0050] In some embodiments, the envelope protein comprises a non-viral envelope protein.

[0051] In an aspect, provided herein is a chimeric protein comprising: (a) a plasma membrane recruitment element; (b) a payload; and (c) one, two, three, or all of : (i) a particle budding motif; (ii) a membrane penetrating peptide; (iii) a post-translational modification motif that promotes assembly of the lipid delivery particle and / or promotes loading of the payload into the lipid delivery particle; or (iv) a multimerization motif.

[0052] In an aspect, provided herein is a chimeric protein comprising: (a) a plasma membrane recruitment element; (b) a payload; and (c) one, two, three, or all of : (i) a particle budding motif; (ii) a membrane penetrating peptide; (iii) a post-translational modification motif selected from the group consisting of: myristoylation motif, acetylation motif, isoprenylation motif, palmitoylation motif, and farnesylation motif; or (iv) a multimerization motif.

[0053] In some emb odiments, the plasma membrane recruitment element is a pleckstrin homology (PH) domain. In some embodiments, the plasma membrane recruitment element is a mutated PH domain or an engineered PH domain.

[0054] In some embodiments, the PH domain is from a protein selected from a group consisting of human phospholipase C51, human Aktl, human Aktl with E17K substitution, human 3 -phosphoinositide-dependent protein kinase 1, human Daapl, mouse Grpl, human Grpl, human OSBP, human Btkl, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, and human MAPKAP1.

[0055] In some embodiments, the plasma membrane recruitment element is linked to a retroviral gag protein. In some embodiments, the plasma membrane recruitment element is linked to an N- terminus of the retroviral gag protein. In some embodiments, the plasma membrane recruitment element is linked to a gag-payload fusion construct. In some embodiments, the plasma membrane recruitment element is linked via its C-terminus to an N-terminus of the payload. In some embodiments, the plasma membrane recruitment element is linked via its N-terminus to a C- terminus of the payload.

[0056] In some embodiments, the payload comprises a therapeutic agent selected from the group consisting of an antibody, a cytokine, a therapeutic protein, a small molecule therapeutic, a therapeutic DNA, and a therapeutic RNA. In some embodiments, the payload comprises a ribonucleoprotein, a zinc-finger, a transcription activator-like effector (TALE), or a CRISPR- based genome editing or modulating protein.

[0057] In some embodiments, the chimeric protein comprises the particle budding motif.

[0058] In some embodiments, the particle budding motif is PTAP, PPXY, LPYX, PPEE, LYPL, LYPSL, PPPY, PPEY, PSAP, LYPAL, PPAP, PPPE, YMYL, YRKL, YQCL, YCYL, LYRTL, YPXnL, or any combination thereof, and n is an integer from 1 to 5.

[0059] In some embodiments, the particle budding motif is linked to the payload or the plasma membrane recruitment element. In some embodiments, the particle budding motif is linked between the payload and the plasma membrane recruitment element. In some embodiments, the particle budding motif is linked to the chimeric protein based on a construct arrangement listed in Table 12 or Table 15.

[0060] In some embodiments, the chimeric protein comprises the membrane penetrating peptide.

[0061] In some embodiments, the membrane penetrating peptide is linked to the payload or the plasma membrane recruitment element. In some embodiments, the membrane penetrating peptide is linked between the payload and the plasma membrane recruitment element. In some embodiments, the membrane penetrating peptide is linked to the chimeric protein based on a construct arrangement listed in Table 13 .

[0062] In some embodiments, the chimeric protein comprises the post-translational modification motif.

[0063] In some embodiments, the chimeric protein comprises the post-translational modification motif to one or more amino acids of the payload or one or more amino acids of the plasma membrane recruitment element. In some embodiments, the chimeric protein comprises the post-translational modification motif to one or more amino acids of a region in the chimeric protein outside of the payload and the plasma membrane recruitment element. In some embodiments, the chimeric protein comprises the post-translational modification motif to one or more amino acids of a region in the chimeric protein between the payload and the plasma membrane recruitment element. In some embodiments, the post-translational modification motif is linked to the chimeric protein based on a construct arrangement listed in Table 14 or Table 15 .

[0064] In some embodiments, the chimeric protein comprises the multimerization motif. In some embodiments, the multimerization motif comprises a leucine zipper oligomerization motif.

[0065] In some embodiments, the multimerization motif is linked to the payload or the plasma membrane recruitment element. In some embodiments, the multimerization motif is linked between the payload and the plasma membrane recruitment element. In some embodiments, the multimerization motif is linked to the chimeric protein based on a construct arrangement listed in Table 16.

[0066] In some embodiments, the chimeric protein comprises the particle budding motif and the membrane penetrating peptide.

[0067] In some embodiments, the chimeric protein comprises the particle budding motif and the post-translational modification motif.

[0068] In some embodiments, the chimeric protein comprises the particle budding motif and the multimerization motif.

[0069] In some embodiments, the chimeric protein comprises the membrane penetrating peptide and the post-translational modification motif.

[0070] In some embodiments, the chimeric protein comprises the membrane penetrating peptide and the multimerization motif.

[0071] In some embodiments, the chimeric protein comprises the post-translational modification motif and the multimerization motif.

[0072] In some embodiments, the chimeric protein comprises the particle budding motif, the membrane penetrating peptide, and the post-translational modification motif.

[0073] In some embodiments, the chimeric protein comprises the particle budding motif, the membrane penetrating peptide, and the multimerization motif.

[0074] In some embodiments, the chimeric protein comprises the particle buddingmotif, the post- translational modification motif, and the multimerization motif.

[0075] In some embodiments, the chimeric protein comprises the post-translational modification motif, the membrane penetrating peptide, and the multimerization motif.

[0076] In some embodiments, the chimeric protein comprises the particle budding motif, the membrane penetrating peptide, the post-translational modification motif, and the multimerization motif.

[0077] In some embodiments, the chimeric protein comprises a tandem repeat of the particle budding motif, the membrane penetrating peptide, or the post -translational modification motif. In some embodiments, the tandem repeat of the particle budding motif further comprises a linker sequence.

[0078] In some embodiments, the chimeric protein further comprises a nuclear localization signal (NLS) or a nuclear export signal (NES).

[0079] In some embodiments, the nuclear localization signal is linked to the payload or the plasma membrane recruitment element.

[0080] In some embodiments, the nuclear localization signal comprises a sequence having atleast about 80% sequence identity to SEQ ID NOs: 268-280. In some embodiments, the nuclear localization signal comprises a sequence having at least one but not more than 8 modifications of an amino acid sequence as setforthin SEQ ID NOs: 268-280. In some embodiments, the nuclear localization signal comprises a sequence as set forth in SEQ ID NOs: 268-280.

[0081] In some embodiments, wherein the chimeric protein comprises 2, 3, 4, 5, or more NLS sequences.

[0082] In some embodiments, the nuclear export signal is linked to the payload or the plasma membrane recruitment element. In some emb odiments, the nuclear export signal is linked between the payload and the plasma membrane recruitment element. In some embodiments, the nuclear export signal is linked to the particle budding motif, the membrane penetrating particle, the post- translational modification motif, and / or the multimerization motif. In some embodiments, the nuclear export signal is linked to a cleavage site.

[0083] In some emb odiments, the nuclear export signal comprises a sequence having at least about 80% sequence identity to SEQ ID NOs: 87-267. In some embodiments, the nuclear export signal comprises a sequence having at least one but not more than 8 modifications of an amino acid sequence as set forth in SEQ ID NOs: 87-267. In some embodiments, the nuclear export signal comprises a sequence as set forth in SEQ ID NOs: 87-267.

[0084] In some embodiments, the chimeric protein comprises 2, 3, 4, 5, or more NES sequences.

[0085] In some embodiments, the chimeric protein further comprises a linker sequence. In some embodiments, the linker sequence comprises a cleavable linker.

[0086] In some embodiments, the post-translational modification comprises myristoylation motif, acetylation motif, isoprenylation motif, palmitoylation motif, farnesylation motif, or any combination thereof.

[0087] In some embodiments, the particle budding motif comprises a sequence having at least about 80% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 1 -32. In some embodiments, the particle budding motif comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 1 - 32. In some embodiments, the particle budding motif comprises a sequence as set forth in any one of SEQ ID NOs: 1-32.

[0088] In some embodiments, the membrane penetrating peptide comprises a sequence having at least about 80% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 33 -56. In some embodiments, the membrane penetrating peptide comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 33-56. In some embodiments, the membrane penetrating peptide comprises a sequence as set forth in any one of SEQ ID NOs: 33-56.

[0089] In some embodiments, the post-translational modification motif comprises a sequence having at least about 80% sequence identity to SEQ ID NOs: 57-86. In some embodiments, the post-translational modification motif comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 57-86. In some embodiments, the post-translational modification motif comprises a sequence as set forth in SEQ ID NOs: 57-86.

[0090] In some embodiments, the multimerization motif comprises a sequence having at least about 80% sequence identity to SEQ ID NOs: 281 -285. In some embodiments, the multimerization motif comprises a sequence having at least one but not more than 10 modifications of an amino acid sequenceas setforth in any one of SEQ ID NOs: 281 -285. In some embodiments, the multimerization motif comprises a sequence as set forth in SEQ ID NOs: 281- 285.

[0091] In some embodiments, the particle budding motif interacts with an endosomal sorting complex required for transport (ESCRT).

[0092] In an aspect, provided herein is a method for preparing a lipid delivery particle comprising a payload, the method comprising: (a) providing a producer cell that produces the lipid delivery particle described herein; and (b) collecting the lipid delivery particle from the producer cell.

[0093] In some embodiments, the producer cell comprises a nucleic acid sequence encoding the chimeric protein.

[0094] In some embodiments, the method further comprises expressing in the producer cell the nucleic acid molecule encoding the chimeric protein. In some embodiments, the method further comprises transiently expressing in the producer cell the nucleic acid molecule encoding thechimeric protein. In some embodiments, the method further comprises permanently expressing in the producer cell the nucleic acid molecule encoding the chimeric protein .

[0095] In some embodiments, the method further comprises culturing the producer cell in a medium and maintaining the producer under conditions to produce the lipid delivery particle .

[0096] In some embodiments, the method further comprises harvesting the medium and purifying the extracellular vesicle. In some embodiments, the purifying retains the structural integrity of the extracellular vesicle.

[0097] In an aspect, provided herein is a lipid delivery particle produced by the method described herein.

[0098] In an aspect, provided herein is a nucleic acid sequence encoding the chimeric protein described herein.

[0099] In an aspect, provided herein is a composition comprising: (a) a firstnucleic acid sequence encoding an envelopeprotein; and(b) a second nucleic acid sequence encoding a chimeric protein, wherein the chimeric protein comprises: (i) a plasma membrane recruitment element; (ii) a payload; and (iii) one, two, there, or all of: a particle budding motif; a membrane penetrating peptide; a post-translational modification motif; or a multimerization motif.

[0100] In an aspect, provided herein is a vector comprising the nucleic acid sequence described herein.

[0101] In some embodiments, thevector is a vector selected from a plasmid, a cosmid, a bacterial vector, a viral vector, an artificial chromosome.

[0102] In an aspect, provided herein is a cell comprising the lipid delivery particle described herein.

[0103] In an aspect, provided herein is a cell produced by the method described herein.

[0104] In an aspect, provided herein is a pharmaceutical formulationcomprisingthe lipid delivery particle described herein and a pharmaceutically acceptable carrier, excipient, adjuvant or diluent.

[0105] In an aspect, provided herein is a kit comprising: (a) the lipid delivery particle described herein, orthe pharmaceutical formulation described herein; and (b) instructions for use of the lipid delivery particle or the pharmaceutical formulation.INCORPORATION BY REFERENCE

[0106] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0107] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0108] FIGs. 1A-1E are schematic representations of constructs encoding exemplary chimeric protein accordingto some embodiments of the present disclosure. FIG. 1 A shows a construct with a promoter sequence (pCMV), sequence encoding a payload, and sequence encoding a plasma membrane recruitment element such as a PH domain (new PH or mutant PH), which is located upstream of (5 ’ to) ordownstream of (3 ’ to) the nucleic acid encodingthe payload . FIG. IB shows a construct with nucleic sequences encoding a payload, a PH domain, and a particle buddingmotif (also named as budding domain, or BD). FIG. 1C shows a construct with nucleic acid sequences encoding a pay load, a PH domain, and a nuclear localization signal (NLS) or nuclear export signal (NES). FIG. ID shows a construct with nucleic acid sequences encoding a payload, a PH domain, and a membrane penetrating peptide (MPP) motif. FIG. IE shows a construct with nucleic acid sequences encoding a payload, a PH domain, and a post -translational modification (PTM) motif.

[0109] FIG. IF shows a chimeric protein containing a plasma membrane recruitment element (PMRE), a repeat of two particle budding motifs (BD), a multimerization motif, such as leucine zipper oligomerization motif (LZ), a third particle budding motif, and a payload.

[0110] FIG. 2 is an exemplary process diagram for manufacturing lipid delivery particles of the present disclosure that can be used to deliver a payload.

[0111] FIG. 3 is a schematic representation of an exemplary construct for a pleckstrin homology domain (PH domain of protein lipase C, PLC PH) fused to a late domain via a linker.

[0112] FIG. 4 is a schematic representation of a lipid delivery particle. The particle comprises a VSVG envelope protein and a pleckstrin homology domain (PH) fused to a payload (e.g., ABE8e, which is coupled with sgRNA).

[0113] FIG. 5A is a schematic representation of constructs for the example lipid delivery particle of FIG. 4. The three constructs comprise (i) a pU6 plasmid encoding sgRNA, (ii) a plasmid with a CMV promoter, followed by sequences encoding adenine base editor payload (ABE8e) and PH domain, and (iii) a plasmid with a CMV promoter followed by sequence encoding VSVG envelope.

[0114] FIG. 5B is a schematic representation of the mechanism for formation of the lipid delivery particle. The PH domain fused to the nuclease buds from the 293 T cell forming a particle with VSVG envelope protein and delivers its payload into the transduced cell.

[0115] FIG. 6A is a schematic representation of an exemplary chimeric protein comprising a payload and PH domain (e.g., AKT (also named as Aktl) with E17K substitution). FIG. 6Bdepicts results of experiments testing effects of various nuclear localization signal (NLS) / nuclear export signal (NES) on base editing efficiency of the resultant lipid delivery particles. Percent adenine to guanine change of Bel la gene in 293 T cells was measured as a readout of the tests following delivery of ABE8e.

[0116] FIGs. 7A-7B depict results of experiments assessing effects of various budding domains on base editing efficacy. FIG. 7A depicts results of high dose of delivery particles (30 pl lOOx PEG-concentrated particles). FIG. 7B depicts results of low dose of delivery particles (1 pl 50x PEG-concentrated particles). Both the high-dose and low-dose of delivery particles showed that PPPY + PSAP motif had the greatest editing percentage.

[0117] FIG. 8A-8B depict the structure and results of an experiment assessing effects of budding domains on delivery particles on base editing efficacy. FIG. 8A shows schematic representations of a subset of tested constructs. ABE8e-PH-NLS, having wildtype AKT PH domain, corresponds to AKT WT in FIG. 8B, ABE8e-PH(E17K)-NLS, having AKT PH domain with E17K substitution, corresponds to AKT E17K in FIG. 8B, and ABE8e-PH(E17K)-BD-NLS, having Aktl PH domain with El 7K substitution and budding domain, corresponds to AKT El 7K 3x BD in FIG. 8B. FIG. 8B shows the results of the experiment measuring A to G conversion. At 1.0 pl, addition of the budding domain (BD) improved editing percentage compared to controls (ABE8e with no PH and AKT WT).

[0118] FIG. 9 depicts a schematic representation of an exemplary payload construct comprising a multimerization domain (MD). The multimerization domain has a fusion at the C-terminus.

[0119] FIGs. 10A-10B depict the structure and results of an experiment to assess effects of multimerization domains on delivery particles on base editing efficacy. FIG. 10 A shows schematic representations of a subset of tested constructs. ABE8e-PH-NLS, having wildtype AKT PH domain, corresponds to AKT WT in FIG. 10B, ABE8e-PH(E17K)-NLS, having AKT PH domain with E17K substitution, corresponds to AKT El 7K in FIG. 10B, and ABE8e-PH(E17K)- MD-NLS, having wildtype AKT PH domain with E17K substitution and a multimerization domain, corresponds to MD Dimer in FIG. 10B. FIG. 10B shows the results of the analysis measuring A to G conversion with MD domains. At 1.0 pl dose, addition of the dimerization domain (MD) improved editing percentage compared to controls (ABE8e with no PH and AKT WT).

[0120] FIGs 11A-11B depict constructs and results examining effects of addition of post- translational modifications to delivery particles on base editing efficacy. FIG. 11 A shows an exemplary construct comprising a post-translational modification (PTM) motif at the N-terminus of the payload. The construct comprises from N-terminus to C-terminus PTM, payload (ABE8e), AKT PH domain with E17K mutation, and NLS. FIG. 11B shows results of the experiment toassess editing efficiency across original delivery particles (Original), particles with multimerization domain (MD), particles with budding domain (BD), and particles with post- translational modification (e.g., myristoylation; PTM). Particles were tested at doses of 0.2, 2.0 and 20.0 pl. At 2.0 pl dose, addition of PTM, BD, and MD to the particle increased the editing percentage compared to that from the original particle (far right bars).DETAILED DESCRIPTION

[0121] Disclosed herein, in some aspects, are lipid delivery particles comprising a phospholipid bilayer and a chimeric protein. The chimeric protein can comprise a plasma membrane recruitment element and a payload, and can further comprise: (i) a particle budding motif; (ii) a membrane penetrating peptide motif; or (iii) a post-translational modification motif, or (iv) a multimerization motif. In some cases, the lipid delivery particle is an extracellular versicle. In some cases, the lipid delivery particle is an ectosome, an exosome, or a viral-like particle. In some cases, the chimeric protein can comprise one, two, or all of: (i) a particle budding motif; (ii) a membrane penetrating peptide motif; (iii) a post-translational modification motif; or (iv) a multimerization motif.

[0122] Without wishing to be bound by a certain theory, presence of one or more of (i) a particle budding motif; (ii) a membrane penetrating peptide motif; (iii) a post-translational modification motif; or(iv) a multimerization motif in the chimeric protein of the present disclosure can improve production of the lipid delivery particles. Without wishing to be bound by theory, presence of (i) a particle budding motif; (ii) a membrane penetrating peptide motif; (iii) a post-translational modification motif; or (iv) a multimerization motif in the chimeric protein of the present disclosure can increase the total number of particles produced, increase the amount of payload encapsulated per particle, and / or increase the efficiency of payload release. In some cases, additional motifs are present in the chimeric protein of the lipid delivery particle. The motifs of the present disclosure can also include polypeptide sequences that are additionally fused to the fusion of the plasma membrane recruitment element and the payload, or in some cases replacing or modifying elements of the fusion of the plasma membrane recruitment element and payload.

[0123] The plasma membrane recruitment elements and motifs can be placed at the N-terminus, C-terminus, or between the N- and C-terminus of the construct. In some cases, the plasma membrane recruitment elements and motifs can be present in repeating fashion or at various segments of the DNA.

[0124] In some cases, the plasma membrane recruitment element comprises a pleckstrin homology (PH) domain. In some cases, the PH domain is an engineered PH domain or a mutated PH domain. Without wishingto beboundby theory, the PH domain can assist recruitinga payload into the lipid delivery particle and enhance release of the payload into a target cell. Accordingly, the PH domain can be fused to the payload. In some cases, the chimeric protein further comprisesa gag protein, e.g. , a retroviral gag protein. In some cases, in the chimeric protein, the PH domain is fused to a gag-payload fusion construct.

[0125] The payload disclosed herein can comprise therapeutic agents. In some cases, the payload comprises gene editing agent including, but not limited to, a ribonucleoprotein, a zinc-finger, a transcription activator-like effector (TALE), or a CRISPR-based genome editing or modulating protein. Alternatively or additionally, the payload can comprise a biomarker, an imaging agent, a sugar or polysaccharide, or a small molecule.

[0126] The lipid delivery particle can comprise an envelope protein. In some cases, the envelope protein is of a viral origin, e.g., having a sequence identical to a wildtype viral protein, or being a variantthereof (e.g., havingatleast 80% sequence identity to the wildtype viral protein sequence). In some cases, the envelope protein is a VSV-G glycoprotein, a human immunodeficiency vims (HIV) GP160 glycoprotein, or a human T-cell lymphotropic virus type 1 (HTLV-1) glycoprotein. In some cases, the envelope protein is derived from a mammalian endogenous retrovirus. In some cases, the mammalian endogenous retrovirus is a koala retrovirus (KoRV) or a Jaagsiekte sheep retrovirus (enJSRV). In some cases, the envelope protein is derived from a human endogenous retrovirus. In some cases, the envelope protein is a non -viral protein.

[0127] In another aspect, the disclosure features a chimeric protein comprising a plasma membrane recruitment element, a payload, and one, two, or all of: (i) a particle budding motif; (ii) a membrane penetrating peptide; (iii) a post -translational modification motif; or (iv) a multimerization motif.

[0128] In another aspect, the disclosure features a nucleic acid molecule encoding a chimeric protein described herein.

[0129] In another aspect, the disclosure features a vector, e.g., an expression vector, comprising the nucleic acid molecules described herein.

[0130] In some cases, the vector comprises a plasmid, a cosmid, a bacterial vector, a viral vector, an artificial chromosome, a liposome, a lipid nanoparticle, or an exosome.

[0131] In another aspect, the disclosure features a cell comprising a lipid delivery particle, a nucleic acid molecule or a vector described herein.

[0132] In another aspect, the disclosure features a method of making, e.g., producing, a lipid delivery particle described herein, comprising providing a producer cell that produces the lipid delivery particle as described in the present disclosure.

[0133] In another aspect, the disclosure features a method of making, e.g., producing, a lipid delivery particle described herein, comprising culturing a producer cell described herein under suitable conditions, e.g., conditions suitable for gene expression.

[0134] In another aspect, the disclosure features a pharmaceutical formulation comprising a lipid delivery particle described herein.

[0135] In another aspect, the disclosurefeatures a kit comprising a lipid delivery particle, a nucleic acid molecule or a pharmaceutical formulation as described herein.

[0136] Accordingly, provided herein are, inter alia, lipid delivery particles (e.g., ectosomes, exosomes, or viral-like particles) that include the aforesaid motifs, nucleic acids encoding the same, and methods of producing the aforesaid particles.DEFINITIONS

[0137] Certain specific details of this description are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand thatthe present disclosure may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the disclosure.

[0138] Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure.

[0139] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The use of the words “a” or “an” when used in conjunction with the term “comprising” herein may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0140] It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0141] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below.

[0142] The term “about” when referringto a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0. 1% from the specified value, as such variations are appropriate to perform the disclosedmethods. As used herein, “about’ and “approximately” generally mean an acceptable degree of error forthe quantity measured giventhe nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given range of values.

[0143] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non -homologous sequences can be disregarded for comparison purposes). In some cases, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

[0144] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some cases, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg. com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In some cases, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http : / / www. gcg.com), using a NWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5.

[0145] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11 -17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecule of the disclosure. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the disclosure. To obtain gapped alignments for comparisonpurposes, GappedBLAST canbeutilized as described in Altschul et al., (1997) Nucleic Acids Res. 25 :3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0146] It is understood that the molecules of the present disclosure may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on their functions.

[0147] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at leasttwo amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0148] The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.

[0149] The term “promoter” refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0150] An example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. In some cases, a polynucleotide comprises a CMV promoter. In some cases, a polynucleotide may comprise a promoter selected from the group consisting of, but not limited to, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV), a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia vims promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus (RSV) promoter, an actin promoter, a myosin promoter, an elongation factor- la promoter, a hemoglobin promoter, and a creatine kinase promoter. In some cases, a polynucleotide comprises a sequence encoding a poly(A) tail. In some cases, a polynucleotide comprises a 3’ UTR sequence. In some cases, a polynucleotide comprises a 5 ’ UTR sequence. In some cases, a polynucleotide comprises multiple smaller and discrete nucleotide sequences that are typically heterologous and exhibit different and measurable function(s), a promoter sequence, a translation initiating sequence, a start codon, a polyadenylation sequence, a stop codon, and / or a linker sequence.

[0151] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human). Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. A “patient” is a subject suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.

[0152] The term “therapeutic” as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.

[0153] The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state.

[0154] The term “transfected” or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny. The term “transformed cell” or “transfected cell” can be a transformed or transfected cell in which inserted DNA can replicate either as an autonomously replicating plasmid or as part of the host chromosome. In some cases, a transfected cell or transformed cell can express the inserted DNA or RNA transiently (e.g., for brief periods of time).

[0155] The terms “nucleic acid,” “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence,” and “polynucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, oranalogs thereof. The polynucleotide may be either single -stranded or double-stranded, and if single-stranded may be the coding strand or non-coding (antisense) strand. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. Apolynucleotidemay be further modified after polymerization, such as by conjugation with a labeling component. The nucleic acid may be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a non-natural arrangement.

[0156] The term “cell line” or “producer cell line”, as used herein, refers to cultured cells that can be passed (e.g., divided) more than once. In some cases, a cell like can be passed more than 2 times, more than 5 times, more than 10 times, more than 50 times, more than 100 times, or more than 150 times. In some cases, a cell line can be passed between 2-250 times. In some cases, a cell line can be passed up to 200 times.

[0157] The term “lipid delivery particle” refers to lipid vesicles secreted by the plasma membrane of a cell having a membrane or envelope that surrounds a central internal space. Lipid delivery particles can be produced via budding out of the cell’s plasma membrane, secretion from lysosomes, or export by the Golgi complex. Lipid delivery particles can promote intercellular communication in vitro and in vivo. Exemplary lipid delivery particles can include, but are not limited to, exosomes, ectosomes (e.g., microvesicles and shedding vesicles), apoptotic bodies, microsomes, micelles, oncosomes, and liposomes.

[0158] Lipid delivery particles can have a range of size, such as a cross-sectional diameter, wherein the cross-sectional diameter has a range from about 1 nm to about 1200 nm, about 10 nm to about 1200 nm, about 25 nm to about 1200 nm, about 50 nm to about 1200 nm, about 100 nm to about 1200 nm, about 10 nm to about lOOO nm, about 25 nm to about 1000 nm, about 50 nm to about 1000 nm, about 100 nm to about 1000 nm, about 10 nm to about 500 nm, about 25 nm to about 500 nm, about 50 nm to about 500 nm or about 100 nm to about 500 nm (all inclusive). Lipid delivery particles can comprise a vesicle size of greater than 10 nm. Lipid delivery particles can comprise a vesicle size of less than 1000 nm.

[0159] The term “module” or “modification”, used interchangeably, can refer to an addition or a motif that is added to a payload or a chimeric protein within lipid delivery particles (e.g., extracellular vesicles and / or virus-like particles). The additions can enhance characteristics of particle assembly and / or payload recruitment. A module can occur in combination or alone. A module can be linked to a payload or another element (e.g., domain) in a construct.

[0160] The term “purifying,” “purified,” and “purify,” can be used interchangeably and refer to a preparation phaseof a lipid delivery particle. A preparation of lipid delivery particles can comprisea known or unknown concentration of a substance. In some cases, purifying produced lipid delivery particles comprises removing, either partially removing or wholly removing, a portion of the produced lipid delivery particles from a sample containing one or more biological components (e.g., producer cells). In some cases, a composition comprising lipid delivery particles, as described herein, that have been purified are enriched as compared to a starting sample. Enrichment of the lipid delivery particles can comprise 5%, 10%, 25%, 50%, 75%, 80%,, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% enrichment compared to a starting sample. In some cases, purified lipid delivery particles can be free or substantially free of residual biological products. Residual biological products can include, but are not limited to, unwanted nucleic acids, proteins, lipids, and / or or metabolites or abiotic materials such as including chemicals. In some cases, lipid delivery particles free of residual biological products comprise lipid delivery particles containing no detectable producer cells in the composition.

[0161] Ranges: throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values withinthat range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97- 99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.LIPID DELIVERY PARTICLE

[0162] In an aspect, described herein is a lipid delivery particle comprising a membrane and a chimeric protein. The chimeric protein can promote targeting of a payload to a recipient cell (e.g, target cell). The lipid delivery particle can comprise an extracellular vesicle secreted by a cell. A lipid delivery particle can be secreted by cells in vivo or if cultured in vitro. Following secretion, the lipid delivery particle can deliver a payload and release its components into a recipient cell. In some cases, the lipid delivery particle can comprise an exosome, an ectosome (e.g., a microvesicle or a shedding vesicle), an apoptotic body, a microsome, a micelle, an oncosome, or a liposome. In some cases, the lipid delivery particle can comprise a nanoparticle.

[0163] Ectosomes can be small heterogenous membrane-bound particles that are shed from the surface of the cell membrane in the extracellular space. Ectosomes can be shed by budding oroutward blebbing from the plasma membrane of the cell. Ectosomes can be shed from plasma membrane micro-domains (e.g., lipid rafts or caveolae domains). Ectosomes have a series of biological functions, including, but not limited to, regulation of nucleic acid metabolism, signal transduction, transport, protein metabolism, intercellular communication, apoptosis, and cell growth. As described herein, ectosomes can comprise a payload that can be delivered to a target cell. Ectosomes can be variable in size. In some cases, an ectosome can be between 25 and 1000 nm. In some cases, the ectosome can be 30 nm, 50 nm, 75 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 1000 nm in size. In some cases, the ectosome can be between about 80 and about 200 nm in size. In some cases, the ectosome is about 80 nm, about 90 nm, about 100 nm, about 1 lO nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, or about 200 nm in size. Formation of ectosomes can be regulated via the endosomal sorting complex required for transport (ESCRT), and / or by non-ESCRT-related mechanisms, including tetraspanins and membrane lipids.

[0164] Exosomes can be small vesicular particles formed by intraluminal vesicles within multivesicular bodies. Exosome can range from 30-125 nm. In some cases, an exosome can comprise 30 nm, 40 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, or 100 nm in size. Formation of exosomes can be regulated via the endosomal sorting complex required for transport (ESCRT), and / or by non-ESCRT-related mechanisms, including tetraspanins and membrane lipids.

[0165] Virus-like particles (VLPs) can be assemblies ofviral proteins that can infect cells but lack viral general material. VLPs can be synthesized through expression of viral structural proteins. VLPs can be formed in vivo through assembly inside a producer cell via recombinant protein expression or in vitro through self-assembly in a vessel using quantities of purified protein. VLPs can be designed to deliver payloads to targeted cells and tissues. VLPs can be used to deliver to a recipient cell an agent (e.g., prophylactic agent, therapeutic agent or diagnostic agent) or an enclosed circular or linear DNA or RNA molecule. Formation of VLPs can be regulated via the endosomal sorting complex required for transport (ESCRT), and / or by non-ESCRT-related mechanisms, including tetraspanins and membrane lipids.

[0166] Nanoparticles can be synthesized particles between 1 and 200 nm in size that can encapsulate payload for delivery to a recipient cell. In some cases, nanoparticles are engineered for a specific cell type and trigger endocytosis and later endosome lysis . In some cases, a nanoparticle can be about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 105 nm, about 1 lO nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 160 nm,about 170 nm, about 180 nm, about 190nm, or about 200 nm in size. With out wishing to be bound by theory, the small size can allow a higher intracellular uptake compared to larger molecules.

[0167] For example, a lipid delivery particle can comprise: (a) a lipid membrane; and (b) a chimeric protein, wherein the chimeric protein comprises a plasma membrane recruitment element and a payload; wherein the chimeric protein further comprises: (i) a particle budding motif; (ii) a membrane penetrating peptide; (iii) a post-translational modification motif that promotes assembly of the lipid delivery particle and / or promotes loading of the payload into the lipid delivery particle; (iv) a multimerization motif, or (v) any combination thereof.

[0168] As another example, a lipid delivery particle can comprise: (a) a lipid membrane; and (b) a chimeric protein, wherein the chimeric protein comprises a plasma membrane recruitment element and a payload; wherein the chimeric protein further comprises: (i) a particle budding motif; (ii) a membrane penetrating peptide; (iii) a post -translational modification motif selected from the group consisting of: myristoylation motif, acetylation motif, isoprenylation motif, palmitoylation motif, and famesylation motif; (iv) a multimerization motif, or (v) any combination thereof.

[0169] As shown in FIG. 4, a lipid delivery particle may comprise an envelope protein (e.g., VSV- G), a payload (e.g., base editor ABE8e), and a plasma membrane recruitment element (e.g., PH). The payload and plasma membrane recruitment element may be fused.ENVELOPE PROTEIN

[0170] In some aspects, the lipid delivery particle provided herein comprises an envelope protein. The envelope protein can be associated with the outside boundary or the surface of the lipid delivery particle, for example, the membrane or envelope of the lipid delivery particle.

[0171] The membrane of the lipid delivery particle can comprise a lipid layer, such as a single layer or a lipid bilayer. In some cases, the membrane of the lipid delivery particle is from plasma membrane, endoplasmic reticulum, or a combination thereof. In some cases, the membrane of the lipid delivery particle is from Golgi complex, ER Golgi intermediate compartment, or nuclear envelope. In some cases, the membrane of the lipid delivery particle is from plasma membrane. In some cases, the membrane of the lipid delivery particle is a phospholipid bilayer.

[0172] The envelope protein canbe associated with the membrane of the lipid delivery particle in various manners. For example, the envelope protein can be anchored or attached to the external membrane of the particle or anchored or attached to the internal membrane of the particle. The envelope protein canbe embedded or inserted in the membrane, spanning through the membrane, with certain portions located at the outside of the membrane, or certain portions extendi ng to the inside of the particle, or both. The envelope protein within the lipid delivery particle describedherein can be overexpressed from an exogenous source, such as plasmids or stably integrated transgenes, in the production cells.

[0173] The envelope protein can play a role in the delivery of the lipid delivery particle to a target cell and release of the components of the lipid delivery particle within the target cell. The envelope protein can contact with the surfaceof atargetcell and participate in the fusion ofthe lipid delivery particle and the membrane of the target cell. The envelope protein can participate in the fusion of the lipid delivery particle with the membrane of the target cell via any appropriate mechanism, such as those described in White et al. Crit Rev Biochem Mol Biol. 2008; 43(3): 189-219. One example of the fusion mechanismsis unifying Trimer-of-Hairpins Fusion Mechanism. Membrane fusion can occur after allosteric priming by binding to a target receptor. In some cases, membrane fusion occurs after proteolysis. In some cases, membrane fusion occurs after isomerization of disulfide bridges. In some cases, membrane fusion occurs by internalization and then priming of fusion via (i) cathepsin -mediatedproteolysis, or (ii) low pH / acidification. The cathepsin-mediated proteolysis can be pH dependent or pH independent. Other fusion triggering mechanisms can include low PH, binding to target cell receptors, and a receptor followed by low pH. The envelope protein can also play a role in the formation of the lipid delivery particle. The envelope protein can interact with another component within the lipid delivery particle and participate in the assembly of the lipid delivery particle, for example, in a producer cell. The envelope protein can make contact with another envelope protein and form an oligomer embedded within the membrane. The envelope protein can be a glycoprotein, for example, a transmembrane glycoprotein. In some cases, envelope protein comprises multiple membrane -spanning regions. These multiple membrane-spanning regions can oligomerize and form channels in the membrane.

[0174] In some cases, the envelope protein is fused with a targeting moiety. In some cases, the targeting moiety recognizes a specific molecule (e.g , antigen, receptor, or other membrane protein) on the surface of a target cell to allow targeted cell entry with more specificity. In some cases, the targeting moiety is specific for a certain cell type or is specific for a certain target cell. The targeting moiety can be fused to the envelope protein at a position that is located at an outside of the lipid delivery particle. For example, the targeting moiety includes scFvs, antibody variable regions, nanobodies, T-cell receptor variable regions, other antigen -binding fragments or their mimetics, such as DARPins. In some cases, the targeting moiety is a protein ligand from the human ligandome. The targeting moiety can be a natural peptide or a synthetic peptide. In some cases, the targeting moiety is not fused with the envelope protein and is attached to the membrane of the lipid delivery particle from the outside, for example, via a transmembrane domain.

[0175] A targeting moiety can include, e.g., an antibody or an antigen-binding fragment thereof (e.g., Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fdfragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), nanob odies, or camelidVHH domains), an antigen -binding fibronectin type III (Fn3) scaffold such as a fibronectin polypeptide minibody, a ligand, a cytokine, a chemokine, or a T cell receptor (TCRs). Membrane-fusion proteins can be re-targeted by noncov alently conjugating a targeting moiety to the membrane -fusion protein or targeting protein (e.g. the hemagglutinin protein). For example, the membrane-fusion protein can be engineered to bind the Fc region of an antibody that targets an antigen on a target cell, redirecting the membrane fusion activity towards cells that display the antibody’s target.

[0176] In some cases, the targeting moiety linked to the membrane-fusion protein binds a cell surf acemarkeronthetarget cell, e.g., a protein, glycoprotein, receptor, cell surface ligand, agonist, lipid, sugar, class I transmembrane protein, class II transmembrane protein, or class III transmembrane protein.

[0177] In some cases, the lipid delivery particles disclosed herein display targeting moieties that are not conjugated to the membrane -fusion protein or other proteins in order to redirect the fusion activity of the lipid delivery particle towards a cell that is bound by the targeting moiety, or to affect tropism of the lipid delivery particle toward the target cell.Envelope protein of viral origin

[0178] In some cases, an envelope protein has a viral origin. For example, a suitable envelope protein is from a DNA virus, anRNA virus, or a retrovirus. The envelope protein can be envelope protein from Herpesviruses, Avian sarcoma leukosis virus, Poxviruses, Hepadnaviruses, Asfarviridae, Flaviviruses, Alphaviruses, Togaviruses, Coronaviruses, Hepatitis D, Orthomyxoviruses, Rhabdovirus, Bunyaviruses, Filoviruses, Oncoretroviruses, lentiviruses, Spumaviruses. In some cases, envelope protein can be envelope protein from lentiviruses, for example, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV). In some cases, an envelope protein is a fusion of two different envelope proteins, wherein each comes from a differentvirus. Additional suitable envelope proteins that are from viral origins and their functions are described in White JM et al. , Crit Rev Biochem Mol Biol. 2008 May-Jun;43(3):189-219.

[0179] In some cases, the envelope protein is a vesicular stomatitis virus glycoprotein (VSVG) or a biologically active mutant thereof. A “biologically active mutant” disclosedherein in connection with a reference protein can refer to a mutant of the reference protein that remains displaying one or more biological activities that are of same nature as the reference protein, which are relevant to the context in which the reference protein is used in the lipid delivery particle disclosed herein, while the level of the one or more biological activities of the biologically active mutant can be either similar as or different than the reference protein. For instance, the biologically active mutantof a VSVG in the context of an envelope protein remains displaying the biological activities of an envelope protein, e.g., mediating membrane fusion, tropism of the lipid delivery particle toward a target cell, or both. Unless otherwise noted, a mutant as described in the present disclosure is equivalent to a biologically active mutant. In some cases, the envelope protein is a Human immunodeficiency virus GP160 or a biologically active mutant thereof. In some cases, the envelope protein is a Baboon Endogenous Retrovirus (BaEVTR) glycoprotein or a biologically active mutant thereof. In some cases, the envelope protein is a modified Baboon Endogenous Retrovirus (BaEVTRless) glycoprotein or a biologically active mutant thereof. In some cases, the envelope protein is the fusion protein of Vesicular stomatitis Indiana virus and Rabies virus Glycoproteins (FuG-E) or a biologically active mutantthereof. In some cases, the envelope protein pantropic murine leukemia virus envelope protein (MLV) or a biologically active mutantthereof. In some cases, the envelope protein is a modified Fusion protein of Vesicular stomatitis Indiana virus and Rabies virus Glycoproteins (FuG-E P440E) or a biologically active mutantthereof. In some cases, the envelope protein is an ecotropic Murine Leukemia Virus envelope protein (MLV ENV ecotropic) or a biologically active mutant thereof. In some cases, the envelope protein is an amphotrophic Murine Leukemia Virus envelope protein (MLV ENV amphotropic) or a biologically active mutant thereof. In some cases, the envelope protein is a Moloney murine leukemia virus envelope protein (MMLV) or a biologically active mutant thereof. In some cases, the envelope protein is a Moloney murine sarcoma virus envelope protein (MoMSVg) or a biologically active mutant thereof. In some cases, the envelope protein is a moloney murine leukemia virus 10 Al strain Glycoprotein (MLV 10A1 ) or a biologically active mutant thereof. In some cases, the envelope protein is a xenotropic murine leukemia virus envelope protein (MLV ENV xenotropic) or a biologically active mutant thereof. In some cases, the envelope protein is a xenotropic murine leukemia virus-related envelope protein (XMRV) or a biologically active mutant thereof. In some cases, the envelope protein is a Baculovirus envelope glycoprotein (GP64) or a biologically active mutant thereof. In some cases, the envelope protein is an endogenous feline virus envelope protein (RD 114 ENV) or a biologically active mutantthereof. In some cases, the envelope protein is a mammalian endogenous retrovirus protein, or a biologically active mutantthereof. The mammalian endogenous retrovirus protein can be a koala retrovirus protein (KoRV) or a Jaagsiekte sheep retrovirus protein (enJSRV), or a biologically active mutant thereof.

[0180] In some cases, the envelope protein is a simian endogenous type D retrovirus protein (RD- 114) or a biologically active mutantthereof. In some cases, the envelope protein is a gibbon ape leukemia virus envelope protein (GALV) or a biologically active mutantthereof. In some cases, the envelope protein is a feline leukemia virus envelope protein (FLV) or a biologically activemutant thereof . In some cases, the envelope protein is a mouse mammary tumor virus envelope protein (MMTV) or a biologically active mutant thereof. In some cases, the envelope protein is an avian leukosis virus envelope protein or a biologically active mutant thereof. In some cases, the envelope protein is a rous sarcoma virus envelope protein or a biologically active mutant thereof.

[0181] In some cases, the envelope protein can direct the lipid delivery particles to fuse with a certain type of target cells rather than other cells. For example, based on the specific type of envelope protein associated with the membrane of the lipid delivery particle, the lipid delivery particle can preferentially target different cell types (z.e. , tropisms of the lipid delivery particles), such as liver cells, ocular cells, nerve cells, lung cells, immune cells, muscle cells, and any other cell types of interest. For example, to fuse with a target liver cells, the envelope protein can be a glycoprotein from human hepatitis viruses or a biologically active mutant thereof, e.g. , Hepatitis B virus (HBV) or hepatitis C virus (HCV), VSV-G glycoprotein or a biologically active mutant thereof, a Marburg virus glycoprotein or a biologically active mutant thereof, an Ebola virus glycoprotein or a biologically active mutantthereof. To fuse with a target muscle cell, for example, a skeletal muscle cell, the envelopeprotein canbe aRoss River virus glycoprotein or a biologically active mutantthereof, or a VSV-G or a biologically active mutantthereof. To fuse with a target ocular cell, f or example, a photoreceptor cell or a retinal cell, the envelope protein can b e an Ebola virus glycoprotein or a biologically active mutant thereof, a Marburg virus glycoprotein or a biologically active mutantthereof, or a VSV-G or a biologically active mutantthereof. To fuse with a target immune cell, for example, CD8+ T cell, an HTLV-1 glycoprotein or a biologically active mutantthereof, or a VSV- G glycoprotein or a biologically active mutantthereof. To fuse with a target immune cell, for example, CD4+ T cell, the envelope protein can be a HIV-1 envelope or a biologically active mutantthereof, a HTLV-1 glycoprotein or a biologically active mutantthereof, or a VSV-G glycoprotein or a biologically active mutantthereof. To fuse with a target lung cells, the envelope protein can be a respiratory syncytial virus glycoprotein or a biologically active mutantthereof, or a SARS-CoV glycoprotein or a biologically active mutant thereof. To fuse with a target nerve cell, such as a cell from the central nervous system cell (e.g, neurons, glial cells including oligodendrocytes, astrocytes and microglia), the envelope protein can be a rabies glycoprotein or a biologically active mutant thereof, a Mokola virus glycoprotein or a biologically active mutantthereof, a Semliki Forest virus glycoprotein or a biologically active mutant thereof, a Venezuelan equine encephalitis virus glycoprotein or a biologically active mutantthereof, or a VSV-G or a biologically active mutantthereof. To fuse with a target sensory cell, such as an auditory cell, including hair cells, cochlear cells, etc., the envelope protein canbean Ebola virus glycoprotein or a biologically active mutant thereof, a Marburg virus glycoprotein or a biologically active mutant thereof, or a VSV-G or a biologically active mutant thereof.

[0182] In some cases, the envelope protein comprises the sequences set forth in Table 1. In some cases, the envelope protein comprises the sequences set forth in Table 1 with at least one amino acid substitution, deletion, or insertion. For instance, N-terminal methionine can be absent from the envelope protein of the lipid delivery particle provided herein relative to the wild-type viral envelope protein. In some cases, the envelope protein comprises the sequences set forth in Table 1 and a heterologous peptide sequence fused to the N-terminal or C-terminal.

[0183] In some cases, the envelope protein comprisesone or more of the sequences set forth in Table 1 with at least one amino acid substitution, deletion, or insertion. For instance, N-terminal methionine can be absent from the envelope protein of the lipid delivery particle provided herein relative to the wild-type viral envelope protein. In some cases, the envelope protein comprises one or more of the sequences set forth in Table 1 and a heterologous peptide sequence fused to the N- terminal or C-terminal.

[0184] In some cases, the envelope protein comprises any one of the sequences set forth in Table 1 with at least one amino acid substitution, deletion, or insertion. For instance, N-terminal methionine can be absent from the envelope protein of the lipid delivery particle provided herein relative to the wild-type viral envelope protein. In some cases, the envelope protein comprises any one of the sequences set forth in Table 1 and a heterologous peptide sequence fused to the N- terminal or C-terminal.

[0185] In some cases, the envelope protein comprises an amino acid sequence that has at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 1. In some cases, the envelope protein comprises an amino acid sequence that has at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in any one of SEQ ID NOs: 286-307. In some cases, the envelope protein comprises an amino acid sequence that has at least about 50% sequence identity to a sequence set forth in any one of SEQ ID NOs: 286-307. In some cases, the envelope protein comprises an amino acid sequence that has at least about 60% sequence identity to a sequence set forth in any one of SEQ ID NOs: 286-307. In some cases, the envelope protein comprises an amino acid sequence that has at least about 70% sequence identity to a sequence set forth in any one of SEQ ID NOs: 286-307. In some cases, the envelope protein comprises an amino acid sequence that has at least about 75% sequence identity to a sequence set forth in any one of SEQ ID NOs: 286-307. In some cases, the envelope protein comprises an amino acid sequence that has 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 asequence set forth in any one of SEQ ID NOs: 286-307. In some cases, the envelope protein comprises an amino acid sequence that has at least about 80% sequence identity to a sequence set forth in any one of SEQ ID NOs: 286-307. In some cases, the envelope protein comprises an amino acid sequence that has at least about 85% sequence identity to a sequence set forth in any one of SEQ ID NOs: 286-307. In some cases, the envelope protein comprises an amino acid sequence that has at least about 90% sequence identity to a sequence set forth in any one of SEQ ID NOs: 286-307. In some cases, the envelope protein comprises an amino acid sequence that has at least about 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 286-307. In some cases, the envelope protein comprises an amino acid sequence that has at least about 96% sequence identity to a sequence set forth in any one of SEQ ID NOs: 286-307. In some cases, the envelope protein comprises an amino acid sequence that has at least about 97% sequence identity to a sequence set forth in any one of SEQ ID NOs: 286-307. In some cases, the envelope protein comprises an amino acid sequence that has at least about 98% sequence identity to a sequence set forth in any one of SEQ ID NOs: 286-307. In some cases, the envelope protein comprises an amino acid sequence that has at least about 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 286-307.Table 1. Exemplary envelope proteins from virus originEnvelope protein of human origin

[0186] In some aspects, the envelope protein in the lipid delivery particle described herein has a human origin, e.g., has significant sequence similarity to a human wild -type protein, such as at least 90%, atleast 95%, atleast 98%, or at least99%. Using an envelope protein of ahuman origin can have benefits such as providing a minimized immunogenicity and better tolerance in a human subject receiving the lipid delivery particles. The lipid delivery particle comprising an envelope protein of a human origin can comprise another component that is from human origin or from non-human origin (e.g., a payload or a plasma membrane recruitment element). An envelope protein that is from human origin can include, example, envelope proteins or glycoproteins of human endogenous retroviruses (HERVs), other human endogenous envelope proteins, or other human endogenous proteins that serve a similar function of recognizing and / or fusing with membrane of a target cell (e.g., clathrin adaptor protein complex- 1, CHMP4C, Proteolipid protein 1, TSAP6, immunoglobulin variable domains, or a mutant thereof).

[0187] In some cases, the envelope protein is a HERV envelope protein such as any one of those listed in Table 2. In some cases, the envelope protein is a hENVHl or a mutant thereof. In some cases, the envelope protein is a hENVH2 or a mutant thereof. In some cases, the envelope protein is a hENVH3 or a mutant thereof. In some cases, the envelope protein is a hENVKl or a mutant thereof. In some cases, the envelope protein is a hENVK2 or a mutant thereof. In some cases, the envelope protein is a hENVK3 or a mutant thereof. In some cases, the envelope protein is a hENVK4 or a mutant thereof. In some cases, the envelope protein is a hENVK5 or a mutant thereof. In some cases, the envelope protein is a hENVK6 or a mutant thereof. In some cases, the envelope protein is a hENVT or a mutantthereof . In some cases, the envelope protein is a hENVW or a mutant thereof. In some cases, the envelope protein is a hENVFRD or a mutant thereof. In some cases, the envelope protein is a hENVR or a mutantthereof. In some cases, the envelope protein is a hENVR(b) or a mutantthereof. In some cases, the envelope protein is a hENVR(c)2 or a mutantthereof. In some cases, the envelope protein is a hENVR(c)l or a mutantthereof. In some cases, the envelope protein is a hENVKcon or a mutant thereof. In some cases, the envelope protein is a truncated HERV protein.Table 2. Exemplary human HERV envelope proteins

[0188] In some cases, the envelope protein comprises the sequences set forth in Table 3. In some cases, the envelope protein comprises the sequences set forth in Table 3 with at least one amino acid substitution, deletion, or insertion. For example, forthose amino acid sequences start with a N-terminal methionine, the N-terminal methionine can be absent. In some cases, the envelope protein comprises the sequences set forth in Table 3 and a heterologous peptide sequence fused to the N-terminal or C-terminal.

[0189] In some cases, the envelope protein comprises one or more of the sequences set forth in Table 3 with at least one amino acid substitution, deletion, or insertion. For instance, N-terminal methionine can be absent from the envelope protein of the lipid delivery particle provided herein relative to the wild-type viral envelope protein. In some cases, the envelope protein comprises one or more of the sequences set forth in Table 3 and a heterologous peptide sequence fused to the N- terminal or C-terminal.

[0190] In some cases, the envelope protein comprises any one of the sequences set forth in Table 3 with at least one amino acid substitution, deletion, or insertion. For instance, N-terminal methionine can be absent from the envelope protein of the lipid delivery particle provided herein relative to the wild-type viral envelope protein. In some cases, the envelope protein comprises any one of the sequences set forth in Table 3 and a heterologous peptide sequence fused to the N- terminal or C-terminal.

[0191] In some cases, the envelope protein comprises an amino acid sequence that has at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in SEQ ID NOs: 308-341. In some cases, the envelope protein comprises an amino acid sequence that has at least about 50% sequence identity to a sequence set forth in any one of SEQ ID NOs: 308-341. In some cases, the envelope protein comprises an amino acid sequence that has at least about 60% sequence identity to a sequence set forth in any one of SEQ ID NOs: 308-341. In some cases, the envelope protein comprises an amino acid sequence that has at least about 70% sequence identity to a sequence set forth in any one of SEQ ID NOs: 308-341. In some cases, the envelope protein comprises an amino acid sequence that has at least about 75% sequence identity to a sequence set forth in any one of SEQ ID NOs: 308-341. In some cases, the envelope protein comprises an amino acid sequence that has 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 a sequence set forth in any one of SEQ ID NOs: 308-341 . In some cases, the envelope protein comprises an amino acid sequence that has at least about 80% sequence identity to a sequence set forth in any one of SEQ ID NOs: 308-341. In some cases, the envelope protein comprisesan amino acid sequence that has atleast about 85% sequence identity to a sequence setforth in any one of SEQ ID NOs: 308-341 . In some cases, the envelope protein comprises an amino acid sequence that has atleast about 90% sequence identity to a sequence set forth in any one of SEQ ID NOs: 308-341. In some cases, the envelope protein comprises an amino acid sequence that has at least about 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 308-341. In some cases, the envelope protein comprises an amino acid sequence that has at least about 96% sequence identity to a sequence set forth in any one of SEQ ID NOs: 308-341. In some cases, the envelope protein comprises an amino acid sequence that has at least about 97% sequence identity to a sequence set forth in any one of SEQ ID NOs: 308-341. In some cases, the envelope protein comprises an amino acid sequence that has at least about 98% sequence identity to a sequence set forth in any one of SEQ ID NOs: 308-341. In some cases, the envelope protein comprises an amino acid sequence that has at least about 99% sequence identity to a sequence setforth in any one of SEQ ID NOs: 308-341.

[0192] Table 3. Exemplary sequences for human HERV envelope proteinsPLASMA MEMBRANE RECRUITMENT ELEMENT

[0193] In some aspects, the lipid delivery particle provided herein comprises a plasma membrane recruitment element. The lipid delivery particle disclosed herein can comprise a membrane encapsulating a protein core, which can comprise a plasma membrane recruitment element. The plasma membrane recruitment element can localize itself to the membrane of the lipid delivery particles. The plasma membrane recruitment element can be utilized to recruit a component (e.g, a payload) to the membrane of the lipid delivery particles via forming a chimeric protein of the plasma membrane recruitment element and a component to be localized to the membrane or other mechanisms of attachment. In some cases, the membrane can encapsulate a protein core. In some cases, at least a portion of the plasma membrane recruitment element forms the basic structure of the lipid delivery particle, such as a portion of the protein core inside the lipid delivery particle. In some cases, at least a portion of the plasma membrane recruitment element binds to the membrane of the lipid delivery particle from the inside.

[0194] The plasma membrane recruitment element can play a role in the assembly of the lipid delivery particle, such as packing various components e.g., a payload) into the lipid delivery particles. The plasma membrane recruitment element can direct budding of the lipid delivery particles from a producer cell. In some cases, expressing plasma membrane recruitment element alone ortogether with an envelope protein disclosed herein in a producer cell can lead to formation of the lipid delivery particle.

[0195] In some cases, the plasma membrane recruitment element has a viral origin. For instance, the plasma membrane recruitment element comprises a retroviral gag protein, e.g., a retroviral polyprotein that comprises one or more of a matrix (MA) polypeptide, an RNA-binding phosphoprotein polypeptide, a capsid (CA) polypeptide, or a nucleocapsid (NC) polypeptide. Theplasma membrane recruitment element can comprise HIV gag or a mutant thereof . The plasma membrane recruitment element can comprise a gag from murine leukemia virus (MLV) or a biologically active mutantthereof . The plasma membrane recruitment element can comprise a gag from Moloney murine leukemia virus (MMLV) or a biologically active mutant thereof. The plasma membrane recruitment element can comprise Respiratory syncytial virus (RSV) M or a biologically active mutant thereof. The plasma membrane recruitment element can comprise Human Papillomavirus (HPV) LI protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise HPV L2 protein or a mutantthereof. The plasma membrane recruitment element can comprise Hepatitis B virus (HBV) core protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise Hepatitis C virus (HCV) core protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise hepatitis E virus (HeV) M protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise Chikungunya virus (CHIKV) C-E3-E2-6k-El or a biologically active mutant thereof. The plasma membrane recruitment element can comprise RSV NP or a biologically active mutant thereof. The plasma membrane recruitment element can comprise Human metapneumovirus (HMPV) M or a biologically active mutant thereof. The plasma membrane can comprise a glycoprotein from a flavirirus. The flavivirus can comprise Chikungunya virus, Zika virus, Dengue virus, or West Niles virus. The plasma membrane recruitment element can comprise Zika virus (ZIKV) C or a biologically active mutant thereof. The plasma membrane recruitment element can comprise ZIKV prM / M or a mutant thereof. The plasma membrane recruitment element can comprise Dengue virus (DENV) C-prM or a biologically active mutant thereof. The plasma membrane recruitment element can comprise West Nile Virus (WNV) prME protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise WNV CprME protein or a biologically active mutantthereof. The plasma membrane recruitment element can comprise Filovirus VP40 or Z protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise Baculovirus Pl protein or a biologically active mutantthereof. The plasma membrane recruitment element can comprise Rotavirus VP7 or a biologically active mutant thereof. The plasma membrane recruitment element can comprise Rotavirus VP2 protein or a biologically active mutantthereof. The plasma membrane recruitment element can comprise Rotavirus VP6 protein or a biologically active mutantthereof. The plasma membrane recruitment element can comprise Porcine Circovirus Type 2 (PCV2) capsid or a biologically active mutant thereof. The plasma membrane recruitment element can comprise baculovirus VP2 protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise baculovirus VP5 protein or a biologically active mutant thereof. The plasma membranerecruitment element can comprised aculovirus VP3 protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise or baculovirus VP7 protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise Ebola nucleocapsid or a biologically active mutant thereof. The plasma membrane recruitment element can comprise Parovirus VP1 protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise Parovirus VP2 protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise Newcastle disease vims (NDV) M protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise Human polyomavirus 2 (JCPyV) VP1 protein or a biologically active mutant thereof . The plasma membrane recruitment element can comprise Human parainfluenza virus type 3 (HPIV3) M protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise HPIV3N protein or a biologically active mutant thereof . The plasma membrane recruitment element can comprise or Mumps virus (MuV) M proteins or a biologically active mutant thereof. The plasma membrane recruitment element can comprise SARS M protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise SARS E protein or a biologically active mutant thereof. The plasma membrane recruitment element can comprise SARS N protein or a biologically active mutant thereof.

[0196] In some cases, the plasma membrane recruitment element is a mammalian protein or part thereof. For example, the plasma membrane recruitment element can include a pleckstrin homology (PH) domain or a transmembrane domain of a mammalian protein, such as a mouse protein or a human protein. In some cases, the plasma membrane recruitment element has a human origin. Utilizing the plasma membrane recruitment element of a human origin in the lipid delivery particle can give rise to reduced immunogenicity for administration to a human subject. The plasma membrane recruitment element can include a gagfrom human endogenous retrovirus, such as Human Endogenous Retrovirus K (e.g., HERV-K113, HERV-K101, HERV-K102, HERV- K104, HERV-K107, HERV-K108, HERV-K109, HERV-K115, HERV- KI lp22, and HERV- K12ql3) and Human Endogenous Retrovirus-W or a mutant thereof. The plasma membrane recruitment element can include a hGAGKcon or a mutant thereof. The plasma membrane recruitment element can include an endogenous gag of a mammal (e.g., human) from retrotransposons (e.g., Arc from vertebrate lineage of Ty3 / gypsy retrotransposon), which are also ancestral to retroviruses. In some cases, the plasma membrane recruitment element comprises a portion from human Arc. In some embodiments, the plasma membrane recruitment element may be linked to a retroviral gag protein. In some embodiments, the plasma membrane recruitment element may comprise a retroviral gag protein.

[0197] The plasma membrane recruitment element can include a pleckstrin homology (PH) domain from a human protein or a biologically active mutant thereof. The PH domains can play a role in protein-membrane interactions via binding to phosphatidylinositol phosphate (PIP), for example PIP2 or PIP3, or other lipids or proteins within the membrane of the lipid delivery particles. PH domains with different sequences can have varied affinities and selectivity when binding different PIPs. The plasma membrane recruitment element can include a PH domain of human phospholipase C51 or a biologically active mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human Aktl or a biologically active mutant thereof. The plasma membrane recruitment element can comprise a mutant PH domain of human Aktl with E17K substitution or a biologically active mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human 3 -phosphoinositide-dependent protein kinase 1 or a biologically active mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human Daap 1 or a biologically active mutant thereof. The plasma membrane recruitment element can comprise a PH domain of mouse Grp 1 or a biologically active mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human Grpl or a biologically active mutant thereof. The plasma membrane recruitment element can comprise a PH domain of Grpl (e.g., mouse Grpl) or a biologically active mutant thereof . The plasma membrane recruitment element can comprise a PH domain of human OSBP or a biologically active mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human Btkl or abiologically active mutantthereof. The plasma membrane recruitment element can comprise a PH domain of human FAPP1 or a biologically active mutantthereof. The plasma membrane recruitment element can comprise a PH domain of human CERT or a biologically active mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human PKD or abiologically active mutantthereof. The plasma membrane recruitment element can comprise a PH domain of human PHLPP1 or a biologically active mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human SWAP70 or a biologically active mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human MAPKAP1 or a biologically active mutant thereof.

[0198] The plasma membrane recruitment element can also include a membrane protein (e.g., a human membrane protein), a transmembrane domain thereof, or a biologically active mutant thereof. For example, the transmembrane domain of a human protein can b e a tetrasp anin. In some cases, the plasma membrane recruitment element comprises a transmembrane domain of human CD9 or a biologically active mutantthereof. In some cases, the plasma membrane recruitment element comprises a transmembrane domain of human CD47 or a biologically active mutant thereof. In some cases, the plasma membrane recruitment element comprises a transmembranedomain of human CD63 or a biologically active mutant thereof. In some cases, the plasma membrane recruitment element comprises a transmembrane domain of human CD81 . In some cases, the plasma membrane recruitment element comprises a portion from human Arc.

[0199] The plasma membrane recruitment element can comprise a retroviral gag or a biologically active mutant thereof. The mutant of a retroviral gag can include only a portion of the retroviral gag. The plasma membrane recruitment element can include a gag of an alpha retrovirus. The plasma membrane recruitment element can a beta retrovirus or mutant thereof. The plasma membrane recruitment element can include a gamma retrovirus or mutant thereof. The plasma membrane recruitment element can include a delta retrovirus or mutant thereof. The plasma membrane recruitment element can include or mutant thereof. The plasma membrane recruitment element can include an epsilon retrovirus or mutant thereof. The plasma membrane recruitment element can include a spumavirus or mutant thereof. The retroviral gag can include a gag of HIV (e.g., HIV-1), a gag of murine leukemia virus (MLV), a gag of Moloney murine leukemia vims (MMLV), a gag of Simian immunodeficiency virus (SIV), a gag of Rous sarcoma virus (RSV), a gag of human T-cell leukemia virus type- 1 (HTLV), or a gag of bovine leukemia virus (BLV), or mutants thereof. The plasma membrane recruitment element can include a gag of HIV (e.g., HIV- 1 ) or a biologically active mutant thereof. The plasma membrane recruitment element can include a gag of MLV or a biologically active mutant thereof . The plasma membrane recruitment element can include a gag of RSV or a biologically active mutant thereof. The plasma membrane recruitment element can include a gag of Friend murine leukemia virus (FMLV) or mutant thereof.

[0200] In some cases, the plasma membrane recruitment element includes those described in Table 4 with a further truncation onthe N-terminus. For example, forthose amino acid sequences start with a N-terminal methionine, the N-terminal methionine can be absent. In some cases, the plasma membrane recruitment element includes those described in Table 4 with a further truncation on the C-terminus. In some cases, the plasma membrane recruitment element includes those described in Table 4 with one amino acid substitution. In some cases, the plasma membrane recruitment element includes those described in Table 4 with two or more amino acid substitutions. In some cases, the plasma membrane recruitment element includes those described in Table 4 and a heterologous peptide sequence fused to the N-terminal or C-terminal.

[0201] In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in Table 4. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 50% sequence identity to a sequence set forth in Table 4. In some cases, the plasma membranerecruitment element comprises an amino acid sequence that has at least about 60% sequence identity to a sequence set forth in Table 4. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 70% sequence identity to a sequence set f orth in Table 4. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 75% sequence identity to a sequence set forth in Table 4. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has 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 a sequence set forth in Table 4. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 80% sequence identity to a sequence set forth in Table 4. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 85% sequence identity to a sequence set forth in Table 4. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 90% sequence identity to a sequence set forth in Table 4. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 95% sequence identity to a sequence set forth in Table 4. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 96% sequence identity to a sequence set f orth in Table 4. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 97% sequence identity to a sequence set forth in Table 4. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 98% sequence identity to a sequence set forth in Table 4. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 99% sequence identity to a sequence set forth in Table 4.

[0202] In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in any one of SEQ ID NOs: 342-389. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 50% sequence identity to a sequence set forth in any one of SEQ ID NOs: 342-389. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 60% sequence identity to a sequence set forth in any one of SEQ ID NOs: 342-389. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 70% sequence identity to a sequence set forth in any one of SEQ ID NOs: 342-389. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 75% sequence identity to a sequence set forth in any one of SEQ ID NOs: 342-389. In some cases, the plasma membrane recruitmentelement comprises an amino acid sequence that has 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 a sequence setforthin any one of SEQ ID NOs: 342-389. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 80% sequence identity to a sequence set forth in any one of SEQ ID NOs: 342-389. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 85% sequence identity to a sequence set forth in any one of SEQ ID NOs: 342-389. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 90% sequence identity to a sequence set forth in any one of SEQ ID NOs: 342-389. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 342- 389. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 96% sequence identity to a sequence set forth in any one of SEQ ID NOs: 342-389. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 97% sequence identity to a sequence set forth in any one of SEQ ID NOs: 342-389. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 98% sequence identity to a sequence set forth in any one of SEQ ID NOs: 342-389. In some cases, the plasma membrane recruitment element comprises an amino acid sequence that has at least about 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 342-389.Table 4. Exemplary plasma membrane recruitment elements and their sequences*hGAGKcon is a consensus sequence from ten proviral GAG sequences. The GAG sequences used to derive this consensus GAG sequence are from the following HERVs: HERV-K113, HERV-K101, HERV-K102, HERV-K104, HERV-K107, HERV-K108, HERV-K109, HERV- K115, HERV- Kl lp22, and HERV-K12ql3.

[0203] In some cases, the lipid delivery particle disclosed herein comprises a protein core that is composed of at least a structural protein of a viral origin, for instance, a retroviral gag protein. In some of these cases, the lipid delivery particle comprisesa retroviral gag-pro-pol polyprotein, e.g., a gag-pro-pol poly protein from HIV, MMLV, or FMLV, which can help assemble a protein core of the lipid delivery particle. In some of these cases, some of the gag-pro-pol polyprotein is cleaved, e.g. , by pro (protease) present freely or in the gag-pro-pol polyprotein. Without wishing to be bound by any particular theory, the cleavage by pro can be inefficient, and the resultant cleavage products can include gag polyprotein, gag-pro polyprotein, free pro, and free pol (polymerase). In some cases, a retroviral gag polyprotein can be further cleaved into MA, CA, NC, and other small fragments, if any. In some other cases, the lipid delivery particle comprises a retroviral gag-pro polyprotein without the pol component, and the gag-pro polyprotein can help form a protein core of the lipid delivery particle. The gag-pro can also be cleaved by pro, in some cases, inefficiently, into separate gag and pro proteins. In some cases, there can be different plasma membrane recruitment elements in a lipid delivery particle. For instance, a gag-pro or gag-pro-pol polyprotein from one species of virus (e.g. , a retrovirus, e.g. , a HIV) can help assemble form a protein core of the lipid delivery particle, while a chimeric protein in the lipid delivery particle, discussed infra, can comprise a payload fused with a gag protein from a different species of virus (e.g., an MMLV), or from aHERV, or a PH domain or transmembrane domain of a human protein (e.g., a PH domain Aktl with E17K substitution).PAYLOAD

[0204] A payload in a lipid delivery particle of the present disclosure can comprise a protein, a polypeptide, a nucleic acid (e.g., DNA or RNA), or any combinations thereof.

[0205] The payload can be a part of the chimeric protein disclosed herein or can comprise a part of the chimeric protein disclosed herein. Alternatively or additionally, the payload can include an entity in the lipid delivery particle separate from the chimeric protein disclosed herein. For instance, in some cases, the payload is a protein or polypeptide coupled to a plasma membrane recruitment element. In some cases, the payload comprises a first moiety (e.g., a nucleic acidbinding protein) that is fused to a plasma membrane recruitment element, and further comprises a second moiety that is coupled to the first moiety via covalent or non -covalent interaction. For instance, the first moiety can be a nucleic acid binding protein that is fused with the plasma membrane recruitment element, and the second moiety can be a nucleic acid molecule that binds to the nucleic acid binding protein.

[0206] In some cases, a payload is directly packaged within the lipid delivery particles and delivered into a target cell in its free form. In some cases, a payload can be fused to a plasma membrane recruitment element (e.g. , pleckstrin homology domain) and form a chimeric protein as part of the lipid delivery particles, and then delivered into the target cell. In some cases, the plasma membrane recruitment element e.g. , pleckstrin homology domain) forms at least part of a protein core of the lipid delivery particle. In some embodiments, the payload in its free form or as part of a chimeric protein is within the inside cavity of the protein core of the lipid delivery particles disclosed herein. In some cases, the payload in its free form derives from a cleavage of the chimeric protein comprising the payload.

[0207] In some cases, a lipid delivery particle can deliver more than one payload. Each of the payloads can independently comprise nucleic acid-binding moiety, a nucleic acid -modifying moiety, a fusion protein, or a nucleic acid, or any combinations thereof.

[0208] In some embodiments, the plasma membrane recruitment element and the payload are coupled via any suitable method. Covalent coupling between the plasma membrane recruitment element and a payload peptide can include inteins that can form peptide bonds, direct proteinprotein chimeras generated from a single reading frame. In some cases, nucleic acids base pairingto other nucleic acids via hydrogen bonding interactions (e.g., DNA / RNA, DNA / DNA, or RNA / RNA hybrids), protein-protein binding, or protein-nucleic acid molecule binding can be involved for the coupling between the plasma membrane recruitment element and the payload. Examples of protein -nucleic acid molecule binding include an RNA binding protein (RBP) and an RBP binding sequence e.g., an RNA) that binds to the RBP. In some embodiments, each of the plasma membrane recruitment element and the payload is fused to a heterologous sequence, and the two heterologous sequences dimerize or multimerize with or without the need for a chemical compound to induce the protein -protein binding, such as a single-stranded nucleic acid sequence or protein dimerization domains). In some embodiments, each of the plasma membrane recruitment element and the payload is fused to one member of a pair of binding partners (e.g., antibody and its target antigen). In some embodiments, the plasma membrane recruitment element is fused to an RBP, and the payload is fused to a RBP binding sequence. Examples of suitable protein domains or nucleic acid molecules for forming the non -covalent connections include single chain variable fragments, nanobodies, affibodies, DmrA / DmrB / DmrC, FKBP / FRB, dDZFs, Leucine zippers, proteins thatbind to DNA and / or RNA, optogeneti c protein domains that can dimerize or multimerize in the presence of certain light wavelengths, proteins with quaternary structural interactions, and / or naturally reconstituting split proteins. Examples of RBPs and their RBP binding sequences that can be used include a 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 a sequence set forth in Table 5. Examples of RBPs and their RBP binding sequences that can be used include a 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 a sequence set forth in any one of SEQ ID NOs: 390-425. In some cases, the RBP comprises an amino acid sequence as set forth in Table 5. In some cases, the RBP comprises any one of the sequences set forth in Table 5. In some cases, the RBP comprises one or more of the sequences set forth in Table 5. In some cases, the RBP comprises more than one of the sequences set forth in Table 5. In some cases, the RBP comprises multiple sequences set forth in Table 5. In some cases, the RBP binding sequence comprises an amino acid sequence as set forth in Table 5. In some cases, the RBP binding sequence comprises any one of the sequences set forth in Table 5. In some cases, the RBP binding sequence comprises one or more of the sequences set forth in Table 5. In some cases, the RBP binding sequence comprises more than one of the sequences set forth in Table 5. In some cases, the RBP binding sequence comprises multiple sequences set forth in Table 5.Table 5. Exemplary RNA binding proteins (RBP) and corresponding RBP binding sequencesNucleic acid binding domains and nucleic acid modifying domains

[0209] In some cases, the payload comprises a nucleic acid -binding moiety, a nucleic acidmodifying moiety, a fusion protein, or a nucleic acid. In some cases, the payload comprises a nucleic acid-binding domain, e.g., a DNA-binding protein domain or polypeptide or an RNA- binding domain or polypeptide e.g., an RNA-binding protein (RBP). A nucleic acid-binding moiety can be capable of binding a nucleic acid. A nucleic acid -binding domain can bind to a nucleic acid in a nonspecific or a site-specific manner.

[0210] In some cases, the nucleic acid-binding moiety binds to a nucleic acid in a site-specific manner. For example, a nucleic acid-binding moiety can comprise an aptamer binding domain that selectively binds to a specific target. In some cases, a nucleic acid -binding moiety recognizes a specific recognition sequence in the target nucleic acid. In some cases, a nucleic acid-binding moiety comprises an aptamer binding domain. In some cases, a nucleic acid binding moiety selectively binds to a sequence or a structural element in a nucleic acid molecule. In some cases, an RNA-binding domain selectively binds to a specific sequence motif in an RNA molecule. In some cases, a nucleic acid-binding moiety selectively binds to a structural element in a nucleic acid molecule. For example, a nucleic acid-binding domain can bind to a stem-loop in a nucleic acid molecule.

[0211] In some cases, a nucleic acid-binding moiety is or comprises a guidable polypeptide domain, a transcriptional regulatory domain, or a nucleic acid -modifying domain. A guidable polypeptide domain can be capable of binding to a polynucleotide (e.g. an RNA guide) that can direct the guidable polypeptide domain a target site. In some cases, the guidable polypeptide domain forms a complex with the RNA guide and recognizes the target sequence through DNA-RNA base pairing. In some cases, a nucleic-acid binding moiety is or comprises a transcriptional regulatory domain. In other cases, a nucleic -binding moiety can help recruit a transcriptional repressor or activator to a target site. In some cases, a nucleic acid -binding moiety is or comprises a nucleic acid-modifying moiety. In some cases, the present disclosure uses nucleic acid -binding moieties to recruit a nucleic acid-modifying moiety to a target site. In some cases, a nucleic-acid binding moiety comprises catalytic activity. In other cases, a nucleic acid-binding moiety is catalytically inactive. In some cases, a nucleic-acid binding moiety comprising catalytic activity is modified to have a reduced level of activity compared to its wild -type counterpart.

[0212] In some cases, the payload in the present disclosure comprises a nucleic acid modifying domain. A nucleic acid-modifying domain can comprise a polypeptide domain, a nucleic acid or a combination thereof (e.g., a ribonucleoprotein complex). A nucleic acid-modifying domain can be capable of modifying nucleic acid, such as cleaving double-stranded nucleic acid; nicking a single-stranded nucleic acid; introducing a mutation, deletion, or insertion in a nucleic acid; methylating or demethylating a nucleic acid, or altering the structure of DNA (e.g. , changing chromatin structure through modifying histones). For example, a nucleic acid modifying domain can comprise a nuclease domain, a nickase domain, a deaminase domain, a polymerase, reverse transcriptase domain, a recombinase domain, a transposase domain, or an epigenetic modifying domain. Anuclease domain canbe capable of cleavingphosphodiesterbonds between nucleotides in nucleic acids. A nuclease domain can comprise an exonuclease (e.g., a nuclease capable of cleaving nucleic acids from the ends) or an endonuclease (e.g., a nuclease capable of cleaving nucleic acids in the middle). In some cases, a nucleic acid modifying effector or nucleic acid binding domain is a nickase, which canbe capable of cleaving a single -strand in a double-stranded DNA. Nucleic acid modifying domains can be useful for gene editing, or for regulating, activating or inhibiting gene expression.Guidable polypeptide domain

[0213] In some cases, the payload in the present disclosure comprises a guidable polypeptide domain (e.g., a CRISPR-Cas protein domain). In some cases, a guidable polypeptide domain is capable of binding to a polynucleotide (e.g., a RNA guide) that directs it to a target site. In some cases, the guidable polypeptide domain forms a complex with the polynucleotide and recognizes the target sequence through DNA-RNA base pairing. In some cases, a guidable polypeptide domain is a CRISPR / CRISPR-associated (Cas) domain. A CRISPR domain canbe a natural or an engineered domain. A Cas protein or domain can be derived from a CRISPR system or share structural and / or functional similarities to a protein involved in a CRISPR system. A CRISPR system is a system encoding DNA sequence arrays known as clustered regularly interspaced short palindromic repeats (CRISPRs), which can be found in microbial genomes or phage genomes. Insome cases, CRISPR systems comprise genes encoding CRISPR-associated (Cas) proteins and / or small RNA guide molecules (e.g., crRNA or tracrRNA) that assemble with the CRISPR domain. In some cases, the CRISPR-Cas domain forms a complex with one or more RNA guide molecules to form an effector ribonucleoprotein complex. The effector ribonucleoprotein complex can recognize a target sequence through sequence specific DNA-RNA base pairing with a spacer sequence in the RNA guide. In some cases, target recognition activates one or more nuclease domains (e.g., aRuvC domain orHNH domain) in the CRISPR domain to make a double -stranded cut at the target DNA. A CRISPR-Cas domain complexed with an RNA guide can be capable of inactivating target gene through a gene knockout. In some cases, the CRISPR domain is used to enable gene insertion and / or deletion, which can inactivate, modify, or restore the gene ’ s function .

[0214] In some cases, the guidable polypeptide domain is any suitable nuclease, e.g., a CRISPR- associated (Cas) protein or a Cas nuclease which functions in a non -naturally occurring CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated) 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., etal, “The CRISPR / Cas bacterial immune system cleaves bacteriophage and plasmid DNA,” Nature (2010) 468:67-71 ; Sapranauskas, R, et al, “The StreptococcusthermophilusCRISPR / Cas system provides immunity in Esch erichiacoli,” Nucleic Acids Res (2011) 39: 9275-9282).

[0215] Suitable nucleases include CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides (e.g. , Cas9 or Cas 14), type III CRISPR-associated (Cas) polypeptides, typelV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides (e.g., Cpfl / Casl2a, C2cl, or c2c3), and type VI CRISPR-associated (Cas) polypeptides (e.g., C2c2 / Casl3a, Casl3b, Casl3c, Casl3d).

[0216] One or more components of a CRISPR / Cas system (e.g., modified and / or unmodified) delivered by the lipid delivery particles disclosed herein can be utilized as a genome engineering tool in a wide variety of organisms including diverse mammals, animals, plants, and yeast. A CRISPR / Cas system can comprise 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 An RNA-guided Cas protein (e.g., a Cas nuclease such as a Cas9 nuclease) can specifically bind a target polynucleotide (e.g., DNA) in a sequence-dependent manner. The Cas protein, if possessing nuclease activity, can cleave the 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; Iinek, M., etal, “ 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 (201 1) 471 :602-607), and has been widely used for programmable genome editing in a variety of organisms and model systems (Cong, L., et al, “Multiplex genome engineeringusing CRISPR Cas systems,” Science (2013) 339:819-823 ; liang, W., et al, “RNA-guided editing of bacterial genomes using CRISPR-Cas systems,” Nat. Biotechnol. (2013) 31 : 233-239; Sander, I. D. & loung, I. K, “CRISPR-Cas systems for editing regulating and targeting genomes,” Nature Biotechnol. (2014) 32:347-355).

[0217] In some cases, the Cas protein is mutated and / or modified to yield a nuclease deficient protein or a protein with decreased nuclease activity relative to a wild -type Cas protein. A nuclease deficient protein can retain the ability to bind DNA but can lack or have reduced nucleic acid cleavage activity . A protein encodedby a donor sequence comprises a Cas nuclease (e.g. , retaining wild-type nuclease activity, having reduced nuclease activity, and / or lacking nuclease activity) can function in a CRISPR / Cas system to regulate the level and / or activity of a target gene or protein (e.g., decrease, increase, or elimination). The Cas protein can bind to a target polynucleotide and prevent transcription by physical obstruction or edit a nucleic acid sequence to yield non -functional gene products. In some cases, the Cas protein cleave s both strands of DNA. In some cases, the Cas protein cleaves one strand of DNA.

[0218] In some embodiments, the nuclease is a Cas protein that forms a complex with a guide nucleic acid, such as a guide RNA (gRNA). In some embodiments, the donor sequence disclosed herein 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 donor sequence disclosed herein encodes a Cas protein that forms a complex with two separate RNA molecules of a dual guide nucleic acid (dgRNA). In some embodiments, the donor sequence in the lipid delivery 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, dgRNA), which is able to form a complex with a Cas protein. In some embodiments, the gRNA comprises a scaffolding sequences that tethers the gRNA to the Cas protein. In some embodiments, the gRNA comprises a scaffolding sequence and a spacer sequence that directs the Cas protein to a specific locus. In some embodiments, the scaffolding sequence is configured to bind to the positively charged groves in the Cas9 protein. In some embodiments, the scaffolding sequence is configured to bind to the Cas protein in the payload. In some cases, Cas undergoes a conformational change when the gRNA binds to the target locus. In some cases, the conformational change in Cas shifts the molecule from an inactive,non-DNA binding conformation into an active DNA-binding conformation. In some cases, the Cas protein undergoes a confirmational change if the spacer sequence has sufficient homology to the sequence at the target locus. In some embodiments, gRNAs can be modified. Exemplary modifications to the gRNA are provided in United States Patent Number 11,479,767 B2, United States Patent Application Publication Number US2020 / 0339980 Al, and United States Patent Application Publication Number US2021 / 0079389 Al , each of which is incorporated herein by reference in its entirety.

[0219] One or more components of any suitable CRISPR / Cas system can be delivered by the lipid delivery particle described in the present disclosure. A CRISPR / Cas system can be referred to using a variety of naming systems. Exemplary naming systems are provided in Makarova, K.S. et al, “An updated evolutionary classification of CRISPR-Cas systems,” Nat Rev Microbiol (2015) 13 :722-736 and Shmakov, S. etal, “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems,” Mol Cell (2015) 60:1-13. A CRISPR / Cas system can be a type I, a type II, a type III, a type IV, a type V, a type VI system, or any other suitable CRISPR / Cas system. A CRISPR / Cas system as used herein can be a Class 1, Class 2, or any other suitably classified CRISPR / Cas system. Class 1 or Class 2 determination can be based upon the genes encoding the effector module. Class 1 systems generally have a multi-subunit crRNA-effector complex, whereas Class 2 systems generally have a single protein, such as Cas9, Cpfl, C2cl, C2c2, C2c3, or a crRNA-effector complex. A Class 1 CRISPR / Cas system can use a complex of multiple Cas proteins to effect regulation. A Class 1 CRISPR / Cas system can comprise, for example, type I (e.g., I, IA, IB, IC, ID, IE, IF, IU), type III (e.g., Ill, IIIA, IIIB, IIIC, IIID), and type IV (e.g., IV, IVA, IVB) CRISPR / Cas type. A Class 2 CRISPR / Cas system can use a single large Cas protein to effect regulation. A Class 2 CRISPR / Cas systems can comprise, for example, type II (e.g. , H, IIA, IIB) and type V CRISPR / Cas type. CRISPR systems can be complementary to each other, and / or can lend functional units in trans to facilitate CRISPR locus targeting. Examples of Cas proteins that can be used as part of the CRISPR systems described herein include c2cl, Casl3a (formerly C2c2), Casl3b, Casl3c, Casl3d, c2c3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas 8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, Cas 14, Cas 10, CaslOd, CasF, CasG, CasH, Cas 12a (formerly Cpfl), Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csb l, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CasX, Csx3, Csxl, Csxl 5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homolog or modified versions thereof. Examples of mutant Cas9 proteins or Cas9 variants include SpG, SpCas9-NG. Cas9-NRNH, SpG, SpRY, Cas9-VQR, Cas9-EQR, SaCas9-KKH, Nme2Cas9, eNme2-C, eNme2-C.NR, eNme2-T. l, eNme2-T.2, SpRY, eSpCas9(l . l), SpCas9-HFl, nSpCas9,eSpCas9, Sniper-Cas9, HypaCas9, evoCas9, Cas9TX, HscCas9-vl .2, superFi-Cas9, efSaCas9, SaCas9-HF, Cas9-HF, Cas mini, SaCas9, SpCas9(H840A), dSpCas9, SpCas9(N863A), SpCas9(D839A), SpCas9(H983A), as well as others described in Chuang CK etal., IntJMol Set. 2021 Sep 13;22(18):9872 and Li, T. et l,, Sig Transduct Target Ther 8, 36 (2023), each of which is incorporated herein by reference in its entirety.

[0220] A CRISPR system can comprise single subunit or multi-subunit effectors. In some cases, a CRISPR system is a Class 1 CRISPR system. A Class 1 CRISPR system can be a type I, type III, or a type IV system. A Class 1 type I CRISPR system can comprise a multi-subunit effector. In some cases, a Class 1 type I CRISPR system comprises a protein or domain in the Cascade- Cas3 protein complex. A Class 1 type I CRISPR system can comprise a Cas6, Cas7, Cas5, Casll, Cas8, or Cas3 domain. A Class 1 type III CRISPR system can comprise a multi-subunit effector. In some cases, a Class 1 type III CRISPR system comprises a Csm complex or a Cmr complex. In some cases, a Class 1 type III CRISPR system comprises a Cas6, a Cas7 (Csm3 or Cmr4), a Cas7-related (Csm5, Cmrl, orCmr6), aCas5 (e.g., Csm4 orCmr5), a Cast 1 (e.g., Csm2 orCmr3), or a CaslO (e.g., Csml or Cmr2) domain. A Class 1 typelV CRISPR system can comprise a Cas6, a Cas7, a Cas5, a Cast 1, aCas8 (e.g., Csfl), ora DinG or CysH domain. In some cases, a CRISPR system comprises Cmrl, Cmr3, Cmr4, Cmr5, or Cmr6. In some cases, a CRISPR system comprises Csb l, Csb2, or Csb3. A CRISPR system can comprise Csfl, Csf2, Csf3, or Csf4. A CRISPR system can comprise Csn2, Csm2, Csm3, Csm4, Csm5, or Csm6. A CRISPR system can comprise Cscl or Csc2. A CRISPR system can comprise Casl, CaslB, Cas2, or Cas4. A CRISPR system can comprise Csyl, Csy2, or Csy3. A CRISPR system can comprise Csel or Cse2. A CRISPR system can comprise Csn2. A CRISPR system can comprise CsaX, Csxl, Csx3, CsxlO, Csxl4, Csxl5, Csxl6, or Csxl7. In some cases, a CRISPR system comprises a modified version of any one of the foregoing Cas proteins. In some cases, a modified version of the foregoing Cas protein comprises a nickase mutation. In some cases, the nickase mutation corresponds to the D 10 A mutation of the wild type Cas9 protein. In some cases, the nickase mutation corresponds to the H840A mutation of the wild type Cas9 protein. In some cases, the nickase mutation occurs in the RuvC domain of the wild type Cas9 protein. In some cases, the nickase mutation occurs in the HNH domain of the wild type Cas9 protein. In some cases the RuvC domain can be mutated to prevent cleavage of the non-targetDNA strand. In some cases the HNH domain can be mutated to prevent cleavage of the target DNA strand. In some cases, a modified version of the foregoing Cas protein comprises one or more mutations that disrupt cleavage activity. In some cases, a Cas protein with disrupted cleavage activity is catalytically inactive or catalytically dead. In some cases, the catalytically dead mutations occur in the RuvC domain and the HNH domain of the wildtype Cas9 protein. In some cases, the catalytically inactive mutations correspond to the D10A mutation and the H840A mutation of the wild type Cas9 protein

[0221] In some cases, a CRISPR system is a Class 2 CRISPR system. A Class 2 CRISPR system can be a Class 2 type II CRISPR system, a Class 2 type V CRISPR system, or a Class 2 type VI CRISPR system. A Class 2 type II CRISPR system can comprise a Cas9 domain (also known as Csnl and Csxl2). A Cas9 domain can be a SpyCas9, a GeoCas9, a SauCas9, a KhuCas9, a AinCas9, an FmaCas9, a SgaCas9, a ScCas9, a SauriCas9 domain. A Cas9 domain can be a hyperactive Cas9 domain. A Class 2 type V CRISPR system can comprise a Casl2 domain. A Casl2 domain can be a Casl2a, a Casl2b, a Casl2c, a Casl2d, a Casl2e, a Casl2f, a Casl2g a Casl2h, a Casl2i, a Casl2j, a Cast 2k, a Cast 21, or a Cast 2m domain. A Class 2 type VI CRISPR system can comprise a Casl3 domain.

[0222] In some cases, a CRISPR system comprises a circularly permuted Cas9.

[0223] In some cases, a CRISPR system comprises CjCas9, Cast 3 a, Cast 3b, Cast 3c, or Casl3d. In some cases, a CRISPR system comprises Casl4, xCas9, or SpCas9-NG.

[0224] In some cases, a CRISPR-Cas domain comprises one or more subdomains. For example, a Cas9 domain can comprise a Reel, a Rec2, a Rec3, a RuvC, an HNH, or a Wedge / PAM- interacting domain. A Casl2 domain can comprise a Reel, Rec2, a crRNA oligonucleotide binding domain (OBD), a Nuc domain, a PAM-interacting (PI) domain, or a RuvC domain. In some cases, the RuvC domain comprises nuclease activity. In some cases, the HNH domain comprises nuclease activity. The PAM-interacting domain can bind to a protospacer adjacent motif (PAM) sequence that is next to a target sequence in a target nucleic acid molecule. PAM recognition can help activate a nuclease domain to make a cut at the target sequence. In some cases, a CRISPR protein or domain is an engineered or mutated variant of a protein involved in a CRISPR system. An engineered or mutated CRISPR domain can comprise a truncation, a deletion of a part of one or more domains or subdomains, or a mutation of an active site (e.g., a RuvC active site or HNH active site). In some cases, a CRISPR domain with a mutation of one or more active sites is catalytically inactive (e.g., dCas9). In some cases, a CRISPR domain with one or more mutated active sites comprises 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 nuclease activity of its wildtype counterpart. For example, a dCas9 can result from the point mutations D 10 A in the RuvC domain and the point mutation H840A in the HNH domain. In other cases, a mutation can result in a CRISPR nickase. A nickase can generate nick or a single - stranded cut. A nickase can generate a nick in the strand complementary to the RNA guide (e.g., the targeting strand) or in the strand on the non-targeting strand. For example, a RuvC mutation DIOA in a Cas9 domain can produce a Cas9 nickase domain that nicks the targeting strand. AnHNH mutation H840A in a Cas9 domain can produce a Cas9 nickase domain that nicks the nontargeting strand.

[0225] A Cas protein can comprise one or more domains. Examples of domains include, guide nucleic acid recognition and / or binding domain, nuclease domains (e.g., DNase or RNase domains, RuvC, HNH), DNA binding domain, RNA binding domain, helicase domains, proteinprotein interaction domains, and dimerization domains. A guide nucleic acid recognition and / or binding domain can interact with a guide nucleic acid. A nuclease domain can comprise catalytic activity for nucleic acid cleavage. A nuclease domain can lack catalytic activity to prevent nucleic acid cleavage. A Cas protein can be a chimeric Cas protein that is fused to other proteins or polypeptides. A Cas protein can be a chimera of various Cas proteins, for example, comprising domains from different Cas proteins.

[0226] In some cases, a CRISPR system comprises an Argonaute (Ago) domain.

[0227] Another example of a Cas protein that can be used as part of the prime editor includes Casl4. A Casl4 protein or polypeptide (also termed as “CasZ” protein or polypeptide) can bind and / or modify (e.g., cleave, nick, methylate, demethylate, etc.) a target nucleic acid and / or a polypeptide associated with target nucleic acid (e.g. , methylation or acetylation of a histone tail) (e.g., in some cases the CasZ protein includes a chimeric partner with an activity, and in some cases the CasZ protein provides nuclease activity). In some cases, the Cas 14 protein or polypeptide is a naturally occurring protein (e.g., naturally occurs in prokaryotic cells) (e.g., a CasZ protein). In other cases, the Casl4 protein or polypeptide not a naturally occurring polypeptide (c.g , the Cas 14 protein is a variant Cas 14 protein, a chimeric protein, and the like). A Cas 14 protein includes 3 partial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains) that are not contiguous with respectto the primary amino acid sequence of the Casl4 protein but form a RuvC domain once the protein is produced and folds. A naturally occurring Cas 14 protein functions as an endonuclease that catalyzes cleavage at a specific sequence in a targeted nucleic acid (e.g., a double stranded DNA (dsDNA)). The sequence specificity is provided by the associated guide RNA, which hybridizes to a target sequence within the target DNA. The naturally occurring Cas 14 guide RNA is a crRNA, where the crRNA includes (i) a guide sequence that hybridizes to a target sequence in the target DNA and (ii) a protein binding segment that binds to the Cas 14 protein. Examples of Casl 4 proteins include those described U.S. 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 donor sequence disclosed herein encodes Cas 14 polypeptide or a nucleic acid molecule encoding Casl4 polypeptide. In some cases, the donor sequence disclosed herein encodes Cas 14a polypeptide. Insome cases, the donor sequence disclosed herein encodes Casl4b polypeptide. In some cases, the donor sequence disclosed herein encodes Casl4c polypeptide.

[0228] A 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, Strepto sporangium roseum, Strepto sporangium 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, Thermosiphoafricanus, Acaryochloris marina, Leptotrichia shahii, Leptotrichia wadeii, Leptotrichia wadeii F0279, Rhodobacter capsulatus SB 1003, Rhodobacter capsulatus R121, Rhodobacter capsulatus DE442, Lachnospiraceae bacterium NK4A179, Lachnospiraceae bacterium MA2020, Clostridium aminophilum DSM 10710, Paludibacter propionicigenes WB4, Camobacterium gallinarum DMS4847, Carnob acterium gallinarum DSM4847, and Francisella novicida. In some aspects, the organism is Streptococcus pyogenes (S. pyogenes). In some aspects, the organism is Staphylococcus aureus (S. aureus). In some aspects, the organism is Streptococcus thermophilus (S. thermophilus).

[0229] A Cas protein can be derived from a variety of bacterial species including Veillonella atypical, Fusobacterium nucleatum, Filif actor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenib acterium mitsuokai, Streptococcus mutans, Listeria innocua, Listeria seeligeri, Listeria weihenstephanensis FSL R90317, Listeria weihenstephanensisFSLM60635, 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, Acidothermuscellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp. Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobacter eiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella succinogenes, Campylobacter jejuni subsp. Jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworth ensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.

[0230] A Cas protein as disclosed herein can be a wildtype or a modified form of a Cas protein. A Cas protein can be an active variant, inactive variant, or fragment of a wild type or modified Cas protein. A Cas protein can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof relative to a wild-type version of the Cas protein. A Cas protein can be a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a wild type exemplary Cas protein. A Cas protein can be a polypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity to a wild type exemplary Cas protein. Variants or fragments can comprise 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. Variants or fragments can be targeted to a nucleic acid locus in complex with a guide nucleic acid while lacking nucleic acid cleavage activity.

[0231] A Cas protein can comprise one or more nuclease domains, such as DNase domains. For example, a Cas9 protein can comprise a RuvC-like nuclease domain and / or an HNH-like nuclease domain. The RuvC and HNH domains can each cut a different strand of double -stranded DNA to make a double-stranded break in the DNA. A Cas protein can comprise only one nuclease domain (e.g., Cpfl comprises RuvC domain but lacks HNH domain).

[0232] A Cas protein can comprise an amino acid sequence having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a nuclease domain (e.g., RuvC domain, HNH domain) of a wild-type Cas protein.

[0233] A Cas protein can be modified to optimize regulation of gene expression. A Cas protein can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, and / or enzymatic activity. Cas proteins 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 a Cas protein can be truncated to remove domains that are not essential for the function of the protein or to optimize (e.g. , enhance or reduce) the activity of the Cas protein for regulating gene expression.

[0234] In some embodiments, the prime editor delivered by the lipid delivery particles of the present disclosure contain a nuclease-null DNA binding protein derived from a DNA nuclease that can induce transcriptional activation or repression of a target DNA sequence. In some embodiments, the donor sequence encodes a nuclease -null RNA binding protein derived from an RNA nuclease that can induce transcriptional activation or repression of a target RNA sequence. For example, a doner sequence can encode a Cas protein which lacks cleavage activity.

[0235] A Cas protein can be a chimeric protein. For example, a Cas protein can be fused to a heterologous functional domain. A heterologous functional domain can comprise a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. A Cas protein can also be fused to a heterologous polypeptide providing increased or decreased stability. The fused domain or heterologous polypeptide can be located at the N-terminus, the C-terminus, or internally within the Cas protein.

[0236] The regulation of genes can be of any gene of interest. It is contemplated that genetic homologues of a gene described herein are covered. For example, a gene can exhibit a certain identity and / or homology to genes disclosed herein. Therefore, it is contemplated that a gene that exhibits or exhibits about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology (at the nucleic acid or protein level) can be modified. It is also contemplated that a gene that exhibits or exhibits 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) can be modified.

[0237] A Cas protein can be provided in any form. For example, a Cas protein can be provided in the form of a protein, such as a Cas protein alone or complexed with a guide nucleic acid. A Cas protein can be provided in the form of a nucleic acid encoding the Cas protein, such as an RNA (e.g., messenger RNA (mRNA)) or DNA.

[0238] The nucleic acid encoding the Cas protein that is part of the prime editor can be codon optimized for efficient translation into protein in a particular cell or organism.

[0239] In some embodiments, a Cas protein is a dead Cas protein. A dead Cas protein can be a protein that lacks nucleic acid cleavage activity.

[0240] A Cas protein can comprise a modified form of a wild type Cas protein. The modified form of the wild type Cas protein can comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid -cleaving 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-cleaving activity of the wild-type Cas protein (e.g. , Cas9 from S. pyogenes). The modified form of Cas protein can have no substantial nucleic acid-cleaving activity. When a Cas protein is a modified form that has no substantial nucleic acid -cleaving activity, it can be referred to as enzymatically inactive and / or “dead” (abbreviated by “d”). A dead Cas protein (e.g. , dCas, dCas9) can bind to a target polynucleotide but may not cleave the target polynucleotide. In some aspects, a dead Cas protein is a dead Cas9 protein.

[0241] A dCas9 polypeptide can associate with a guide nucleic acid molecule (e.g., PEgRNA) to activate or repress transcription of target DNA. Guide nucleic acid molecules can be introduced into cells expressing the engineered chimeric receptor polypeptide. In some cases, such cells contain one or more different guide nucleic acid molecules that target the same nucleic acid. In other cases, the guide nucleic acid molecules target different nucleic acids in the cell. The nucleic acids targeted by the guide nucleic acid molecule can be any that are expressed in a cell such as an immune cell. The nucleic acids targeted can be a gene involved in immune cell regulation. In some embodiments, the nucleic acid is associated with cancer. The nucleic acid associated with cancer can be a cell cycle gene, cell response gene, apoptosis gene, or phagocytosis gene. The recombinant guide nucleic acid molecule can be recognized by a CRISPR protein, a nuclease-null CRISPR protein, variants thereof, derivatives thereof, or fragments thereof.

[0242] Enzymatically inactive can refer to a polypeptide that can bind to a nucleic acid sequence in a polynucleotide in a sequence-specific manner, but may not cleave a target polynucleotide. An enzymatically inactive site-directed polypeptide can comprise an enzymatically inactive domain (e.g., nuclease domain). Enzymatically inactive can refer to no activity. Enzymatically inactive can refer to substantially no activity. Enzymatically inactive can refer to essentially no activity. Enzymatically inactive can refer to an activity 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 a wild-type exemplary activity (e.g., nucleic acid cleaving activity, wild-type Cas9 activity).

[0243] One or a plurality of the nuclease domains (e.g., RuvC, HNH) of a Cas protein can be deleted or mutated so that they are no longer functional or comprise reduced nuclease activity(e.g. , deactivated or dead Cas, i.e., “dCas”). For example, in a Cas protein comprising at least two nuclease domains (e.g., Cas9), if one of the nuclease domainsis deleted or mutated, the resulting Cas protein, known as a nickase, can generate a single-strand break at a CRISPR RNA (crRNA) recognition sequencewithin a double-stranded DNAbutnot a double-strand break. Such a nickase can cleave the complementary strand or the n on-complementary strand, but may not cleave both. If all of the nuclease domains of a Cas protein (e.g. , both RuvC and HNH nuclease domains in a Cas9 protein; RuvC nuclease domain in a Cpf 1 protein) are deleted or mutated, the resulting Cas protein can have a reduced or no ability to cleave both strands of a double -stranded DNA. An example of a mutation that can convert a Cas9 protein into a nickase is a D10A (aspartate to alanine at position 10 of Cas9) mutation in the RuvC domain of Cas9 from S. pyogenes. H939A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) in the HNH domain of Cas9 from S. pyogenes can convert the Cas9 into a nickase. An example of a mutation that can convert a Cas9 protein into a dead Cas9 is a D 10 A (aspartate to alanine at position 10 of Cas9) mutation in the RuvC domain and H939 A (histidine to alanine at amino acid position 839) or H840 A (histidine to alanine at amino acid position 840) in the HNH domain of Cas9 from S. pyogenes.

[0244] A dead Cas protein can comprise one or more mutations relative to a wild -type version of the protein. The mutation can result in 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-cleaving activity in one or more of the plurality of nucleic acid- cleaving domains of the wild-type Cas protein. The mutation can result in one or more of the plurality of nucleic acid-cleaving domains retainingthe ability to cleave the complementary strand of the target nucleic acid but reducing its ability to cleave the non -complementary strand of the target nucleic acid. The mutation can result in one or more of the plurality of nucleic aci d-cleaving domains retaining the ability to cleave the non -complementary strand of the target nucleic acid but reducing its ability to cleave the complementary strand of thetargetnucleic acid. Themutation can result in one or more of the plurality of nucleic acid-cleaving domains lacking the ability to cleave the complementary strand and the non -complementary strand of the target nucleic acid. The residues to be mutated in a nuclease domain can correspond to one or more catalytic residues of the nuclease. For example, residues in the wild type exemplary S. pyogenes Cas9 polypeptide such as AsplO, His840, Asn854 and Asn856 can be mutated to inactivate one or more of the plurality of nucleic acid-cleaving domains (e.g. , nuclease domains). The residues to be mutated in a nuclease domain of a Cas protein can correspond to residues AsplO, His840, Asn854 and Asn856 in the wild type S. pyogenes Cas9 polypeptide, for example, as determined by sequence and / or structural alignment.

[0245] As examples, residues DIO, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 (or the corresponding mutations of any of the Cas proteins) can be mutated. For example, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A. Mutations other than alanine substitutions can be suitable.

[0246] A D10A mutation can be combined with one or more of H840A, N854A, or N856A mutations to produce a Cas9 protein substantially lacking DNA cleavage activity (e.g., a dead Cas9 protein). A H840 A mutation can be combined with one or more of DI 0A, N854A, orN856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity. A N854A mutation can be combined with one or more of H840A, DIO A, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity. A N856A mutation can be combined with one ormore ofH840A, N854A, or DIOAmutationsto produce a site-directed polypeptide substantially lacking DNA cleavage activity.

[0247] In some cases, a dCas9 can be fused to other proteins. In some cases, dCas9 can be fused to SunTag, KRAB, VPS4, P3000, VPR, VP64, V64-p65-Rta, VP160, VP192, HDAC1, DNMT3 A, TET1, SPH, KRAB-MeCP2, epigenetic regulators, or other proteins. In some cases, a dCas9 fusion comprises a ZIM3 KRAB-Cas9 fusion. In some cases, a Cas9 fusion can be a paired dCas9 system. In some cases, the dCas9 can be part of a SAM system or REDMAP system. Examples of Cas9 variants and fusion proteins can be found in Li, T. et al. , Sig Transduct Target Ther 8, 36 (2023), which is incorporated in its entirety.

[0248] In some embodiments, a Cas protein is a Class 2 Cas protein. In some embodiments, a Cas protein is a type II Cas protein. In some embodiments, the Cas protein is a Cas9 protein, a modified version of a Cas9 protein, or derived from a Cas9 protein. For example, a Cas9 protein lacking cleavage activity. In some embodiments, the Cas9 protein is a Cas9 protein from S. pyogenes (e.g., SwissProt accession number Q99ZW2). In some embodiments, the Cas9 protein is a Cas9 from S. aureus (e.g., SwissProt accession number J7RUA5). In some embodiments, the Cas9 protein is a modified version of a Cas9 protein from S. pyogenes or S. Aureus. In some embodiments, the Cas9 protein is derived from a Cas9 protein from S. pyogenes or S. Aureus. For example, a S. pyogenes or S. Aureus Cas9 protein lacking cleavage activity.

[0249] Cas9 can generally refer to a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity to a wild type exemplary Cas9 polypeptide e.g., Cas9 i ovaS. pyogenes). Cas9 can refer to a polypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity to a wild type exemplary Cas9 polypeptide e.g., from S. pyogenes). Cas9 can refer to the wildtype or a modified form of the Cas9 protein that can comprise an aminoacid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.

[0250] In some embodiments, a guidable polypeptidedomainis a Cas9 or variantthereof . In some embodiments, the Cas9 or variantthereof is a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase domain or a variant thereof. In some embodiments, a guidable polypeptide domain is Cas9, Casl 2e, Casl2d, Casl2a, Casl2bl, Casl3a, Casl2c, or Argonaute (Ago domain), any of which optionally has a nickase activity. In some embodiments, a guidable polypeptide domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 99% identical to any one of sequences listed in Table 17 below. In some embodiments, a guidable polypeptide domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 99% identical to any one of sequences set forth in SEQ ID NOs: 426-446. In some cases, a guidable polypeptide domain is a Cas9 H840 A nickase. In some cases, a guidable polypeptide domain is Cas9 D10A nickase. Cas9-H840A. In some cases, a guidable polypeptide domain is a Casl2a / b nickase.Table 17. Exemplary guidable polypeptide domain sequencesPolymerase

[0251] In some cases, the payload in the present disclosure comprises a polymerase (e.g., reverse transcriptase). A polymerase can comprise a natural or an engineered domain. A polymerase can be capable of synthesizing nucleic acids. A polymerase can be a DNA polymerase or an RNA polymerase. In some cases, a polymerase is a reverse transcriptase. A reverse transcriptase can synthesize DNA from deoxyribonucleotides. In some cases, a reverse transcriptase adds deoxyribonucleotides to the 3 ’ end of a nucleic acid primer to synthesize DNA. In some cases, a reverse transcriptase uses an RNA template and uses base-pairing interactions to synthesize a DNA strand that is complementary to the RNA template. The reverse transcriptase domain can be a reverse transcriptase from any organism, phage, virus, or an engineered or mutated variant. The reverse transcriptase domain can be a reverse transcriptase derived from or sharing structural or sequencing similarity to a reverse transcriptase in a CRISPR system . The reverse transcriptase can be an M-MLV or HIV reverse transcriptase. The reverse transcriptase can be a human LINE-1 reverse transcriptase or a group II intron reverse transcriptase. The reverse transcriptase can be a human endogenous retrovirus reverse transcriptase.Transposase Domain

[0252] In some cases, a nucleic-acid modifying effector or a nucleic acid-binding moiety comprises a transposase domain. A transposase domain can be a natural or an engineered domain. A transposase domain can be capable of aiding the translocation of a transposable element, a nucleic acid sequence that can change its position within a genome. In some cases, a transposase domain comprises a TnsA, a TnsB, a TnsC, or a TnsD domain. In some cases, a transposase domain comprises a TniQ domain. In some cases, a transposase domain is derived from or shares sequence or structural similarity with a transposase in a CRISPR system (e.g., a CRISPR - associated transposase). In some cases, a transposase domain is derived from or share sequence or structural similarity with a transposase domain from a type I CRISPR-associated transposon(CAST) system. In some cases, transposase domain is derived from or share sequence or structural similarity with a transposase domain from a type V CRISPR-associated transposon (CAST) system. A transposase domain can be capable of binding to a guidable polypeptide domain. In some cases, a transposase domain is coupled to a guidable polypeptide domain. In some cases, a transposase domain is capable of bindingto a type I CRISPR-Cas domain (e.g., a Cascade domain, a Cas8 domain, or a Cas5 domain). In some cases, a transposase domain is capable of bindingto a type V CRISPR-Cas domain (e.g., a Casl2 domain). In some cases, a transposase domain is capable of mediating targeted insertion of a nucleic acid into a target nucleic acid. In some cases, a transposase domain is capable of mediating targeted insertion of a nucleic acid that is at least 5 kb, at least 6 kb, atleast 7 kb, atleast 8kb, atleast 9kb, atleast lOkb, atleast 1 Ikb, at least 12kb, at least 13kb, at least 14kb, or at least 15 kb into a target nucleic acid.Transcriptional Regulatory Element

[0253] In some cases, the payload comprises a transcriptional regulatory domain. A transcriptional regulatory domain can be a natural or an engineered domain. A transcriptional regulatory domain can be capable of regulating, activating, or inhibiting gene expression. For example, a transcriptional repressor can silence gene expression by bindingto the promoter of a gene. A transcriptional activator can bind to enhancers or regulatory elements to activate expression of a gene. A transcriptional regulatory domain can comprise a transcription factor. A transcriptional regulatory domain can comprise a transcriptional activation domain or a transcriptional repression domain. For example, a transcriptional activation domain can be or comprise a CAP domain, a VP64 domain, a p65 domain, an Rta domain, a synergistic activation mediator (SAM) domain, a SunTag domain, a VPR domain, a DNA demethylase domain, a histone methyltransferase domain, a histone acetyltransferase domain, or a histone demethylase domain. A transcriptional repression domain can be or comprise a dCas9 domain, a KRAB domain, a Sin3 interacting domain (SID), or a MePC2 domain, a DNA methyltransferase domain, a histone deacetylase domain, a histone methyltransferase domain, or a histone demethylase domain. In some cases, a transcriptional regulatory domain comprises an epigenetic modifying effector domain. For example, an epigenetic modifying effector can be a DNA methyltransferase, a DNA demethylase, a histone methyltransferase, a histone demethylase, a histone acetyltransferase, or a histone deacetylase domain. A DNA methyltransferase domain can be capable of methylating a nucleic acid. A DNA demethylase domain can be capable of demethylating a nucleic acid. A histone methyltransferase domain can be capable of methylating a histone. A histone demethylase domain can be capable of demethylating a histone. A histone acetyltransferase domain can be capable of adding an acetyl group to a histone. A histone deacetylase domain can be capable of removing an acetyl group from a histone.Zinc Finger Domain

[0254] In some embodiments, the payload comprises a zinc finger domain. A zinc finger domain can be a natural or an engineered domain. A zinc finger domain can bind to a specific DNA sequence in a target nucleic acid. A zinc finger domain can comprise from 1 to 10, from 2 to 10, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10 zinc fingers, from 1 to 8, from 2 to 8, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 8 to 8, from 9 to 8 zinc fingers. In some cases, a zinc finger domain comprises a two- handed zinc finger domain. A two-handed zinc finger domain can comprise two clusters of zinc finger domains that are separated by intervening amino acids. A two-handed zinc finger domain can bind to two noncontiguous target DNA sequences. In some cases, the spacing between the two noncontiguous target sequences comprises from 1 to 15, from 1 to 12, from 1 to 10, from 1 to 8, or from 1 to 5 nucleotides. For example, a two-handed type of zinc finger binding protein can be SIP1 . A cluster of zinc finger domains in a two-handed zinc finger domain can be capable of binding to a unique target nucleic acid sequence.TALE Domain

[0255] In some embodiments, the payload comprises a TALE domain. A TALE domain can be a natural or an engineered domain. A TALE domain can bind to a specific DNA sequence. A TALE domain can comprise one or more effector domains. A TALE effector domain can comprise a central repeat domain comprising tandem repeats. A tandem repeat can comprise repeat variable residues (RVD). One or more RVDs can detect a specific DNA base. Different TALE effector domains may have a different numb er of repeats and a different order of their repeats. The C- terminal repeat is usually shorter in length (e.g., about 20 amino acids). Sequential repeats and their RVDs can recognize sequential DNA bases.

[0256] A TALE domain described herein can be derived from a TALE effector from a bacterial species. The TALE domain can be engineered to target a given nucleic acid sequence based on their DNA base specificities. The TALE domain can be engineered to remove or add a TALE effector domain. In some cases, the TALE domain corresponds to a perfect match to a nucleic acid target sequence. In some cases, the TALE domain of an epigenetic effector corresponds to one or more mismatches to a target base in the target nucleic acid.Fusion Protein

[0257] In some cases, the payload in the present disclosure comprises a fusion protein. A fusion protein can comprise two or more polypeptide domains of any of the polypeptide domains described elsewhere herein. A fusion protein can be a natural or an engineered fusion protein. In some cases, the two or more polypeptide domains are coupled together. The two or more polypeptide domains can be coupled together directly or coupled together indirectly. For example,a first polypeptide domain can be coupled directly to a second polypeptide domain. Alternatively, the first polypeptide domain can be coupled indirectly to the second polypeptide domain by coupling with a third polypeptide domain that is coupled directly to the second polypeptide domain. In some cases, a first polypeptide domain is coupled to the N-terminus of a second polypeptide domain. In some cases, a first polypeptide domain is coupled to the C-terminus of a second polypeptide domain. In some cases, a first polypeptide domain is coupled to an internal component of a second polypeptide domain. In some cases, the two or more polypeptide domains are covalently linked. In some cases, the two or more polypeptide domains are noncovalently linked. In some cases, the two or more polypeptide domains are coupled together by a linker. For example, a linker may be a peptide linker. A linker can be a rigid linker, which helps maintain a fixed distance between the polypeptide domains that it links. A linker can be a flexible linker, which can allow some flexibility in movement of one polypeptide domain relative to the other polypeptide domain that it is linked to. In some cases, a linker is a cleavable linker. For example, a cleavable linker can comprise a disulfide bond. Alternatively, a cleavable linker can be an enzymatic cleavable linker, e.g., a linker comprising a protease cleavage site.

[0258] The present disclosure provides fusion proteins comprising a guidable polypeptide domain (e.g., a CRISPR domain). A fusion protein comprising a guidable polypeptide domain (e.g., a CRISPR domain) can comprise one or more of a FokI domain, a deaminase domain, a reverse transcriptase domain, an RNA binding domain, a transcriptional regulatory domain, a plasma membrane recruitment domain, a transmembrane domain, a signaling domain, a receptor domain, a packaging domain, or a targeting domain.

[0259] In some cases, the present disclosure provides a fusion protein comprising a guidable polypeptide domain (e.g., a CRISPR domain) coupled to a deaminase domain. A guidable polypeptide domain (e.g., a CRISPR domain) coupled to a deaminase domain can be used for base editing. A base editor can be capable of editing a nucleic acid sequence in a target nucleic acid molecule. A base editor can be capable of enablingthe generation of base conversions or point mutations in a target nucleic acid. For example, a cytosine base editor can comprise a guidable polypeptide domain (e.g., a CRISPR domain) and a cytidine deaminase domain. An adenine base editor can comprise CRISPR domain and an adenosine deaminase domain. In some cases, a base editor enables the conversion of C to G, A to I, or C to U. A cytosine base editor can be capable of enablingthe conversion of a C-G base pair to a T-A base pair. A glycosylase base editor can be capable of enablingthe conversion of a G-C base pair to a C-G base pair or a G-T base pair. An adenine base editor can be capable of enablingthe conversion of an A-T base pair to a G-C base pair. In some cases, a base editor comprises a catalytically inactive guidable polypeptide domain (e.g., a CRISPR domain) (e.g., dCas9, dCas!2a, or dCas!3b). In other cases, the baseeditor comprises a guidable polypeptide nickase domain (e.g., nCas9). In some cases, the base editor enables a base pair conversion without introducing a double -stranded break. In some cases, the base editor enables base pair conversions in a target window. In some cases, the base editor comprises a targeting window of from 1 to 20 bases, from 1 to 19 bases, from 1 to 18 bases, from 1 to 17 bases, from 1 to 16 bases, from 1 to 15 bases, from 1 to 14 bases, from 1 to 13 bases, from1 to 12 bases, from 1 to 11 bases, from 1 to 10 bases, from 1 to 9 bases, from 1 to 8 bases, from 1 to 7 bases, from 1 to 6 bases, from 1 to 5 bases, from 1 to 4 bases, from 1 to 3 bases, or from 1 to2 bases. In some cases, a base editor has a targeting window of from 3 to 10 bases, from 3 to 9 bases, from 3 to 8 bases, from 3 to 7 bases, from 3 to 6 bases, from 3 to 5 bases, or from 3 to 4 bases. In some cases, the guidable polypeptide domain (e.g., a CRISPR domain) is coupled to the N-terminus of a deaminase domain. In some cases, the guidable polypeptide domain (e.g., a CRISPR domain) is coupled to the C-terminus of a deaminase domain. In some cases, the guidable polypeptide domain (e.g., a CRISPR domain) is coupled to an internal component of a deaminase domain.

[0260] In some cases, the fusion protein comprises a guidable polypeptide domain (e.g., a CRISPR domain) coupled to a reverse transcriptase domain. A guidable polypeptide domain (e.g, a CRISPR domain) coupled to a reverse transcriptase can be capable of enabling prime editing A prime editor can be capable of editing a nucleic acid sequence in a target nucleic acid molecule. A prime editor can be capable of mediating insertion or deletion of a nucleic acid sequence in a target nucleic acid molecule. In some cases, the prime editor enables a sequence insertion or sequence deletion without introducing a double -stranded break. In some cases, the prime editor introduces a nick at the target site. The prime editor can enable insertion of a template sequence in a target nucleic acid molecule. The template sequence can comprise the desired edit. In some cases, a prime editor reverse transcribes a template sequence to synthesize a complementary strand. In some cases, the synthesized complementary strand is inserted in the target nucleic acid molecule. In some cases, the prime editor uses a primer to carry out reverse transcription. The prime editor can install nucleotides to the 3 ’ end of a primer strand. In some cases, a primer strand is generated by nicking the target nucleic acid molecule. In some cases, nicking a strand of the target nucleic acid molecule produces a flap with a 3 ’ OH group. In some cases, the prime editor uses the flap with the 3 ’ OH group as the primer to carry out reverse transcription. In some cases, a guidable polypeptide domain (e.g., a CRISPR domain) is coupled to the N-terminus of a reverse transcriptase domain. In some cases, a guidable polypeptide domain (e.g., a CRISPR domain) is coupled to the C-terminus of a reverse transcriptase domain. In some cases, a guidable polypeptide domain (e.g., a CRISPR domain) is coupled to an internal component of a reverse transcriptase domain.

[0261] In some cases, the fusion protein comprises a guidable polypeptide domain (e.g., a CRISPR domain) coupled to a transcriptional regulatory domain. In some cases, a guidable polypeptide domain (e.g., a CRISPR domain) is coupled to a transcriptional regulatory domain. In some cases, a guidable polypeptide domain (e.g., a CRISPR domain) is coupled to a transcriptional activation domain. In some cases, a guidable polypeptide domain (e.g., a CRISPR domain) is coupled to a transcriptional repression domain. In some cases, guidable polypeptide domain (e.g., a CRISPR domain) is coupled to a transcriptional regulatory domain. A guidable polypeptide domain (e.g., a CRISPR domain) coupled to a transcriptional regulatory domain can be capable of enabling CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa). In some cases, a guidable polypeptide domain (e.g., a CRISPR domain) can be coupled to a transcriptional regulatory domain such as P3000 or DNMT3. In some cases, a guidable polypeptide domain (e.g., a CRISPR domain) is coupled to the C-terminus of a transcriptional regulatory domain. In some cases, a guidable polypeptide domain (e.g., a CRISPR domain) is coupled to an internal component of a transcriptional regulatory domain. Any of the payloads described herein can further comprise a plasma membrane recruitment domain, transmembrane domain, a signaling domain, a receptor domain, a packaging domain, or a targeting domain. Any of payloads described herein can comprise or be engineered to comprise a protein tag, a peptide tag, or small molecule tag. For example, a pay load can comprise a small nuclear localization signal (NLS), a nuclear export signal (NES), a cellpenetratingpeptide (CPP), a mitochondria penetrating peptide (MPP), a solubility tag, or a fluorescent tag.Base Editor

[0262] In some cases, the payload to be delivered by the lipid containing particles of the present disclosure comprises a nucleobase editor (also termed as “b ase editor”) or one or more components of a nucleobase editing (also termed as “base editing”) complex.

[0263] The term “base editor (BE), ” or “nucleobase editor (NBE),” as used herein, can refer to an agent comprising a polypeptide that is capable of making a modification to a base (e.g. , A, T, C, G, or U) within a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, the base editor is capable of deaminating a base within a nucleic acid. In some embodiments, the base editor is capable of deaminating a base within a DNA molecule. In some embodiments, the base editor is capable of deaminating an adenosine (A) in DNA. In some embodiments, the base editor is capable of deaminating a cytosine (C) in DNA. In some embodiments, the base editor is capable of converting a guanine (G) in DNA through a glycosylase.Deaminase Domain

[0264] In some cases, the payload in the present disclosure comprises a deaminase domain. The deaminase domain can be a natural or an engineered domain. A deaminase domain can be capableof carrying out deamination reactions in DNA. A deaminase domain can be capable of enabling the generation of base conversions or point mutations in a target nucleic acid. For example, a deaminase domain can be a cytidine deaminase domain or an adenosine deaminase domain. A cytidine deaminase domain can be capable of converting cytosine to uracil. A cytidine deaminase domain can be capable of enabling the conversion of a C-G base pair to a T-A base pair. For example, a cytidine deaminase can be or comprise a APOBEC1 cytidine deaminase. An adenosine deaminase domain can be capable of converting an adenosine to hypoxanthine. An adenosine deaminase domain can be capable of converting an adenosine to an inosine. An adenosine deaminase can comprise TadA or a TadA mutant. In some embodiments, TadA comprises a monomer. In some embodiments, TadA comprises a heterodimer comprising a wildtype TadA and a mutated Tad A. In some embodiments, TadA comprises a homodimer comprising two wildtype TadA domains or two mutated TadA domains. An adenosine deaminase domain can be capable of enabling the conversion of an A-T base pair to a G-C base pair. A deaminase domain can be a mutated variant. In some cases, a deaminase domain enables the conversion of C to G, A to I, or C to U.

[0265] In some cases, the payload in the present disclosure comprises a glycosylase domain. The glycosylase domain can be a natural or an engineered domain. A glycosylase-based guanine base editor can be designed to remove G, and the AP site generated is repaired by translesion synthesis and / or DNA replication, leading to G-to-C or G-to-T conversion. A glycosylase domain can be capable of enabling the generation of base conversions or point mutations in a target nucleic acid. For example, a glycosylase domain can be a guanine glycosylase domain. Examples of glycosylase base edits can be found in Sun N, et al., Mol Tlier. 2022 Jul 6;30(7):2452-2463 and Huawei Tong, et al., National Science Review , Volume 10, Issue 8, August 2023, each of which is incorporated in its entirety herein.

[0266] In some cases, the base editor disclosed herein comprises a deaminase or a functional domain thereof (“deaminase domain”) that catalyzes deamination reaction.

[0267] The term "deaminase" or "deaminase domain," as used herein, refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase, catalyzing the deamination of adenosine, converting it to the nucleoside hypoxanthine. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase, catalyzing the hydrolytic deamination of cytidine or deoxy cytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase domain, catalyzing 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 someembodiments, the deaminase or deaminase domain is a variant of a naturally -occurring deaminase from an organism, that does not occur in nature. For example, in some embodiments, the deaminase or deaminase domain is atleast 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least75% atleast 80%, atleast 85%, atleast90%, atleast95%, atleast 96%, atleast 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally -occurring deaminase from an organism.

[0268] As used herein, an “adenosine deaminase” is an enzyme that catalyzes the deamination of adenosine, converting it to the nucleoside hypoxanthine. Under standard Watson-Crick hydrogen bond pairing, an adenosine base hydrogen bonds to a thymine base (or a uracil in case of RNA). When adenine is converted to hypoxanthine, the hypoxanthine undergoes hydrogen bond pairing with cytosine. Thus, a conversion of “A” to hypoxanthine by adenosine deaminase will cause the insertion of “C” instead of a “T” during cellular repair and / or replication processes. Since the cytosine “C” pairs with guanine “G”, the adenosine deaminase in coordination with DNA replication causes the conversion of an A»T pairing to a C»G pairing in the double -stranded DNA molecule.

[0269] In some embodiments, the base editor is a chimeric protein comprising 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 broadly be referred to as a “napR / DNAbp -programming nucleic acid molecule” and includes, for example, guide RNA in the case of Cas systems) which direct or otherwise program the protein to localize to a specific target nucleotide sequence (e.g., a gene locus of a genome, or an RNA molecule) that is complementary to the one or more nucleic acid molecules (or a portion or region thereof) associated with the protein, thereby causing the protein to bind to the nucleotide sequence at the specific target site. This term napR / DNAbp embraces CRISPR Cas9 proteins, as well as Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non -naturally occurring (e.g. , engineered or recombinant), and can include a Cas9 equivalent from any type of CRISPR system (e.g., type II, V, VI), including Cpfl (a type-V CRISPR-Cas systems), C2cl (a type V CRISPR-Cas system), C2c2 (a type VI CRISPR-Cas system) and C2c3 (a type V CRISPR- Cas system). Further Cas-equivalents are described in Makarova et al., “C2c2 is a singlecomponent 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 this disclosure are not limited to CRISPR-Cas systems. The present disclosure embraces any suchprogrammable protein, such as the Argonaute protein from Natronobacterium gregoryi (NgAgo) which can also be used for DNA-guided genome editing. NgAgo-guide DNA system does not require aPAM sequence or guide RNAmolecules, whichmeansgenome editing can be performed simply by the expression of generic NgAgo protein and introduction of synthetic oligonucleotides on any genomic sequence. 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.

[0270] In some cases, the napR / DNAbpis derived from a nuclease disclosedherein, such as, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cast 4, Cpfl, C2cl, C2c2, C2c3, Argonaute protein, or a variant thereof. In some embodiments, the base editor comprises a Cas9 (e.g. , dCas9 and nCas9), CasX, CasY, Cpfl, C2cl, C2c2, C2c3, or Argonaute protein fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a Cas9 nickase (nCas9) fused to an 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 a nucleaseinactive 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 Casl4 protein fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a Cpfl protein fused to a deaminase (e.g., cytidine deaminase or adenosine deaminase). In some embodiments, the base editor comprises a C2cl 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).

[0271] In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenosine. In some embodiments, the adenosine deaminases provided herein are capable of deaminating adenosinein a deoxy adenosine residue ofDNA. 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 includes one or more mutations corresponding to any of the mutations provided herein (e.g., mutations in ecTadA). One of skill in the art will be able to identify the corresponding residue in any homologous protein and in the respective encoding nucleic acid by methods well known in the art, e.g. , by sequence alignmentand determination of homologous residues. Accordingly, one of skill in the art would be able to generate mutations in any naturally -occurring adenosine deaminase (e.g., having homology to ecTadA) that corresponds to any of the mutations described herein, e.g., any of the mutations identified in ecTadA. In some embodiments, the adenosine deaminase is from a prokaryote. In some embodiments, the adenosine deaminase is from a bacterium. In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus, or Bacillus subtilis. In some embodiments, the adenosine deaminase is from E. coli.

[0272] In some cases, the deaminase domain of the base editor disclosed herein is derived from a cytidine deaminase. In some cases, the cytidine deaminase domain is derived from the apolipoprotein B mRNA-editing complex (APOBEC) family deaminase, such as APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, or APOBEC3H deaminase. In some cases, the cytidine deaminase is a modification of an APOBEC family deaminase. In some cases, the cytidine deaminase is an evolved derivative of an APOBEC family deaminase.

[0273] In some embodiments, the base editor comprises BE1, BE2, BE3, BE4, BE4max, or another base editor variant. In some embodiments, the base editor comprises BE4max (R33 A) AUGI-hUNG complex (CGBE1).

[0274] In some embodiments, the base editoris fused to, orfurthercomprisesaspartof a chimeric protein, an inhibitor of base excision repair, for example, a uracil clycosylase inhibitor (UGI) domain. In some embodiments, the base editor is fused to one, two, or three UGI domains. In some embodiments, the base editor is fused to one or more UGI domains. In some embodiments, a UGI domain reduces off target effects, specifically the conversion of C to G or C to A.

[0275] In some cases, the base editor disclosed herein is a chimeric protein that comprises a structure such as, NH2-[deaminase domain]-[napR / DNAbp]-[UGI domain]-COOH; NH2- [deaminase domain]-[napR / DNAbp]-[UGI]-[UGI]-COOH; NH2-[deaminase domain]- [napR / DNAbp]-[UGI]-COOH; NH2-[UGI]-[ deaminase domain]-[napR / DNAbp]-COOH; NH2- [deaminase domain]-[UGI]-[napR / DNAbp]-COOH; NH2-[napR / DNAbp]-[UGI]-[deaminase domain]-COOH; or NH2-[napR / DNAbp]-[deaminase domain]-[UGI]-COOH; wherein each instance of comprises an optional linker.

[0276] In some cases, the base editoris fused to, orfurthercomprisesaspartof a chimeric protein, a uracil binding protein (UBP). The term “uracil binding protein” or“UBP,” as used herein, refers to a protein that is capable of binding to uracil. In some embodiments, the uracil binding protein is a uracil modifying enzyme. In some embodiments, the uracil binding protein is a uracil baseexcision enzyme. In some embodiments, the uracil binding protein is a uracil DNA glycosylase (UDG). In some embodiments, a uracil binding protein binds uracil with an affinity that is at least 1%, 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or at least 95% of the affinity that a wild type UDG (e.g., a human UDG) binds to uracil. The term “base excision enzyme” or “BEE,” as used herein, refers to a protein that is capable of removing a base (e.g., A, T, C, G, orU) from a nucleic acid molecule (e.g., DNA orRNA). In some embodiments, a BEE is capable of removing a cytosine from DNA. In some embodiments, a BEE is capable of removing a thymine from DNA. Exemplary BEEs include, without limitation UDG Tyrl47Ala, and UDG Asn204Asp as described in Sang et al., “A Unique Uracil -DNA binding protein of the uracil DNA glycosylase superfamily, ’’Nucleic Acids Research, Vol. 43, No. 172015; the entire contents of which are hereby incorporated by reference.

[0277] In some embodiments, the UBP is a uracil modifying enzyme. In some embodiments, the UBP is a uracil base excision enzyme. In some embodiments, the UBP is a uracil DNA glycosylase. In some embodiments, the UBP is any of the uracil binding proteins provided herein. For example, the UBP can be a UDG, a UdgX, a UdgX*, a UdgX On, or a SMUG1 . In some embodiments, the UBP comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to a uracil binding protein, a uracil base excision enzyme or a uracil DNA glycosylase (UDG) enzyme.

[0278] In some cases, the base editor is fused to, or comprises as a part of the chimeric protein, a nucleic acid polymerase domain (NAP). For instance, the nucleic acid polymerase domain is a eukaryotic nucleic acid polymerase domain. In some cases, the nucleic acid polymerase domain is a DNA polymerase domain. In some cases, the nucleic acid polymerase domain has translesion polymerase activity. In some cases, the nucleic acid polymerase domain is a translesion DNA polymerase. In some cases, the nucleic acid polymerase domain is from Rev7, Revl complex, polymerase iota, polymerase kappa, and polymerase eta. In some cases, the nucleic acid polymerase domain is selected fromthe group of eukaryotic polymerases consisting of alpha, beta, gamma, delta, epsilon, gamma, eta, iota, kappa, lambda, mu, and nu.

[0279] In some cases, the base editor disclosed herein is a chimeric protein that comprises a structure such as, NH2-[deaminase domain]-[napR / DNAbp domain]-[UBP]-[NAP]-COOH; NH2- [ deaminase domain]-[napR / DNAbp]-[NAP]-[UBP]-COOH; NH2-[deaminase domain]-[NAP]- [napR / DNAbp]-[UBP]-COOH; or NH2-[NAP]-[ deaminase domain]-[napR / DNAbp]-[UBP]- COOH; wherein each instance of“-” comprises an optional linker.

[0280] In some cases, the base editor disclosed herein is complexed with a napR / DNAbp- programming nucleic acid molecule. In some cases, the base editing system disclose herein comprises a base editor and a napR / DNAbp -programming nucleic acid molecule, e.g., the baseeditor complexed with the napR / DNAbp-programming nucleic acid molecule. In some cases, the lipid containing particles of the present disclosure deliver a base editing system that comprises both a base editor and a napR / DNAbp-programming nucleic acid molecule, e.g., the base editor complexed with the napR / DNAbp-programming nucleic acid molecule. In some cases, a base editor is delivered separately from the napR / DNAbp-programming nucleic acid molecule through lipid containing particles disclosed herein, or together with other delivery methods, into a cell.

[0281] The term “napR / DNAbp-programming nucleic acid molecule” or equivalently “guide sequence” refers the one or more nucleic acid molecules which associate with and direct or otherwise program a napR / DNAbp protein to localize to a specific target nucleotide sequence (e.g. , a gene locus of a genome) that is complementary to the one or more nucleic acid molecules (or a portion or region thereof) associated with the protein, thereby causing the napR / DNAbp protein to bind to the nucleotide sequence at the specific target site. An example is a guide RNA of a Cas protein of a CRISPR-Cas genome editing system.

[0282] Exemplary configurations, sequences, and mutations thereof for deaminase domains, napR / DNAbp domains, UGI domains, and whole base editor proteins, and exemplary configurations of a base editing system (e.g., comprising both a base editor and a napR / DNAbp- programming nucleic acid molecule) that can be delivered by a lipid containing particle disclosed herein include those describedin U.S. PatentPublicationNos. US20170121693, US20180073012, US20180312828, US20180170984, US2020010835, US2020172931, US20210230577, US20210198330, US20210277379, US2020399626, US2021371858, US2021380955, US2021277379, US2021301274, US20220127622, US20220313799, US20230055682, US20230159913, US20230086199, US20220127622, US20220313799, US20230279373, US20230055682, each of which is incorporated herein by reference in its entirety. Exemplary configurations, sequences, and mutations thereof for deaminase domains, napR / DNAbp domains, UGI domains, and whole base editor proteins, that can be delivered by a lipid containing particle disclosed herein also include those described in Komor AC et al. Nature. 2016 May 19;533(7603):420-4; Kim YB et al. Nat Biotechnol. 2017 Apr;35(4):371-376; Rees HA et al. Nat Cozw w. 2017 Jun6;8:15790;Newby GAetal.A o / 7 . 2021 Nov3;29(l l):3107-3124;Huang TP et al. Nat Protoc. 2021 Feb;16(2):1089-l 128; Lapinaite A et al. Science. 2020 Jul 31;369(6503):566-571; Anzalone AV et al. Nat Biotechnol. 2020 Jul;38(7):824-844; Rees HA et al. Nat Rev Genet. 2018 Dec;19(12):770-788; Koblan LW et al. Nat Biotechnol. 2018 Oct;36(9):843-846; and Gaudelli NM et al. Nature. 2017 Nov 23;551(7681):464-471; each of which is incorporated herein by reference in its entirety.Primer Editor

[0283] In some cases, the lipid delivery particles disclosed herein is capable of delivering a payload, such as a prime editing system, or one or more components thereof, such as a ribonucleoprotein (RNP) complex, into a cell in vitro, ex vivo, or in vivo. In some embodiments, the prime editing system, or one or more components thereof, is within the inside cavity of the protein core of the lipid delivery particles disclosed herein.

[0284] Prime editing system is a ‘search-and-replace’ genome editing technology by which the genome of living organisms can be modified. The term "prime editing system" or "prime editor (PE)" refers the compositions involved in genome editing using target-primed reverse transcription (TPRT) describe herein, can comprise a nucleic acid-guided polypeptide, e.g., nucleic acid-guided polypeptide, a nucleic acid polymerase, chimeric proteins (e.g., comprising guidable polypeptide domain and reverse transcriptase), guide nucleic acid molecule (e.g., guide RNAs), and complexes comprising fusion proteins and guide RNAs, as well as accessory elements, such as second strand nicking components and 5' endogenous DNA flap removal endonucleases (e.g., FEN1) for helping to drive the prime editing process towards the edited product formation.

[0285] In some embodiments, the prime editing system disclosed herein comprises a ribonucleoprotein (RNP) complex. In some cases, the RNP complex comprises a prime editor and a guide nucleic acid molecule. In some cases, the prime editor is formed between one or more proteins and one or more polynucleotides. The prime editor can comprise a nucleic acid-guided polypeptide. The guidable polypeptide domain can comprise a nucleic acid-guided polypeptide, for example a nuclease (e.g. , a Cas protein). For instance, the prime editor can comprise a fusion protein, comprising a nucleic acid programmable R / DNA binding protein (e.g. , a nuclease, such as a Cas protein) and a nucleic acid polymerase (e.g., a reverse transcriptase or any suitable DNA polymerase). In some cases, the nucleic acid polymerase is coupled to the nucleic acid -guided polypeptide. In some cases, the guide nucleic acid molecule can comprise a guide nucleic acid molecule, e.g. , a guide RNA. In some cases, the prime editor is operably linkedto the guide nucleic acid molecule via a linker, forming the RNP complex. In some cases, the prime editor is directly linked to the guide nucleic acid molecule, forming the RNP complex.

[0286] In a specific instance, prime editing system comprises a fusion protein that comprises an engineered Cas9 nickase and a reverse transcriptase, and the fusion protein is paired with an engineered prime editing guide RNA (PEgRNA). In some cases, the PEgRNA can direct Cas9 to a target site within a host cell where the lipid delivery particles are delivered. In some cases, the peg RNA can encode the information for installing the desired edit. In some cases, the prime editing system can function through a multi-step process: 1) the Cas9 domain can bind and nickthe target genomic DNA site, which is specified by a spacer sequence in the PEgRNA; 2) the reverse transcriptase can use the nicked genomic DNA as a primer to initiate synthesis of an edited DNA strand using an engineered extension on the PEgRNA as a template for reverse transcription, which can generate a single-stranded 3 ' flap containingthe edited DNA sequence; 3 ) cellular DNA repair mechanism can resolve the 3' flap intermediate by the displacement of a 5' flap species that occurs via invasion by the edited 3' flap, excision of the 5' flap containing the original DNA sequence, and ligation of the new 3' flap to incorporate the edited DNA strand, forming a heteroduplex of one edited and one unedited strand; and 4) cellular DNA repair mechanism can replace the unedited strand within the heteroduplexusingthe edited strand as a template for repair, which completes this editing process. In some embodiments, the prime editing machinery edits a target DNA molecule. In some embodiments, the prime editing machinery edits a target RNA molecule. Examples of targeting RNA molecules using prime editing are described in international patent application WO2021072328 and U.S. Patent Application number US20230357766, each of which is incorporated in its entirety. In other instances, a prime editing system is a multi -flap prime editing system that can simultaneously edit both DNA strands. For example, a dual-flap prime editing system comprises two PEgRNAs, which can be used to target opposite strands of a genomic site and direct the synthesis of two complementary 3’ flaps containing edited DNA sequence. The pair of edited DNA strands (3 ’ flaps) does not need to directly compete with 5’ flaps in endogenous genomic DNA, as the complementary edited strand is available for hybridization instead. In this instance, both strands of the duplex are synthesized as edited DNA, the dual -flap prime editing system obviates the need for the replacement of the non-edited complementary DNA strand. Instead, cellular DNA repair machinery can only excise the paired 5’ flaps (original genomic DNA) and ligate the paired 3 ’ flaps into the locus.

[0287] In some embodiments, a prime editing system can be paired with a separate Cas9 nickase and a separate gRNA that nicks the DNA at a locus that is different than the locus targeted by the PEgRNA. In some embodiments, one or more prime editing systems can be paired, each targeting a different locus. In some cases, pairing of two prime editing systems, each of which targets a different locus on the same chromosome, can install large insertions, deletions, or modifications. In some cases, pairing of two prime editing systems, each of which targets a different locus, can install large structural modifications. In some embodiments, a prime editor can install up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 modifications. In some embodiments, a prime editor can install 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more modifications. In some embodiments, a prime editor can install up to about 25, about 30, about 35, about40, about45, about50, about55, about60, about65, about70, about75, about80, about 85, about 90, about 95, or about 100 modifications. In some embodiments, a prime editor caninstall more than about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 modifications. In some cases, a prime editor can install more than 100 modifications. In some embodiments, more than one prime editor can be used to install mutations more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more modifications. In some embodiments, more than one prime editor can be used to install mutations more than about 25, about 30, about 35, about 40, about45, about50, about55, about60, about65, about70, about75, about80, about85, about 90, about 95, or about 100 modifications. In some embodiments, more than one prime editor can be used to install mutations more than about Ikb, 5kb, lOkb, 20kb, 30kb, 40kb, 50kb, 60kb, 70kb, 80kb, 90kb, or more.

[0288] Different variants of prime editors have been developed, such as prime editors (PE) PEI, PE2, PE3, PE4, or PE5, some of which are described in Liu, D. et al., Nature 2019, 576, 149-157 and Huang Z, Liu G. Front Bioeng Biotechnol. 2023:11 :1039315, U.S. Patent Application numbers US20210292769, US20230090221, US2022078655, US20230220374, each ofwhichis hereby incorporated by reference herein in its entirety. In some cases, the prime editor comprises a reverse transcriptase (RT) fused with Cas9 H 840A nickase (Cas9n (H840A)) and a primeediting guide RNA (pegRNA). In some cases, the RT comprises an RNA-dependent DNA polymerase. In some cases, the RT comprises a protein derived from a retrovirus. In some embodiments, the RT comprises Moloney Murine Leukemia Virus (M-MMLV) RT. In some embodiments, the RT comprises a RT from HFV, LtrA, HERV-Kcon, Tel4c, Marathon, Gst-IIC, MA-INT5, or another RT ortholog. In some cases, the RT is modified, mutated, truncated, or evolved. In some cases, the RT comprises a full length RT protein. In some cases, the RT comprises a truncated RT. In some cases, the RT is fused to the Cas protein. In some cases, the RT is fused to the Cas protein at the N terminus to the Cas protein. In some cases, the RT is fused to the Cas protein at the C terminus of the Cas protein. In some cases, the RT is fused to the Cas protein as an inlaid fusion. In some cases, the RT is untethered to the Cas protein. Examples of prime editing architecture are described in Grunewald, J., et al.. Nat Biotechnol 41, 337-343 (2023) and Gao Z, et al., Mol Ther. 2022, 30(9):2942-2951, each ofwhich is incorporated herein in its entirety.

[0289] In some cases, the prime editor comprises (a) a fusion protein having the followingN- terminus to C-terminus structure: [NLS]-[Cas9(H840A)]- [linker] -[MMLV_RT(wt)] and (b) a PEgRNA. In some cases, the prime editor comprises (a) a fusion protein havingthe followingN- terminus to C-terminus structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)] and (b)aPEgRNA. In some cases, the prime editor comprises (a) a fusion protein having the following N-terminus to C-terminusstructure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)]; (b) a PEgRNA; and (c) a nicking guide RNA that introduces a nick in the non-edited DNA strand. In some cases, the addition of nicking guide RNA increases the chances of the unedited strand to be repaired rather than the edited strand. In some cases, the prime editor comprises (a) a fusion protein having the following N-terminus to C-terminus structure: [NLS]- [Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K) (W313F)]; (b) a PEgRNA; and (c) a nicking guide RNA that is designed with a spacer that matches only the edited strand but not the original allele before editing, so that the nicking guide RNA is not introduced until after the desired edit is installed. In some cases, the prime editor comprises (a) a fusion protein havingthefollowingN-terminus to C-terminus structure: [NLS]-[Cas9(H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K) (W313F)]; (b) a PEgRNA; and (c) evading specific DNA mismatch repair (MMR) protein, such as co -expression of a dominant negative MMR protein, such as MLHldn (e.g., MLH1 A754-756). In some cases, the prime editor comprises (a) a fusion protein havingthe following N-terminus to C-terminus structure: [NLS]- [Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K) (W313F)]; (b) a PEgRNA; (c) a nicking guide RNA that introduces a nick in the non-edited DNA strand; and (d) evading specific DNA mismatch repair (MMR) protein, such as co -expression of a dominant negative MMR protein, such as MLHldn (e.g., MLH1 A754-756). Evading MMR protein, such as by co-expression of MMR protein MLHldn can increase efficiency of prime editing, as described in International Publication No., WO2023102538 and Chen et al., Cell Volume 184, Issue 22, 28 October 2021, Pages 5635-5652.e29, each of which is hereby incorporated by reference herein in its entirety. An exemplary sequence for MLHldn (MLH1 A754-756) is: MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLKLIQI QDNGTGIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTADG KCAYRASYSDGKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNPSEEYGKILEVVG RYSVHNAGISFSVKKQGETVADVRTLPNASTVDNIRSIFGNAVSRELIEIGCEDKTLAFK MNGYISNANYSVKKCIFLLFINHRLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISPQN VDVNVHPTKHEVHFLHEESILERVQQHIESKLLGSNSSRMYFTQTLLPGLAGPSGEMVK STTSLTSSSTSGSSDKVYAHQMVRTDSREQKLDAFLQPLSKPLSSQPQAIVTEDKTDISSG RARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEMSEKRGPTSSNPRKRHREDSDVE MVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINEQGHEVLREMLHNHSFVGCVNPQW ALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLALDSPESGWT EEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIFILRL ATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIV YKALRSHILPPKHFTEDGNILQLANLPDLYKVF. In some cases, other strategies for evadingMMR protein can be adopted, such as installing silent mutations next to the desired edit or coexpressing an antibody targeting the MMR protein. In some cases, the foregoing prime editor comprises (a) a fusion protein having the following N-terminus to C-terminus structure: [bipartite NLSI-[Cas9(R221K)(N394K)(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)]- [bipartite NLS]-[NLS] instead. In some cases, the components in the foregoing prime editors are packaged in a single lipid delivery particle. In some cases, the components in the foregoing prime editors are packaged in two or more lipid deliver particles that are delivered to the recipient cell simultaneously. In some cases, the components in the foregoing prime editors are packaged in two or more lipid deliver particles that are delivered to the recipient cell sequentially.

[0290] In some cases, the prime editing system can comprise a flap endonuclease (e.g., FEN1 or variant thereof) that is delivered as a part of the lipid delivery particle (e.g., fused to a plasma membrane recruitment element as a chimeric protein). The flap endonuclease can comprise naturally occurring enzymes that process the removal of 5' flaps formed during cellular processes, includingDNAreplication. The flap endonucleaseincludes those described in Patel et al., Nucleic Acids Research, 2012, 40(10): 4507-4519 and Tsutakawac / a / ., Cell, 2011, 145(2): 198-211, each of which is incorporated herein by reference in its entirety.

[0291] Additional elements that can be delivered as a part of the prime editing system via the lipid delivery particles (e.g. , fused to the nucleic acid-guided polypeptide, or fused to plasma membrane recruitment element) described herein include inhibitor of base repair (e.g., proteins that inhibit a nucleic acid repair enzyme, for example, a base excision repair enzyme), uracil glycosylase inhibitor domains (e.g., protein that inhibits a uracil -DNA glycosylase base-excision repair enzyme), epitope tags, and reporter gene sequences, including those described in International Publication No. WO2023205744, which is incorporated herein by reference in its entirety.Epigenetic Editor

[0292] In some cases, the payload to be delivered by the lipid containing particles of the present disclosure comprises an epigenetic editor or one or more components of an epigenetic editing complex (e.g., comprising an epigenetic editor and a nucleic acid molecule that guides the epigenetic editor to bind and / or modify one or more specific target sequences).

[0293] In some cases, the epigenetic editor or epigenetic editing complex disclosed herein has epigenetic activities, such as, methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming 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, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity,ribosylation activity, derib osylation activity, myristoylation activity, remodelling activity, protease activity, oxidoreductase activity, transferase activity, hydrolase activity, lyase activity, isomerase activity, synthase activity, synthetase activity, or demyristoylation activity. In some cases, the epigenetic editor or epigenetic editing complex disclosed herein has a chromosome modification enzyme, or a functional domain that has the functional activity equivalent to a chromosome modification enzyme, such as a methylase, demethylase, acetylase, deacetylase, deaminase, phosphorylase, dephosphorylase, histone modifying enzyme, or nucleotide modifying enzyme. In some cases, the epigenetic editor or epigenetic editing complex disclosed herein has a histone modifying enzyme, or a functional domain that has the functional activity equivalent to a histone modifying enzyme. In some cases, the epigenetic editor or epigenetic editing complex disclosed herein has a nucleotide modifying enzyme, or a functional domain that has the functional activity equivalent to a nucleotide modifying enzyme.

[0294] In some embodiments, the epigenetic editor or epigenetic editing system comprises a protein domain that represses expression of the target gene. For example, the epigenetic editor or epigenetic editing system can comprise a functional domain derived from a zinc finger repressor protein. Sequences of exemplary functional domains of an epigenetic editor or epigenetic editing system that can reduce or silence target gene expression are provided can be found in PCT / US2021 / 030643 and Tycko etal. (Tycko J, DelRosso N, Hess GT, Aradhana, Banerjee A, Mukund A, Van MV, Ego BK, Yao D, Spees K, Suzuki P, Marinov GK, Kundaje A, Bassik MC, Bintu L. High-Throughput Discovery and Characterization of Human Transcriptional Effectors. Cell. 2020 Dec 23;183(7):2020-2035.el6. doi: 10.1016 / j .cell.2020.11.024. Epub 2020 Dec 15. PMID: 33326746; PMCID: PMC8178797.), each ofwhich is incorporated here by reference in its entirety.

[0295] In some embodiments, the epigenetic editor or epigenetic editing system makes an epigenetic modification at a target gene that activates expression of the target gene. In some embodiments, the epigenetic editor or epigenetic editing system modifies the chemical modification of DNA or histone residues associated with the DNA at a target gene harboring the target sequence, thereby activating or increasing expression of the target gene. In some embodiments, the epigenetic editor or epigenetic editing system comprises a DNA demethylase, a DNA dioxygenase, a DNA hydroxylase, or a histone demethylase domain.Nucleic acids and polynucleotides

[0296] In some cases, the lipid delivery particle of the present disclosure comprises a payload comprising a nucleic acid. The nucleic acid as a payload can comprise or be composed of one or more nucleotides. Nucleotides are referred to by their commonly accepted single-letter codes: A represents adenine, C represents cytosine, G represents guanine, T represents thymine, Urepresents uracil, I represents inosine. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' orientation. In some cases, the nucleic acid as a payload comprises a polynucleotide. The nucleic acid as a payload can comprise DNA or RNA. In some cases, the nucleic acid as a payload comprises or encodes a gene. The nucleic acid as a payload can comprise or encode any of the polynucleotides described elsewhere herein. The nucleic acid as a payload can be a vector encoding any of the polypeptide domains described elsewhere herein. In some cases, the nucleic acid as a payload is an engineered polynucleotide.

[0297] In some cases, the payload does not comprise a repair template. In some cases, the double stranded break is repaired through non -homologous end joining. In some cases, the payload comprises a repair template. The repair template can be double-stranded or single-stranded. The repair template can comprise a template sequence comprising a desired edit to be introduced in a target nucleic acid molecule. In some cases, the repair template is a homology -directed repair template. A homology -directed repair template can comprise a homology arm that is homologous to a sequence in the target nucleic acid. In some cases, the payload comprises a DNA-synthesis template comprising a DNA-synthesis template sequence. The DNA-synthesis template can comprise a desired edit to be introduced in a target nucleic acid molecule. The DNA-synthesis template can be a template for a DNA polymerase or a reverse transcriptase to carry out DNA synthesis. For example, in prime editing, a prime editor can use the DNA synthesis template sequence to synthesize a DNA strand that is complementary to the DNA synthesis template sequence. In some cases, the DNA strand is inserted into the target nucleic acid. In some cases, the nucleic acid comprising the DNA synthesis template sequence also comprises a primerbinding sequence. A primer-binding sequence can be complementary to a sequence in a primer strand to which a DNA polymerase or reverse transcriptase can add nucleotides. In some cases, the primer strand is part of a target nucleic acid molecule. A primer-binding sequence can be complementary to a sequence in the target nucleic acid.

[0298] In some cases, the payload comprises a double-stranded DNA containing a desired gene sequence to be inserted in the target nucleic acid molecule. In some cases, the double -stranded DNA is configured to couple to a transposase domain. In some cases, the payload is delivered in the same particle as the transposase domain. In some cases, the payload is delivered in a separate particle as the transposase domain.

[0299] In some cases, the payload comprises a polynucleotide that is configured to bind to a guidable polypeptide domain. In some cases, the polynucleotide directs a guidable polypeptide domain to a sequence in a target nucleic acid molecule. In some cases, the polynucleotide comprises a scaffold segment configured to bind to a guidable polypeptide domain (e.g., Cas9 or Cast 2). In some cases, polynucleotide comprises a spacer sequence that is complementary to atarget sequence in the target nucleic acid molecule and is capable of hybridizing to the target sequence. The polynucleotide can be a natural molecule or an engineered or synthetic molecule. The polynucleotide can be derived or share sequence or structural similarities to CRISPR RNA (crRNA), a tracrRNA, or a scoutRNA encoded in a CRISPR system. In some cases, the polynucleotide is engineered to be a single RNA guide (sgRNA) comprising elements of the crRNA and the tracrRNA. In some cases, the polynucleotide comprises a scaffold segment and a spacer sequence. The scaffold segment can be configured to bind to a guidable polypeptide domain. The scaffold segment can be specific to a specific type of guidable polypeptide (e.g, Cas9 or Casl2). In some cases, the spacer sequence is programmed to be any sequence. In some cases, the spacer sequence is programmed to a sequence complementary to a target nucleic acid sequence.

[0300] In some cases, the payload comprises a polynucleotide that is a guide nucleic acid molecule for a prime editing system, e.g., a prime editing guide RNA (PEgRNA). In some cases, the PEgRNA is capable of (i) identifying a target nucleotide sequence to be edited, and (ii) encoding new genetic information that replaces the targeted sequence. In some cases, a guide nucleic acid molecule for a prime editing system comprises two or more guide RNAs. In some cases, a guide nucleic acid molecule for a prime editing system comprises a nicking guide RNA. In some cases, a guide RNA comprises (A) a primer binding site, (B) a clamp segment, (C) a sequence encoding new genetic information that replaces the targeted sequence, (D) an aptamer, (E) spacer, or (F) scaffold, or any combinations thereof. In some cases, a guide RNA comprises a sequence encoding new genetic information thatreplacesthe targeted sequence, a spacer, and scaffold. In some cases, a guide RNA comprises a spacer and scaffold. In some cases, the guide nucleic acid molecule is heterologous to the cell or host receiving the lipid delivery particle.

[0301] In some cases, the PEgRNA comprises an extended single guide RNA (sgRNA) containing a primer binding site (PBS) and a template sequence for nucleic acid polymerase (e.g., reverse transcriptase or DNA polymerase). For example, a PEgRNA can comprise an architecture corresponding to 5'-[spacer]-[guide RNA core]-[extension arm]-3'. The spacer sequence can comprise about 20 nucleotides in length. The spacer sequence can bind to a protospacer in a target nucleic acid molecule. The spacer sequence can guide the nucleic acid -guided polypeptide (e.g., Cas9) to the target nucleic acid molecule. The guide RNA core can be responsible for binding of the nucleic acid-guided polypeptide (e.g., Cas9). The extension arm can comprise aprimer binding site, an edit template, and a homology arm, in a 3' to 5' direction. The PEgRNA can further comprise, optionally, a 3’ end modifier region, 5’ end modifier region, a transcriptional signal at the 3’ end. The PEgRNA can optionally comprise a secondary structure, such as, hairpins, stem / loops, toe loops, RNA-binding protein recruitment domains (e.g. , the MS2 aptamer whichrecruits and binds to the MS2cp protein). In some cases, the PEgRNA comprises an aptamer and the prime editor further comprises an aptamerbindingprotein (e.g. , fusedto Cas protein or reverse transcriptase). Guide RNAs including an aptamer include those described in International Publication No. W02023205708, which is hereby incorporated herein by reference in its entirety. Homology arm can encode a portion of a resulting reverse transcriptase -encoded single strand DNA flap to be integrated into the target DNA site by replacing the endogenous strand. The portion of the single strand DNA flap encoded by the homology arm is complementary to the non- edited strand of the target DNA sequence, which facilitates the displacement of the endogenous strand and annealing of the single strand DNA flap in its place, thereby installing the edit. The edit template can comprise a sequence corresponding to new genetic information that replaces the targeted sequence, i.e., a single strand RNA of the PEgRNA that codes for a complementary single strand DNA that is either the sense or the antisense strand of the new genetic information that replaces the targeted sequence and which is incorporated into the genomic DNA target locus through the prime editing process.

[0302] In some cases, during genome editing, the primer binding site allows the 3 ’ end of the nicked DNA strand to hybridize to the PEgRNA, while the reverse transcriptase template serves as a template for the synthesis of edited genetic information. A prime editing system can allow DNA synthesis based on the reverse transcriptase template at a nick site a single 3' flap, which becomes integrated into a target nucleic acid on the same strand. In other embodiments, a prime editing system can be a multi-flap prime editing system that generate pairs or multiple pairs of 3' flaps on different strands, which form duplexes comprising desired edits and which become incorporated into target nucleic acid molecules, e.g., at specific loci or edit sites in a genome. In some embodiments, the pairs or multiple pairs of 3' flaps form duplexes because they comprise reverse complementary sequences which anneal to one another once generated by the prime editors described herein. The duplexes can be incorporated into the target site by cell -driven mechanisms that naturally replace the endogenous duplex sequences located between adjacent nick sites. In certain embodiments, the new duplex sequences can be introduced at one or more locations (e.g., at adjacent genomic loci or on two different chromosomal locations), and can comprise one or more sequences of interest, e.g., protein-encoding sequence, peptide-encoding sequence, or RNA-encoding sequence.

[0303] In some cases, the payload comprises a polynucleotide comprising a scaffold segment, a spacer sequence, a DNA synthesis template, and a primer-binding sequence. In some cases, the scaffold segment and a spacer sequence are on a first nucleic acid molecule andthe DNA synthesis template and the primer-binding sequence are on a second nucleic acid molecule.

[0304] In some cases, the guide RNA further comprises a clamp segment. In some cases, the guide RNA comprising, from 3’ to 5’, a primer binding site, a sequence encoding at least a portion of the first recombinase recognition sequence, a clamp segment, scaffold, and spacer. The clamp segment comprises a sequence that, after being reverse transcribed is at least partially complementary to a genomic site close to the primer binding site and where the spacer binds. Without wishing to be bound by a certain theory, the clamp segment can enhance integration efficiency of the new genetic material that replaces the target sequence at the double -stranded target DNA sequence relative to a guide RNA without the clamp segment. The clamp segment can allow for a reduced number of nucleotides in the primer binding site need to bind its genomic site and facilitate reverse transcription, which in turn enables design of a guide RNA that is shorter than conventional guide RNAs used for other gene editing methods. The clamp segment is described in International Publication No. WO2023215831, which is hereby incorporated herein by reference in its entirety.

[0305] In some cases, a guide RNA can complete the insertion of new genetic material that replaces the target sequence without another guide RNA when delivered to a cell together with a prime editor described herein. The guide RNA can complete the insertion of the new genetic material that replaces the target sequence with a second guide RNA that is a nicking guide RNA when delivered together with a prime editor described herein.

[0306] In some cases, a guide RNA comprises two or more guide RNAs. In some cases, the two or more guide RNAs comprise a first guide RNA encoding at least a first portion of new genetic material that replaces the target sequence. In some cases, the two or more guide RNAs comprise a second guide RNA encoding at least a second portion of the new genetic material that replaces the target sequence. In some cases, the first guide RNA and the second guide RNA work in a pair and collectively encode the new genetic material that replaces the target sequence, thereby inserting the new genetic material that replaces the target sequence into the genome of a cell receiving the lipid delivery particles in a site-specific manner. In some cases, the first and the second portion of the new genetic material that replaces the target sequence have at least 6bp overlap. In some cases, the first portion of the new genetic material that replaces the target sequence is 46 bp. In some cases, the first portion of the new genetic material that replaces the target sequence is 42 bp. In some cases, the first portion or the second portion of the new genetic material that replaces the target sequence is 36 bp, 38 bp, 40 bp, 42 bp, 44 bp, or 46 bp. The first guide RNA comprises a first spacer. The second guide RNA comprises a second spacer. The first spacer and the second spacer bind to two genomic target sites that are within 5 -100 bp from each other. When the two or more guide RNAs are delivered to a cell together with a prime editor, the double strand DNA between the two genomic target sites are deleted and the full sequence of thenew genetic material that replaces the target sequence is inserted instead. The deletion can be mediated by the following steps: (a) reverse transcription of the sequence encoding the first portion of the new genetic material that replaces the target sequence in the first guide RNA and the sequence encoding the second portion of the new genetic material that replaces the target sequence in the second guide RNA, wherein the first and the second portion of the new genetic material that replaces the target sequence having at least 6bp overlap, (b) annealing of the two overlapped portion of the new genetic material that replaces the target sequence, (c) synthesis of the second strand comprising the full sequence of the new genetic material that replaces the target sequence, (d) excision of the original DNA sequence, and (e) ligation of the pair nicks. The mechanism, process, and components of this process include those described in International Publication Nos. WO2023122764, W02023205710, and WO2023225670, each of which is hereby incorporated herein by reference in its entirety .

[0307] In some cases, the payload comprises a polypeptide domain described herein coupled to a polynucleotide domain described herein. In some cases, the payload comprises a polypeptide domain described herein complexed to a polynucleotide domain described herein. In some cases, the payload comprises a ribonucleoprotein. For example, the payload may comprise a guidable polypeptide domain complexed to a polynucleotide configured to bind to the guidable polyp eptide domain (e.g., Cas9 complexed with an RNA guide).

[0308] Any ofthepayloadsdescribed herein can further comprisea plasma membranerecruitment element, a transmembrane domain, a signaling domain, a receptor domain, a packaging domain, or a targeting domain. Any of payloads described herein can comprise or be engineered to comprise a protein tag, a peptide tag, or small molecule tag. For example, a payload can comprise a nuclear localization signal (NLS), a nuclear export signal (NES), a cell penetrating peptide (CPP), a mitochondria penetrating peptide (MPP), a solubility tag, a fluorescent tag, or any combinations thereof.Other Payloads

[0309] In some cases, the payload in the lipid delivery particle of the present disclosure comprises a recombinant protein. The payload can be a diagnostic imaging agent, such as a contrast agent. In some cases, the payload comprises a therapeutic agent, including, butnotlimited to, a nuclease, a recombinase, a growth factor, an antibody, a chimeric antigen receptor, a T cell receptor, a cytokine, a cytokine inhibitor or agonist, a transcription factor, an organelle, a nucleic acid molecule, a therapeutic DNA, a therapeutic RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, or a ribozyme, a generic or specific kinase inhibitor, a small molecule drug, an immunomodulator, a tumor suppressor, a developmental regulator, a cancer vaccine, an anesthetic, an enzyme, a hormone, a ligand, a receptor, a T cell receptor, atransposon, a retrotransposon, a DNA polymerase, a RNA dependent DNA polymerase, a homing endonuclease, interferons, chemokines, insulin, growth factors, an antisense oligonucleotide, an RNAi, a shRNA, and any combination thereof. The payload can be a prophylactic agent. In some cases, the payload comprises a biomarker. The payload can also comprise an exogenous antigen or an enzyme. In some cases, the payload comprises a metabolite molecule. In some cases, the payload comprises a lipid molecule. In some cases, the payload comprises a structural protein. In some cases, the payload comprises a hormone or a hormonal protein.CHIMERIC PROTEIN

[0310] In aspects, the present disclosure provides a chimeric protein comprising a plasma membrane recruitment element and a payload that is a protein or a fragment thereof. In some aspects, the lipid delivery particle comprises a chimeric protein comprising a plasma membrane recruitment element and a payload that is a protein or a fragment thereof. In some cases, the plasma membrane recruitment element and the payload are fused directly in the chimeric protein. In other cases, the plasma membrane recruitment element and the pay load are fused indirectly via a linker. In some cases, the linker between the plasma membrane recruitment element and the payload is a cleavable linker that is recognized by a protease.

[0311] The chimeric protein (e.g., comprising a gag protein) can form at least part of a protein core of the lipid delivery particle. A lipid delivery particle can comprise two or more chimeric proteins. The chimeric protein can includea structural protein. The structural protein can comprise a plasma membrane recruitment element (e.g., retroviral gag protein). The plasma membrane recruitment element can be fused to a payload. In some cases, the two or more chimeric proteins comprise the same structural protein. In some cases, the two or more chimeric proteins comprise different structural proteins. In some cases, the two or more chimeric proteins comprise different payloads. In some cases, the chimeric protein comprises a payload that comprises a nucleic acidbinding moiety. In some cases, the payload further comprises a guide nucleic acid molecule that forms a ribonucleoprotein complex with the nucleic acid -binding moiety. In some cases, the chimeric protein is suitable for delivery by a lipid delivery particle disclosed herein.

[0312] In some cases, the lipid delivery particle of the present disclosure further comprises a protease that recognizes the cleavable linker in the chimeric protein and cuts the chimeric protein at the cleavable linker. In some cases, the payload is present as a "free" entity separate from the plasma membrane recruitment element. As a result of the cleavage at the cleavable linker by the protease, the payload can be separated from the plasma membrane recruitment element. In some cases, the payloadispresentas a “free” entity separatefromtheplasmamembranerecruitmentelement. For instance, the payload can be free and present within an inside of the protein core of the lipiddelivery particle. In some cases, the protease is part of a second chimeric protein comprising a second plasma membrane recruitment element and the protease, where the second plasma membrane recruitment element can be either different from or same as the plasma membrane recruitment element that is fused with the payload.

[0313] In some cases, the chimeric protein disclosed herein also comprises one or more non- cleavable linkers that operably link components together. The non -cleavable linker can be any suitable linker sequence that is used for chimeric protein construction, such as peptide linkers that consist 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 (SEQ ID NO: 426), (GGS)x (SEQ ID NO: 427), (GGGGS)x (SEQ ID NO: 428), (GGSG)x (SEQ ID NO: 429), and (SGGG)x (SEQ ID NO: 430), and wherein x is an integer from 1 to 50In some cases, the chimeric protein of the present disclosure comprises a nuclear export signal sequence that can direct transport of the chimeric protein out of the nucleus of a cell, e.g., a producer cell.

[0314] In some cases, the chimeric protein disclosed herein has one of the following configurations of components positioned in an order from N-terminus to C-terminus:[plasma membrane recruitment element]-[cleavable linker]-[payload];[plasma membrane recruitment element]-[n * NES]-[cleavable linker]-[payload];[plasma membrane recruitment element]-[cleavable linker]-[payload]-[n * NES];[plasma membrane recruitment element]-[cleavable linker]-[n * NES]-[payload];[plasma membrane recruitment element]-[cleavable linker l]-[payload]-[cleavable linker 2]-[n * NES];[payload]-[cleavable linker]-[n * NES]-[plasma membrane recruitment element];[payload]-[n * NES]-[cleavable linker]-[plasma membrane recruitment element];[n * NES]-[payload]-[cleavable linker]-[plasma membrane recruitment element];[n * NES]-[cleavable linker l]-[payload]-[cleavable linker 2]-[plasma membrane recruitment element]; and[payload]-[cleavable linker]-[plasma membrane recruitment element]; wherein n is an integer in the range of from 1 to 10, and denotes the number of repeats of the NES sequence. Non-cleavable linker sequence can be present or absent in any of the foregoing configurations between any two neighboring components. As provided herein, the payload sequence in the chimeric protein can have one or more NLS sequences, at its N -terminus, C- terminus, or both.

[0315] In some cases, a chimeric protein described herein can comprise: (i) a budding motif, (ii) a multimerization domain, (iii) a post-translation modification (e.g., a post-translational motif), or (iv) any combination thereof. In some cases, a chimeric protein can comprise a budding motif anda multimerization domain. In some cases, a chimeric protein can comprise a budding motif and a post-translation modification. In some cases, a chimeric protein can comprise a multimerization domain and a post-translation modification. In some cases, a chimeric protein can comprise a budding motif, a multimerization domain, and a post -translation modification.Motifs and Modifications in Lipid Delivery Particles

[0316] In some aspects, provided herein are short polypeptide motifs and / or modifications that can be present within the chimeric protein as described herein. Without wishing to be bound by theory, the motifs in the chimeric proteins and / or modifications to the chimeric protein disclosed herein can enhance one or multiple characteristics of particle assembly and / or payload delivery, including, but not limited to, payload expression, payload stability, payload loading and offloading, particle formation and / or budding, and payload translocation to and from the nucleus.

[0317] In some cases, in the construct encoding the chimeric proteins disclosed herein, there are zero, or one or more codons, optionally neutral structural codons such as alanine, in between the sequences encoding the different components of the chimeric proteins, including, e.g., the motifs disclosed herein (particle budding motif, membrane penetrating peptide, post -translational modification motif, multimerization motif, nuclear export signal, and / or nuclear localization signal), plasma membrane recruitment element, and payload.Particle Budding Motif

[0318] A particle budding motif can be a short polypeptide fused to the payload or plasma membrane recruitment element. Viral particles and / or viral-like particles may bud through limiting host cell membranes and acquire a lipid envelope to exit a cell. In some cases, viral budding may utilize short peptide motifs (e.g., late domains) located within the viral protein that function by recruiting cellular factors. Withoutwishingto be boundby theory, the particle budding motif as described herein can enhance the interaction with host factors that are responsible for the creation of lipid delivery particles. In some cases, the budding motif can be a PPXY, LPXY, PTAP, YPXnL, or any combination thereof. In some embodiments, the budding motif can be YPXnL, wherein n can be an integer from 1 to 5. In some cases, n = 1 , n =2, or n =3. The particle budding motif can bindNEDD4, TSG101, and / or Alix. In some cases, the particle budding motif promotes the efficiency of particle assembly or budding (e.g., increase particle titer). In some cases, the particle budding motif increases the particle titer by at least 1 -fold, at least 2-fold, at least 3 -fold, at least 4-fold, or at least 5 -fold. In some cases, the particle budding motif increase the particle titer by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. The particle budding motif can comprise a late assembly (L) domain. L domains are conserved sequences that can engage the endosomal sorting complex required for transport(ESCRT) components to promote budding. The particle budding motif can be found in retroviral gag proteins which recruit members of the ESCRT complex. The ESCRT complex is located on the cytoplasmic surface of the multivesicular body and can recognize ubiquitinated proteins into vesicles. The vesicles can then bud into the multivesicular body and can be released into the extracellular space. The ESCRT also plays a role in endolysosomal membrane and plasma membrane repair, neuronal pruning, nuclear envelope maintenance, and autophagy . The ESCRT complex comprises three complexes (ESCRT-I, ESCRT-II, ESCRT -III) which are active in the cytosol and the plasma membrane to assist in assembly and fission from the membrane. The particle budding motif can comprise PTAP, PPXY, LPYX, PPEE, LYPL, LYPSL, PPPY, PPEY, PSAP, LYPAL, PPAP, PPPE, YMYL, YRKL, YQCL, YCYL, LYRTL, YPXnL, or any combination thereof.

[0319] The lipid delivery particle can comprise the particle budding motif as described herein. In some cases, the particle budding motif is linked to the C -terminus of the chimeric protein. In some cases, the particle buddingmotif is linked to the N -terminus of the chimeric protein. In some cases, the particle budding motif is linked to the payload. In some cases, the particle budding motif is linked to a plasma membrane recruitment element (e.g., a PH domain). In some cases, the particle budding motif is linked between the payload and the plasma membrane recruitment element. In some cases, the particle budding motif is linked to a nuclear export signal domain. In some cases, the particle budding motif is linked to a non-endogenous cleavage site. In some cases, the particle budding motif is linked to a post -translational modification motif. In some cases, the particle budding motif is present as tandem repeats in the chimeric protein. In some cases, the chimeric protein can comprise one, two, three, four, five, or more particle budding motifs. In some cases, addition of two, three, four, or five budding motifs to the chimeric protein may enhance a lipid delivery particle compared to a lipid delivery particle with a chimeric protein comprising only one budding motif . In some cases, flexible linkers can be present in-between the particle budding motifs. In some cases, a particle budding motif can have a linker to the N-terminus and / or the C- terminus.

[0320] Addition of budding motif to a chimeric protein described herein may increase an efficacy of a lipid delivery particle, for instance, increasing amount of payload loaded in the lipid delivery particle (e.g., average amount per each particle among a population of particles), increasing amount of payload released by the lipid delivery particle upon fusion to a target cell (e.g., average amount per each particle among a population of particles), increasing titer of the lipid delivery particle (e.g. , amount of lipid delivery particles per each preparation, e.g. , amount of particles per unit volume (e.g , m ) of solution containing the particles that is prepared from a certain amount of production cells), or any combination thereof. In some cases, fusion of a budding motif in aconstruct described herein may improve a base editing percentage (e.g., improve a capacity for a lipid delivery particle to deliver base editing payload to atransduced cell). In some cases, addition of budding motif can increase a mean base editing in a cell compared to a level of base editing in a cell transduced with a lipid delivery particle with no budding motif. Addition of one or more budding motifs to a chimeric protein of a lipid delivery particle described herein may increase a mean base editing percentage from at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 99% greater than a mean base editing percentage from a lipid delivery particle with no budding motif. Addition of one or more budding motifs to a chimeric protein of a lipid delivery particle described herein may increase a mean base editing percentage from at most about 99%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 5%, or less than about 5% greater than a mean base editing percentage from a lipid delivery particle with no budding motif . Addition of one or more budding motifs to a chimeric protein of a lipid delivery particle described herein may increase a mean base editing percentage between about 10% to about 99% compared to a mean base editing percentage from a lipid delivery particle without a budding motif. Addition of one or more budding motifs to a chimeric protein of a lipid delivery particle described herein may increase a mean base editing percentage between about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 65%, about 30%to about 70%, about 30% to about 75%, about 30% to about 80%, about 30% to about 85%, about 30% to about 90%, about 30 % to ab out 95 % , ab out 30 % to ab out 99% , ab out 40% to ab out 50 % , ab out 40 % to ab out 60%, about 40% to about 65%, about 40% to about 70%, about40% to about 75%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 40% to about 99%, about 50% to about 60%, about 50% to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 99%, about 60% to about 65%, about 60% to about 70 % , ab out 60 % to ab out 75 %, ab out 60 % to ab out 80% , ab out 60 % to ab out 85 % , ab out 60% to about 90%, about 60% to about 95%, about 60% to about 99%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 85%, about 65% to about 90%, about 65% to about 95%, about 65% to about 99%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 99%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75 % to ab out 95 % , ab out 75 % to ab out 99% , ab out 80% to ab out 85 % , ab out 80 % to ab out 90%,about 80% to about 95%, about 80% to about 99%, about 85% to about 90%, about 85% to about 95%, about 85% to about 99%, about 90% to about 95%, about 90% to about 99%, or about 95% to about 99% compared to a mean base editing percentage from a lipid delivery particle without a budding motif.

[0321] In some cases, the particle budding motif is sourced from a virus. The virus can include, but is not limited to, HIV, EIAV, RSV, ALV, FUJSV, HTLV-1 , MLV, EBOV, HTLV-2, SRV-2, CHMP4A, AVISY, fMLV, influenza, zika virus, dengue virus, WNV, and Tupaia. In some cases, the particle budding motif comprises a sequence with atleast 80% sequenceidentity to a sequence as set forth in any one of SEQ ID NOs: 1-32. In some cases, the particle budding motif comprises a sequence with at least 90% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 1-32. In some cases, the particle budding motif comprises a sequence with at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 1-32. In some cases, the particle budding motif comprises a sequence as set forth in any one of SEQ ID NOs: 1 - 32. In some cases, the particle budding motif comprises any of the sequences or a minimal sequence drawn from any of the sequences as set forth in Table 6.Table 6. Exemplary particle budding motif sequences

[0322] In some cases, the construct for the lipid delivery particle or the chimeric protein with a particle budding motif is designed with any of the following arrangements in Table 12.Membrane Penetrating Peptide

[0323] A membrane penetrating peptide (e.g., a cell penetrating peptide) can be a short polypeptide fused to the payload or plasma membrane recruitment element that can facilitate protein translocation across a phospholipid bilayer. As referred herein, a “membrane penetrating peptide” can comprise a short peptide (e.g., 5-30 amino acids) comprising a charge, which can facilitate penetration into a cell across a cell membrane. Without wishing to be bound by theory, a membrane penetrating peptide may facilitate delivery of cargo through endocytosis. Amembrane penetrating peptide may have a positive charge. A membrane penetrating peptide may have a negative charge. A membrane penetrating peptide may have a neutral charge. In some cases, the membrane penetrating peptide can enhance delivery of cargo through energy dependent mechanisms (e.g., endocytosis), energy independent mechanisms (e.g., direct penetration), or any combination thereof.

[0324] In some cases, the membrane penetrating peptide is fewer than 40 amino acids. In some cases, the membrane penetrating peptide is fewer than 30 amino acids. In some cases, the membrane penetrating peptide is fewer than 20 amino acids. In some cases, the membrane penetrating peptide is about 40 amino acids. In some cases, the membrane penetrating peptide is about 35 amino acids. In some cases, the membrane penetrating peptide is about 30 amino acids. In some cases, the membrane penetrating peptide is about 25 amino acids. In some cases, the membrane penetrating peptide is about 20 amino acids.

[0325] In some cases, the membrane penetrating peptide promotes recruitment of the payloadto the plasma membrane. In some cases, the membrane penetrating peptide increases the efficiency of particle assembly and / or budding. In some cases, the membrane penetrating peptide functions in a pH-inducible manner. In some cases, the pHthatcan modulate membrane penetrating activity of the membrane penetrating peptide is between 3 and 10, such as 3, 4, 5, 6, 7, 8, 9 or 10. Without wishing to be bound by theory, membrane penetrating peptides facilitate payload translocation via penetration of the membrane, endocytosis-mediated entry, or translocation through a transitory structure.

[0326] Membrane penetrating peptides can have amino acid compositions that either contain a high relative abundance of positively charged amino acids such as lysine or arginine or have sequences that contain an alternating pattern of polar, charged amino acids and non-polar, hydrophobic amino acids, as described in Derakhshankhah and Jafari (2018) Biomedicine and Pharmacotherapy 108:1090-1096. Membrane penetrating peptides can also contain only apolar residues with low net charge or hydrophobic amino acid groups that are helpful for cellular uptake. As described herein, the membrane penetrating peptides can be cationic, amphipathic, or hydrophobic.

[0327] The chimeric protein can comprise the membrane penetrating peptide as described herein. In some cases, the membrane penetrating peptide is linked to the C -terminus of the chimeric protein. In some cases, the membrane penetrating peptide is linked to the N-terminus of the chimeric protein. In some cases, the membrane penetrating peptide is linked to the payload. In some cases, the membrane penetrating peptide is linked to a plasma membrane recruitment element (e.g., a PH domain). In some cases, the membrane penetrating peptide is linked between-Il l-the payload and the plasma membrane recruitment element. In some cases, the membrane penetrating peptide is present as tandem repeats in the chimeric protein.

[0328] The construct for the lipid delivery particle or the chimeric protein with a membrane penetrating peptide will be designed with any of the following arrangements in Table 13. PH domain is abbreviated PH, membrane penetrating peptide is abbreviated MPP, and a protease cleavage site is abbreviated CS.

[0329] Addition of a membrane penetrating peptide to a chimeric protein described herein may increase an efficacy of a lipid delivery particle, for instance, increasing amount of payload loaded in the lipid delivery particle (e.g., average amount per each particle among a population of particles), increasing amount of payload released by the lipid delivery particle upon fusion to a target cell (e.g., average amountper each particle amonga population of particles), increasingtiter of the lipid delivery particle (e.g., amount of lipid delivery particles per each preparation, e.g., amount of particles per unit volume (e.g. , mL) of solution containing the particles that is prepared from a certain amount of production cells), or any combination thereof. In some cases, fusion of a membrane penetrating peptide in a construct described herein may improve a base editing percentage (e.g., improve a capacity for a lipid delivery particle to deliver base editing payload to a transduced cell). In some cases, addition of amembranepenetratingpeptide can increase a mean base editing in a cell compared to a level of base editing in a cell transduced with a lipid delivery particle with no budding motif. Addition of one or more membrane penetrating peptides to a chimeric protein of a lipid delivery particle described herein may increase a mean base editing percentage from at least about 5%, atleast about 10%, atleast about20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 99% greater than a mean base editing percentage from a lipid delivery particle with no membrane penetrating peptide. Addition of one or more membrane penetrating peptides to a chimeric protein of a lipid delivery particle described hereinmay increase a mean base editingpercentage from atmost about 99%, atmost about95%, atmost about90%, atmostabout85%, atmost about80%, atmost about 75%, atmost about70%, atmost about60%, atmostabout50%, atmost about40%, atmost about 30%, at most about 20%, at most about 10%, at most about 5%, or less than about 5% greater than a mean base editing percentage from a lipid delivery particle with no membrane penetrating peptide. Addition of one or more membrane penetrating peptides to a chimeric protein of a lipid delivery particle described herein may increase a mean base editing percentage between about 10% to about 99% compared to a mean base editing percentage from a lipid delivery particle without a membrane penetrating peptide. Addition of one or more membrane penetrating peptides to a chimeric protein of a lipid delivery particle described herein may increase a mean base editingpercentage between about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 65%, about 30% to about 70%, about 30% to about 75%, about 30% to about 80 % , ab out 30 % to ab out 85 %, ab out 30 % to ab out 90% , ab out 30 % to ab out 95 % , ab out 30% to about 99%, about 40% to about 50%, about 40% to about 60%, about 40% to about 65%, about 40% to about 70%, about 40% to about 75%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 40% to about 99%, about 50% to about 60%, about 50% to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 99%, about 60% to about 65%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 99%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 85%, about 65% to about 90%, about 65% to about 95%, about 65% to about 99%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 99%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 75% to about 99%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 99%, about 85% to about 90%, about 85% to about 95%, about 85% to about 99%, about 90% to about 95%, about 90% to about 99%, or about 95% to about 99% compared to a mean base editing percentage from a lipid delivery particle without a membrane penetrating peptide.

[0330] In some cases, the membrane penetrating peptide comprises a sequence with at least 80% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 33-56. In some cases, the membrane penetrating peptide comprises a sequence with at least 90% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 33-56. In some cases, the membrane penetrating peptide comprises a sequence with at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 33-56. In some cases, the membrane penetrating peptide comprises a sequence as setforth in any one of SEQ ID NOs: 33-56. In some cases, the membrane penetrating peptide comprises a sequence as set forth in Table 7.Table 7. Exemplary membrane penetrating peptide sequencesPost-Translational Modifications Motif

[0331] A post-translational modification motif disclosed herein can be a short peptide present within the chimeric protein that leads the payload to be modified following translation (e.g., a post-translational modification). A post-translational modification can be any modification following translation. For example, the post-translational modification may be any chemical modification ofthe amino acids in a protein product after the protein productis translated . In some cases, the post-translational modification motif is between about 10 and about 40 amino acids in length. Without wishing to be bound by theory, the post-translational modification alters the payload in a way that can promote recruitment to the plasma membrane. In some cases, the post- translational modification motif, upon the corresponding post-translational modification to the chimeric protein, can alter localization of the payload. The post-translational modification can be a covalent modification of one or more amino acids within the chimeric protein following biosynthesis. The post-translational modification motif can promote plasma membrane localization of cytosolic proteins. The post -translational modification motif can be enzymatic. Asdescribed herein, a post-translational modification motif can be to one or more amino acids within the payload, the plasma membrane recruitment element, or both. In some cases, a post- translational modification comprisesphosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, lipidation, acetylation, or proteolysis. In some cases, a post -translational modification comprises geranylgeranylation, myristoylation, glypiation, isoprenylation, palmitoylation, farnesylation, or any combinationthereof. Acetylation can comprise transfer of an acetyl group to nitrogen. Myristoylation can comprise an attachment of a myristoyl group to a N- terminal glycine residue of a protein. Isoprenylation can comprise an attachment of a prenyl group {e.g., a hydrophobic molecule) to a protein. Farnesylation can comprise an attachment of a famesyl group to a C-terminal cysteine residue of a protein. Phosphorylation can comprise addition of a phosphate group and can occur on serine, threonine, or tyrosine residues. Glycosylation can comprise addition of a sugar moiety {e.g., monosaccharide, polysaccharide, oligosaccharide, or carbohydrate) to a residue. Ubiquitination can comprise the addition of a ubiquitin polypeptide to a lysine residue of a protein. Nitrosylation can comprise incorporation of a nitrosyl moiety of nitric oxide to a protein. Nitrosylation can occur on free cysteine residues to produce S -nitrothiols. Methylation can comprise the transfer of one -carbon methyl groups to a protein and can increase hydrophobicity of the protein. Lipidation can comprise incorporation of a lipid moiety to a protein. Proteolysis can comprise cleaving of peptide bonds of a protein and can assist antigen processing apoptosis, surface protein shedding, and cell signaling. Geranylgeranylation can comprise attachment of at least one lipophilic geranylgeranyl isoprene unit from geranylgeranyl diphosphate to at least one cysteine residue. Glypiation can comprise covalent bonding of a glycosylphosphatidylinositol (GPI) anchor to a protein. Palmitoylation can comprise the attachment of at least one fatty acid {e.g., palmitic acid) to a cysteine residue of a protein.

[0332] In some cases, the construct for the lipid delivery particle or the chimeric protein with a particle budding motif and a post-translational modification is designed with any of the following arrangements in Table 15. PH domain is abbreviated PH, particle budding motif is abbreviated BD, post-translational modification motif is abbreviated PTM, and a protease cleavage site is abbreviated CS.

[0333] The chimeric protein can comprise the post -translational modification motif as described herein. In some cases, the post-translational modification motif is linked to the C-terminus of the chimeric protein. In some cases, the post-translational modification motif is linked to the N- terminus of the chimeric protein. In some cases, the post -translational modification motif is linked to the payload. In some cases, the post-translational modification motif is linked to a plasma membrane recruitment element {e.g., a PH domain). In some cases, the post-translational modification motif is linked to both a payload and a plasma membrane recruitment element {e.g.,a PH domain). In some cases, the post-translational modification motif is linked to some other part(s) of the chimeric protein than a payload and a plasma membrane recruitment element (e.g., a PH domain). In some cases, the post-translational modification motif is linked to some other part(s) of the chimeric protein besides a payload and a plasma membrane recruitment element (e.g. , a PH domain). In some cases, the post-translational modification motif is linked between the payload and the plasma membrane recruitment element. In some cases, the post-translational modification motif is linked to a nuclear export signal domain. In some cases, the post- translational modification motif is linked to a non-endogenous cleavage site. In some cases, the post-translational modification motif is linked to a particle buddingmotif sequence. In some cases, the post-translational modification motif is present as tandem repeats in the chimeric protein.

[0334] In some cases, the post-translational modification motif comprises a sequence with at least 80% sequence identity to a sequence as setforth in any one of SEQ ID NOs: 57-86. In some cases, the post-translational modification motif comprises a sequencewith atleast 90% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 57-86. In some cases, the post-translational modification motif comprises a sequence with at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 57-86. In some cases, the post-translational modification motif comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 57- 86, with at least one but not more than 8 nucleic acid differences. In some cases, the post- translational modification motif comprises a sequence as set forth in any one of SEQ ID NOs: 57- 86. In some cases, the post-translational modification motif comprises a sequence as setforth in Table 8.

[0335] The con struct for the lipid delivery particle orthe chimeric protein with a post -translational modification motif will be designed with any of the following arrangements in Table 14. PH domain is abbreviated PH, post-translational modification motif is abbreviated PTM, and a protease cleavage site is abbreviated CS.

[0336] Addition of a post-translational modification motif to a chimeric protein described herein may increase an efficacy of a lipid delivery particle, for instance, increasing amount of payload loaded in the lipid delivery particle (e.g., average amount per each particle among a population of particles), increasing amount of payload released by the lipid delivery particle upon fusion to a target cell (e.g. , average amount per each particle among a population of particles), increasing titer of the lipid delivery particle (e.g., amount of lipid delivery particles per each preparation, e.g., amount of particles per unit volume (e.g. , m ) of solution containing the particles that is prepared from a certain amount of production cells), or any combination thereof. In some cases, fusion of a post-translational modification motif in a construct described herein may improve a base editingpercentage (e.g., improve a capacity for a lipid delivery particle to deliver base editing payload to a transduced cell). In some cases, addition of a post -translational modification motif can increase a mean base editing in a cell compared to a level of base editing in a cell transduced with a lipid delivery particle with no budding motif. Addition of one or more post -translation al modification motifs to a chimeric protein of a lipid delivery particle described herein may increase a mean base editing percentage from at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, atleast about80%, atleastabout85%, atleastabout90%, atleastabout95%, or99% greater than a mean base editing percentage from a lipid delivery particle with no post -translational modification motif. Addition of one or more post -translational modification motifs to a chimeric protein of a lipid delivery particle described herein may increase a mean base editing percentage from atmost about99%, atmostabout95%, atmost about90%, atmostabout85%, atmost about 80%, atmost about75%, atmost about70%, atmostabout60%, atmost about50%, atmost about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 5%, or less than about 5% greater than a mean base editing percentage from a lipid delivery particle with no post- translational modification motif. Addition of one or more post-translational modification motifs to a chimeric protein of a lipid delivery particle described herein may increase a mean base editing percentage between about 10% to about 99% compared to a mean base editing percentage from a lipid delivery particle without a post-translational modification motif . Addition of one or more post-translational modification motifs to a chimeric protein of a lipid delivery particle described herein may increase a mean base editing percentage between about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 65%, about 30% to about 70%, about 30 % to ab out 75 % , ab out 30 % to ab out 80% , ab out 30% to ab out 85 % , ab out 30 % to ab out 90%, about 30% to about 95%, about 30% to about 99%, about 40% to about 50%, about 40% to about 60 % , ab out 40 % to ab out 65 %, ab out 40 % to ab out 70% , ab out 40 % to ab out 75 % , ab out 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 40% to about 99%, about 50% to about 60%, about 50% to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 99%, about 60% to about 65%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 99%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 85%, about 65% to about 90%, about 65% to about 95%, about 65% to about 99%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 99%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%,about 75% to about 95%, about 75% to about 99%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 99%, about 85% to about 90%, about 85% to about 95%, about 85% to about 99%, about 90% to about 95%, about 90% to about 99%, or about 95% to about 99% compared to a mean base editing percentage from a lipid delivery particle without a post-translational modification motif.Table 8. Exemplary post-translational modification motif sequencesMultimerization Motif

[0337] The chimeric protein described herein can comprise a multimerization motif. In some cases, the multimerization motif can perform protein-protein interactions. As referred to herein, a “multimerization motif’ can comprise any amino acid sequence wherein the sequence recognizes another amino acid sequence of a monomer and promotes multimerization (e.g., formation of a multimer). For example, multimerization may occur at a plasma membrane and enhance assembly and budding of particles described herein. In some cases, the multimerization motif forms a parallel homodimer, such as a coiled-coil. In some cases, the multimerization motif forms dimers, trimers, tetramers, or other oligomers. In some cases, the multimerization motif is a leucine zipper oligomerization motif. The leucine zipper oligomerization motif can comprise a series of leucines spaced at intervals of every seventh amino acid along an a-helix. In some cases, a leucine zipper oligomerization motif can mediate dimerization or oligomerization. In some cases, the coiled-coil is a leucine zipper oligomerization motif (e.g., a GCN4 leucine zipper or a mutant thereof). Upon dimerization, the leucine zipper oligomerization motif can form a parallel-coiled coil. In some cases, the leucine zipper oligomerization motif (e.g., the GCN4 leucine zipper motif). Without wishing to be bound by theory, the GCN4 leucine zipper can be used to induce multimerization that can assist in assembly of the lipid delivery particles described herein. As such, the GCN4 leucine zipper can complement removal of nucleic acid binding domains that are necessary for multimerization in native gag proteins. Multimerization motifs can form dimers, trimers, tetramers, or any combination thereof.

[0338] In some cases, the leucine zipper oligomerization motif is linked to the C-terminus of the chimeric protein. In some cases, the leucine zipper oligomerization motif is linked to the N- terminus of the chimeric protein. In some cases, the leucine zipper oligomerization motif is linked to the payload. In some cases, the leucine zipper oligomerization motif is linked to a plasma membrane recruitment element (e.g., a PH domain). In some cases, the leucine zipper oligomerization motif is linked to both a payload and a plasma membrane recruitment element (e.g., a PH domain). In some cases, the leucine zipper oligomerization motif is linked to some other part(s) of the chimeric protein than a payload and a plasma membrane recruitment element (e.g., a PH domain). In some cases, the leucine zipper oligomerization motif is linked to someother part(s) of the chimeric protein besides a payload and a plasma membrane recruitment element (e.g., a PH domain). In some cases, the leucine zipper oligomerization motif is linked between the payload and the plasma membrane recruitment element. In some cases, the leucine zipper oligomerization motif is linked to a nuclear export signal domain. In some cases, leucine zipper oligomerization motif is linked to a non-endogenous cleavage site. In some cases, the leucine zipper oligomerization motif is linked to a particle budding motif sequence. In some cases, leucine zipper oligomerization motif is present as tandem repeats in the chimeric protein.

[0339] The construct for the lipid delivery particle or the chimeric protein with a multimerization motif will be designed with any of the following arrangements in Table 16. In this Example, a leucine zipper domain will be the multimerization motif. PH domain is abbreviated PH, particle budding motif is abbreviated BD, post-translational modification motif is abbreviated PTM, leucine zipper oligomerization motif is abbreviated LZ, and a protease cleavage site is abbreviated CS. The notation (BD-Payload) will be any one of the combinations of budding and payload configurations. The notation (PH / PTM) will be any one of the combinations of PTMs and payloads, either singly or in combination.

[0340] Addition of multimerization motif to a chimeric protein described herein may increase an efficacy of a lipid delivery particle, for instance, increasing amount of payload loaded in the lipid delivery particle (e.g, average amount per each particle among a population of particles), increasing amount of payload released by the lipid delivery particle upon fusion to a target cell (e.g., average amount per each particle among a population of particles), increasing titer of the lipid delivery particle (e.g. , amount of lipid delivery particles per each preparation, e.g. , amount of particles per unit volume (e.g , mL) of solution containing the particles that is prepared from a certain amount of production cells), or any combination thereof. In some cases, fusion of a multimerization motif in a construct described herein may improve abase editing percentage (e.g, improve a efficacy for a lipid delivery particle to deliver base editing payloadto a transduced cell). In some cases, addition of multimerization motif can increase a mean base editing in a cell compared to a level of base editing in a cell transduced with a lipid delivery particle with no multimerization motif. Addition of one or more multimerization motifs to a chimeric protein of a lipid delivery particle described herein may increase a mean base editing percentage from at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 99% greater than a mean base editing percentage from a lipid delivery particle with no multimerization motif. Addition of one or more multimerization motifs to a chimeric protein of a lipid delivery particle described herein may increase a mean base editing percentage from at most about 99%, at most about 95%, at mostabout 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 5%, or less than about 5% greater than a mean base editing percentage from a lipid delivery particle with no multimerization motif. Addition of one or more multimerization motifs to a chimeric protein of a lipid delivery particle described herein may increase a mean base editing percentage between about 10% to about 99% compared to a mean base editing percentage from a lipid delivery particle without a multimerization motif. Addition of one or more multimerization motifs to a chimeric protein of a lipid delivery particle described herein may increase a mean base editing percentage between about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 65%, about 30% to about 70%, about 30 % to ab out 75 % , ab out 30 % to ab out 80% , ab out 30% to ab out 85 % , ab out 30 % to ab out 90%, about 30% to about 95%, about 30% to about 99%, about 40% to about 50%, about 40% to about 60 % , ab out 40 % to ab out 65 %, ab out 40 % to ab out 70% , ab out 40 % to ab out 75 % , ab out 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 40% to about 99%, about 50% to about 60%, about 50% to about 65%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 99%, about 60% to about 65%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 99%, about 65% to about 70%, about 65% to about 75%, about 65% to about 80%, about 65% to about 85%, about 65% to about 90%, about 65% to about 95%, about 65% to about 99%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 99%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 75% to about 99%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 99%, about 85% to about 90%, about 85% to about 95%, about 85% to about 99%, about 90% to about 95%, about 90% to about 99%, or about 95% to about 99% compared to a mean base editing percentage from a lipid delivery particle without a multimerization motif

[0341] In some cases, the multimerization motif comprises a sequence with atleast 80% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 281 -285. In some cases, the multimerization motif comprises a sequence with at least 90% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 281-285. In some cases, the multimerization motif comprises a sequence with at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 281 -285. In some cases, the multimerization motif comprises an amino acidsequence as set forth in any one of SEQ ID NOs: 281-285, with at least one but not more than 8 nucleic acid differences. In some cases, the multimerization motif comprises a sequence as set forth in any one of SEQ ID NOs: 281 -285. In some cases, the multimerization motif comprises a sequence as set forth in Table 9.Table 9. Exemplary multimerization motif sequencesTable 12. Exemplary Construct Arrangements for Chimeric Protein with Budding MotifTable 13. Exemplary Construct Arrangements for Chimeric Protein with Membrane Penetrating PeptideTable 14. Exemplary Construct Arrangements for Chimeric Protein with Post-Translational ModificationTable 15. Exemplary Construct Arrangements for Chimeric Protein with Post-Translational Modification Motif and Budding MotifTable 16. Exemplary Construct Arrangements for Chimeric Protein withMultimerization MotifNuclear Export Signal

[0342] Direction of nuclear transport within the cell can be governed by nuclear targeting signals within freight proteins. As used herein, the term “nuclear export signal” refers to a sequence of amino acids that targets a freight protein for export from the nucleus. In some cases, a nuclear export signal (NES) is a short target peptide sequence containing four hydrophobic residues. These residues target the protein for export from the nucleus to the cytoplasm through the nuclear pore complex. A chimeric protein provided herein can comprise 1 NES, 2 NESs, 3 NESs, 4 NESs, 5 NESs, 6 NESs, 7 NESs, 8 NESs, 9 NESs, or 10 NESs. In some cases, the NES is located at the N- terminus, C-terminus, or in an internal region of the chimeric protein. In some cases, a NES is coupled between the plasma membrane recruitment element and the payload in the chimeric protein. In some cases, there is a cleavable linker between the plasma membrane recruitment element and the payload in the chimeric protein, and one or more NESs present on the same of the cleavable linker as the plasma membrane recruitment element.

[0343] In some cases, the NES sequence that is used in the chimeric protein comprises LQLPPLERLTL derived from HIV-1 Rev protein, or any of the sequences having at least 80% identity thereto. In some cases, the NES sequence comprises LALKLAGLDI derived from PKIa, or any of the sequences having at least 80% identity thereto. In some cases, the NES sequence that is used in the chimeric protein comprises an amino acid sequence as set forth in Tables 10A-10E. In some cases, the NES sequence comprises an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any sequence listed in Tables 10A-10E . In some cases, the NES sequence described herein comprises a sequence with greaterthan 80% sequence identity to any sequence listed in Tables 10A-10E. In some cases, the NES sequence that is used in the chimeric protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 87-267. In some cases, the NES sequence comprisesan amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to the sequence listed set forth in any one of SEQ ID NOs: 87-267. In some cases, the NES sequence described herein comprises a sequence with greaterthan 80% sequence identity to the sequence listed set forth in any one of SEQ ID NOs: 87-267. The transport of payload proteins within a cell is enabled through both NES and nuclear export receptors. In some cases, the NES described herein is associated with a nuclear export receptor (e.g., CRM-1). In some cases, the NES may be conditionally active or inactive. In some cases, the NES sequence disclosed herein comprises a sequence such as those described in T la Cour, et al., Nucleic Acids Res. 2003 ;3 l(l):393-396; and Xu D, et al. Mol Biol Cell. 2012 Sep;23(18):3673-6, each of which is incorporated hereinby reference in its entirety. Any of the NES sequences described in the NESsequence database (NESdb®; prodata.swmed.edu / LRNes) or (NESbase; services. healthtech.dtu.dk / datasets / NESbase-1.0) can be used in a chimeric protein disclosed herein, e.g., for the purpose of packaging a payload into the molecular assembly, e.g., the lipid delivery particle.Table 10A. Exemplary nuclear export signal sequencesTable 10B. Exemplary nuclear export signal sequencesTable IOC. Exemplary nuclear export signal sequencesTable 10D Exemplary nuclear export signal sequencesTable 10E Exemplary nuclear export signal sequences

[0344] In some cases, a chimeric protein disclosed herein includes at least one NES sequences, such as, 2 or more, 3 or more, 4 or more, or 5 or more NES sequences. In some cases, one or more NES sequences (2 or more, 3 or more, 4 or more, or 5 or more NES sequences) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and / or the C- terminus of the chimeric protein. In some cases, the chimeric protein disclosed herein comprises only one NES sequence. In some cases, the chimeric protein disclosed herein comprises two NES sequences. In some cases, the chimeric protein disclosed herein comprises three NES sequences. In some cases, one or more NES sequences (2 or more, 3 or more, 4 or more, or 5 or more NES sequences) are positioned at or near (e.g., within 50 amino acids of) the N-terminus of the chimeric protein. In some cases, one or more NES sequences (2 or more, 3 or more, 4 or more, or 5 or more NES sequences) are positioned at or near (e.g., within 50 amino acids of) the C-terminus of the chimeric protein. In some cases, one or more NES sequences (3 or more, 4 or more, or 5 or more NES sequences) are positioned at or near (e.g. , within 50 amino acids of) both the N-terminus and the C-terminus of the chimeric protein. In some cases, an NES sequence is positioned at the N-terminus and an NES sequence is positioned at the C-terminus of the chimeric protein.

[0345] In some cases, a payload is a protein that is delivered as part of the chimeric protein disclosed herein, e.g., operably linked to a structural protein (e.g., human endogenous retroviral structural protein or a Plasma membrane recruitment element). In some embodiments, the one or more NES sequences are positioned at or nearthe one orboth ends of the payload protein sequence inside the chimeric protein. For example, in some cases, one or more NES sequences (2 or more, 3 or more, 4 or more, or 5 or more NES sequences) are positionedat or near (e.g., within 50 amino acids of) the N-terminus and / or the C- terminus of the payload protein sequence. In some cases, one or more NES sequences (2 or more, 3 or more, 4 or more, or 5 or more NES sequences) are positioned at or near (e.g., within 50 amino acids of) the N-terminus of the payload protein sequence. In some cases, one or more NES sequences (2 or more, 3 or more, 4 or more, or 5 or more NES sequences) are positioned at or near (e.g., within 50 amino acids of) the C-terminus of the payload protein sequence. In some cases, one or more NES sequences (3 or more, 4 or more, or 5 or more NES sequences) are positioned at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus of the pay load protein sequence. In some cases, an NES sequence is positioned atthe N-terminus and an NES sequence is positioned atthe C-terminus of the payloadprotein sequence. In some cases, the chimeric protein disclosed herein comprises only one NES sequence. In some cases, the chimeric protein comprises only one NES sequence, and the NES sequence is positioned at or near (e.g., within 50 amino acids of) the N-terminus of the pay load protein.

[0346] In eukaryotic cells, transport of proteins between the nucleus and the cytoplasm can be mediated by transport factors in the karyopherin-P family, which are also known as importins and exportins. The direction of nuclear-cytoplasmic transport can be dictated by nuclear targeting signals within the payload proteins. Nuclear export sequences (NESs) can direct export of proteins from the nucleus to the cytoplasm. NESs can bind directly to the export karyopherin CRM1 (also known as exportin 1), which can escort payload proteins through the nuclear pore complex.Nuclear Localization Signal

[0347] In some instances, a payload described herein comprises one or more nuclear localization sequences (NLS). As used herein, the term “nuclear localization signal” refers to a sequence of amino acids thattargets a pay load (e.g., a protein or a short polypeptide) to localize to the nucleus. In some cases, an NLS facilitates the import of a polypeptide comprising an NLS into the cell nucleus. A polypeptide may comprise 1 NLS, 2 NLSs, 3 NLSs, 4 NLSs, 5 NLSs, 6NLSs, 7NLSs, 8 NLSs, 9 NLSs, or 10 NLSs. In some cases, the NLS is located at the N-terminus, C-terminus, or in an internal region of the polypeptide. In some cases, a NLS is coupled to a nucleic acid binding domain described elsewhere herein. In some cases, a NLS is coupled to a nucleic acid modifying domain described elsewhere herein. In some cases, a NLS is coupled to a guidable polypeptide domain, a deaminase domain, or a reverse transcriptase domain. In some cases, aNLS is covalently linked to a nucleic acid binding domain described elsewhere herein. In some cases, a NLS is covalently linked to a nucleic acid modifying domain described elsewhere herein. In some cases, a NLS is covalently linked to a guidable polypeptide domain, a deaminase domain, or a reverse transcriptase domain. In some cases, a nucleic acid binding domain does not comprise an NLS. In some cases, a nucleic acid binding domain does not comprise an NLS. In some cases, a guidable polypeptide domain, a deaminase domain, or a reverse transcriptase domain does not comprise an NLS. Examples of NLS are provided in Table 11 below. In some cases, NLS sequence can comprise an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any sequence listed in Table 11. In some cases, the NLS sequence described herein can comprise a sequence with greater than 80% sequence identity to any sequence listed in Table 11. In some cases, the NLS sequence described herein can comprise any of the sequences listed in Table 11. In some cases, NLS sequence cancomprise an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% sequence identity to any sequence as set forth in SEQ ID NOs: 268-280.Table 11. Exemplary Nuclear Localization Signal Sequences

[0348] In some cases, a chimeric protein disclosed herein includes a nuclear localization sequence (NLS). In some cases, the NLS facilitates delivery of the chimeric protein, or a payload released from the chimeric protein (for instance, released from the chimeric protein following cleavage of a cleavable linker), into the nucleus of a target cell.

[0349] In some cases, a payload is a protein and is delivered as part of the chimeric protein disclosed herein, e.g., operably linked to a structural protein (e.g., plasma membrane recruitment element). In some embodiments, the one or more NLS sequences are positioned at or near the one or both ends of the payload protein sequence of the chimeric protein. In some cases, a chimeric protein includes (e.g., is fused to) between 2 and 5 NLS sequences (e.g., 2-4, or 2-3 NLSs). Examples of NLS sequences include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 271); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 270); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 277) orRQRRNELKRSP (SEQ ID NO: 435); the hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 436); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 437) of the IBB domain from importin -alpha; the sequences VSRKRPRP (SEQ ID NO: 438) and PPKKARED (SEQ ID NO: 439) of the myoma T protein; the sequence PQPKKKPL(SEQ ID NO: 440) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 441) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 442) and PKQKKRK (SEQ ID NO: 443) of the influenza virus NS1 ; the sequence RKLKKKIKKL (SEQ ID NO: 444) of the Hepatitis virus delta antigen;the sequence REKKKFLKRR (SEQ ID NO: 445)of the mouse Mxl protein; the sequence KRKGDE VDGVDEV AKKKS KK (SEQ ID NO: 446) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 447) of the steroid hormone receptors (human) glucocorticoid, and sequences having at least 80% identity to the foregoing. In some cases, an NLS comprises the amino acid sequence MDSLLMNRRKFLY QFKNVRWAKGRRETYLC (SEQ ID NO: 448).

[0350] Other examples of an NLS sequence include KRTADGSEFESPKKKRKV (SEQ ID NO: 449), KKTELQTTNAENKTKKL (SEQ ID NO: 450), KRGINDRNFWRGENGRKTR (SEQ ID NO: 451), RKSGKIAAIVVKRPRK (SEQ ID NO: 452), andMDSLLMNRRKFLY QFKNVRWAKGRRETYLC (SEQ ID NO: 453), SPKKKRKVEAS (SEQ ID NO: 454), encoded by AGCCCCAAGAAgAAGAGaAAGGTGGAGGCCAGC (SEQ ID NO: 455), GPKKKRKVAAA (SEQ ID NO: 456), as well as 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.

[0351] In some embodiments, the chimeric protein comprises one NES sequence and two NLS sequences. In some cases of these embodiments, the NES sequence, NLS sequences, and the payload protein sequence are positioned in an order from N-terminus to C-terminus as follows: NES-NLS-payload protein-NLS. In some embodiments, the chimeric protein comprises two or more NES sequences and two NLS sequences. In some cases of these embodiments, the NES sequences, NLS sequences, and the payload protein sequence are positioned in an order from N- terminus to C-terminus as follows: n X NES (n >=2)-NLS-payload protein-NLS.Cleavable Linker

[0352] In some cases, the chimeric protein comprises a cleavable linker in between two or more components. For instance, the chimeric protein can comprise a cleavable linker between a pay load protein sequence and a plasma membrane recruitment element sequence (e.g., retroviral gag protein sequence). In some cases, the cleavable linker separates the plasma membrane recruitment element sequence from a NLS sequence, and / or a NES sequence at its N -terminus or C-terminus. The cleavable linker can separate the payload protein sequence from the plasma membrane recruitment element sequence, NLS sequence, and / or NES sequence at its N-terminus or C-terminus. Examples of cleavable linker sequences that can be used in the chimeric protein include TSTLLMENSS (SEQ ID NO: 431), PRSSLYPALTP (SEQ ID NO: 432), VQALVLTQ (SEQ ID NO: 433), and PLQ VLTLNIERR (SEQ ID NO: 434), and sequences having at least 80% identity to any one of the foregoing.

[0353] The cleavable linker sequence provided herein can be a cleavable sequence that is recognized and cleaved by any applicable protease, such as a viral protease, a bacterial protease, or a eukaryotic protease (e.g., a protease derived from a plant, an animal, or a fungus). In some cases, the cleavable sequence is recognized by a retroviral protease (pro), such as pro protein derived from Moloney murine leukemia virus (MMLV) or Friend murine leukemia virus (FMLV). In some cases, the cleavable sequence is recognized by a mammalian endogenous protease (e.g., furin, cathepsin, metalloprotease, ADAMI 0, or presenilin). In some cases, the lipid delivery particle further comprises a protease that recognizes the cleavable linker sequence, such as pro protein derived from Moloney murine leukemia virus (MMLV) or Friend murine leukemia vims (FMLV), or protease that is of other viral origin, bacterial origin, or eukaryotic origin. Examples of cleavable linker sequences that can be present in the chimeric protein include TSTLLMENSS, PRSSLYPALTP, VQALVLTQ, and PLQ VLTLNIERR, as well as variant sequences having at least 80% identity to the foregoing sequences.NUCLEIC ACIDS

[0354] Nucleic acids encoding the aforementioned lipid delivery particles are also disclosed.

[0355] In some cases, the invention features a nucleic acid comprising nucleotide sequences that encode the lipid delivery particle, plasma membrane recruitment element, or other component of the chimeric protein disclosed herein. The nucleic acid molecule can encode one, two, or all of: (i) a particle budding motif; (ii) a membrane penetrating peptide; (iii) a post-translational modification motif; and / or (iv) a multimerization motif.

[0356] In some cases, the nucleic acid is an isolated nucleic acid or a non -naturally occurring nucleic acid. Non-naturally occurring nucleic acids are well known to those of skill in the art. In some cases, the nucleic acid is an in vitro transcribed nucleic acid. The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non -native environment such as, for example, a host cell. In some cases, provided herein is an isolated nucleic acid molecule encoding a lipid delivery particle comprising a lipid membrane, a plasma membrane recruitment element, and a chimeric protein comprising a particle budding motif, a membrane penetratingpeptide, and / or a post-translational modification motif. In some cases, provided herein is an isolated nucleic acid molecule encoding a chimeric protein comprising a particle budding motif, a membrane penetrating peptide, and / or a post-translational modification motif.

[0357] In some cases, the nucleic acid molecule is a recombinant nucleic acid molecule. In some cases, provided herein is a recombinant nucleic acid molecule encoding a lipid delivery particle comprising a lipid membrane, a plasma membrane recruitment element, and a chimeric protein comprising a particle budding motif, a membrane penetrating peptide, and / or a post-translational modification motif. In some cases, provided herein is a recombinant nucleic acid molecule encoding a chimeric protein comprising a particle buddingmotif, a membrane penetrating peptide, and / or a post-translational modification motif. In some instances, the recombinant nucleic acid further comprises a leader sequence. In some instances, the recombinant nucleic acid further comprises a promoter sequence. In some instances, the recombinantnucleic acid further comprises a sequence encoding a poly(A) tail. In some instances, the recombinant nucleic acid further comprises a 3 ’UTR sequence.

[0358] In some cases, the lipid delivery particle is encoded by a messenger RNA (mRNA). In one aspect the mRNA encoding the lipid delivery particle is introduced into a cell for production of a lipid delivery particles. In one embodiment, the in vitro transcribed RNA lipid delivery particle can be introduced to a cell as a form of transient transfection. The RNA is produced by in vitro transcription using a polymerase chain reaction (PCR) -generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. The desired template for in vitro transcription is a lipid delivery particle of the present disclosure. In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the nucleic acid can include some or all of the 5’ and / or 3’ untranslated regions (UTRs). The nucleic acid can include exons and introns.

[0359] In some cases, the chimeric protein described herein is encoded by a messenger RNA (mRNA). In one aspect the mRNA encoding the chimeric protein is introduced into a cell for production of a lipid delivery particles comprising the chimeric protein. In one embodiment, the in vitro transcribed RNA chimeric protein can be introduced to a cell as a form of transient transfection. The RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA,synthetic DNA sequence or any other appropriate source of DNA. The desired template for in vitro transcription is a chimeric protein of the present disclosure. In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the nucleic acid can include some or all of the 5 ’ and / or 3 ’ untranslated regions (UTRs). The nucleic acid can include exons and introns.

[0360] In another aspect, the applicationfeatures producer cells and vectors containing the nucleic acids described herein. The nucleic acids may be present in a single vector or separate vectors present in the same host cell or separate host cell.VECTORS

[0361] In various aspects, the lipid delivery vesicles can be embodied as plasmid vectors useful for transient or permanent protein expression in mammalian cells.

[0362] Further provided herein are vectors comprising the compositions or nucleotide sequences encoding a lipid delivery particle or a chimeric protein describ ed herein. In some cases, the vectors comprise nucleotides encoding a lipid delivery particle described herein. In some cases, the vectors comprise nucleotides encoding a chimeric protein described herein. The vectors include, but are not limited to, a virus, plasmid, cosmid, lambda phage or a yeast artificial chromosome (YAC).

[0363] Numerous vector systems can be employed. For example, one class of vectors utilizes DNA elements which are derived from animal viruses such as, fo...

Claims

CLAIMSWhat is claimed is:

1. A lipid delivery particle that comprises:(a) a lipid membrane; and(b) a chimeric protein, wherein the chimeric protein comprises a plasma membrane recruitment element and a payload; wherein the chimeric protein further comprises:(i) a particle budding motif;(ii) a membrane penetrating peptide;(iii) a post-translational modification motif that promotes assembly of the lipid delivery particle and / or promotes loading of the payload into the lipid delivery particle;(iv) a multimerization motif, or(v) any combination thereof.

2. A lipid delivery particle that comprises:(a) a lipid membrane; and(b) a chimeric protein, wherein the chimeric protein comprises a plasma membrane recruitment element and a payload; wherein the chimeric protein further comprises:(i) a particle budding motif;(ii) a membrane penetrating peptide;(iii) a post-translational modification motif selected from the group consisting of : myristoylation motif, acetylation motif, isoprenylation motif, palmitoylation motif, and famesylation motif;(iv) a multimerization motif, or(v) any combination thereof.

3. The lipid delivery particle of claim 1 or 2, wherein the lipid delivery particle is an ectosome, an exosome, or a viral-like particle.

4. The lipid delivery particle of any one of claims 1-3, wherein the plasma membrane recruitment element comprises a pleckstrin homology (PH) domain.

5. The lipid delivery particle of any one of claims 1-4, wherein the plasma membrane recruitment element comprises a mutated PH domain or an engineered PH domain.

6. The lipid delivery particle on any one of claims 4 or 5, wherein the PH domain is from a protein selected from the group consisting of human phospholipase C51, human Aktl, human Aktl with E17K substitution, human 3 -phosphoinositide-dependent proteinkinase 1, human Daapl, mouse Grp 1, human Grpl, human OSBP, human Btkl, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, and human MAPKAP1 .

7. The lipid delivery particle of any one of claims 4-6, wherein the plasma membrane recruitment element further comprises a retroviral gag protein.

8. The lipid delivery particle of claim 7, wherein the PH domain is linked to an N-terminus of the retroviral gag protein.

9. The lipid delivery particle of any one of claims 1-8, wherein the PH domain is linked to a gag-payload fusion peptide.

10. The lipid delivery particle of any one of claims 1-9, wherein the plasma membrane recruitment element is linked via its C-terminus to an N-terminus of the payload.

11. The lipid delivery particle of any one of claims 1-9, wherein the plasma membrane recruitment element is linked via its N-terminus to a C-terminus of the payload.

12. The lipid delivery particle of any one of claims 1-11, wherein the payload comprises a therapeutic agent selected from the group consisting of an antibody, a cytokine, a therapeutic protein, a small molecule therapeutic, a therapeutic DNA, and a therapeutic RNA.

13. The lipid delivery particle of any one of claims 1-12, wherein the payload comprises a ribonucleoprotein, a zinc-finger, a transcription activator-like effector (TALE), a nucleic acid modifying domain, or a guidable polypeptide domain.

14. The lipid delivery particle of any one of claims 1-13, wherein the chimeric protein comprises the particle budding motif.

15. The lipid delivery particle of any one of claims 1-14, wherein the particle budding motif is PTAP, PPXY, LPYX, PPEE, LYPL, LYPSL, PPPY, PPEY, PSAP, LYPAL, PPAP, PPPE, YMYL, YRKL, YQCL, YCYL, LYRTL, YPXnL, or any combination thereof, and wherein n is an integer from 1 to 5.

16. The lipid delivery particle of claim 14 or 15, wherein the particle budding motif is linked to the payload or the plasma membrane recruitment element.

17. The lipid delivery particle of claim 14 or 15, wherein the particle budding motif is linked between the payload and the plasma membrane recruitment element.

18. The lipid delivery particle of claim 14 or 15, wherein the particle budding motif is linked to the chimeric protein based on a construct arrangement listed in Table 12 or Table 15.

19. The lipid delivery particle of any one of claims 1-18, wherein the chimeric protein comprises the membrane penetrating peptide.

20. The lipid delivery particle of claim 19, wherein the membrane penetrating peptide is linked to the payload or the plasma membrane recruitment element.

21. The lipid delivery particle of claim 19, wherein the membrane penetrating peptide is linked between the payload and the plasma membrane recruitment element.

22. The lipid delivery particle of claim 19, wherein the membrane penetrating peptide is linked to the chimeric protein based on a construct arrangement listed in Table 13.

23. The lipid delivery particle of any one of claims 1-22, wherein the chimeric protein comprises the post-translational modification motif.

24. The lipid delivery particle of claim 23, wherein the chimeric protein comprises the post- translational modification motif linked to one or more amino acids of the payload or one or more amino acids of the plasma membrane recruitment element.

25. The lipid delivery particle of claim 23, wherein the chimeric protein comprises the post- translational modification motif linked to one or more amino acids of a region in the chimeric protein outside of the payload and the plasma membrane recruitment element.

26. The lipid delivery particle of claim 23, wherein the chimeric protein comprises the post- translational modification motif linked to one or more amino acids of a region in the chimeric protein between the payload and the plasma membrane recruitment element.

27. The lipid delivery particle of claim 23 wherein the post translational modification motif is linked to the chimeric protein based on a construct arrangement listed in Table 14 or Table 15.

28. The lipid delivery particle of any one of claims 1-27, wherein the chimeric protein comprises the multimerization motif.

29. The lipid delivery particle of any one of claims 1-28, wherein the multimerization motif comprises a leucine zipper oligomerization motif.

30. The lipid delivery particle of claim 29, wherein the multimerization motif is linked to the payload or the plasma membrane recruitment element.

31. The lipid delivery particle of claim 29, wherein the multimerization motif is linked between the payload and the plasma membrane recruitment element.

32. The lipid delivery particle of claim 29, wherein the multimerization motif is linked to the chimeric protein based on a construct arrangement listed in Table 16.

33. The lipid delivery particle of any one of claims 1-32, wherein the chimeric protein comprises the particle budding motif and the membrane penetrating peptide.

34. The lipid delivery particle of any one of claims 1-32, wherein the chimeric protein comprises the particle budding motif and the post -translational modification motif.

35. The lipid delivery particle of any one of claims 1-32, wherein the chimeric protein comprises the particle budding motif and the multimerization motif.

36. The lipid delivery particle of any one of claims 1-32, wherein the chimeric protein comprises the membrane penetrating peptide and the post -translation al modification motif.

37. The lipid delivery particle of any one of claims 1-32, wherein the chimeric protein comprises the membrane penetrating peptide and the multimerization motif.

38. The lipid delivery particle of any one of claims 1-32, wherein the chimeric protein comprises the post-translational modification motif and the multimerization motif.

39. The lipid delivery particle of any one of claims 1-32, wherein the chimeric protein comprises the particle budding motif, the membrane penetrating peptide, and the post- translational modification motif.

40. The lipid delivery particle of any one of claims 1-32, wherein the chimeric protein comprises the particle budding motif, the membrane penetrating peptide, and the multimerization motif.

41. The lipid delivery particle of any one of claims 1-32, wherein the chimeric protein comprises the particle budding motif, the post-translational modification motif, and the multimerization motif.

42. The lipid delivery particle of any one of claims 1-32, wherein the chimeric protein comprises the post-translational modification motif, the membrane penetrating peptide, and the multimerization motif.

43. The lipid delivery particle of any one of claims 1-32, wherein the chimeric protein comprises the particle budding motif, the membrane penetrating peptide, the post- translational modification motif, and the multimerization motif.

44. The lipid delivery particle of any one of claims 1-43, wherein the chimeric protein comprises a tandem repeat of the particle budding motif, the membrane penetrating peptide, the post-translational modification motif, or any combination thereof.

45. The lipid delivery particle of claim 44, wherein the tandem repeat of the particle budding motif further comprises a linker sequence.

46. The lipid delivery particle of any one of claims 1-44, wherein the chimeric protein further comprises a nuclear localization signal (NLS) or a nuclear export signal (NES).

47. The lipid delivery particle of claim 46, wherein the nuclear localization signal is linked to the payload or the plasma membrane recruitment element.

48. The lipid delivery particle of claim 46 or 47, wherein the nuclear localization signal comprises a sequence having at least about 80% sequence identity to the sequence set forth in any one of SEQ ID NOs: 268-280.

49. The lipid delivery particle of any one of claims 46-48, wherein the nuclear localization signal comprises a sequence having at least one but not more than 8 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 268-280.

50. The lipid delivery particle of claim 48 or 49, wherein the nuclear localization signal comprises a sequence as set forth in any one of SEQ ID NOs: 268-280.51 . The lipid delivery particle of any one of claims 46-50, wherein the lipid delivery particle comprises 2, 3, 4, 5, or more NLS sequences.

52. The lipid delivery particle of claim 46, wherein the nuclear export signal is linked to the payload or the plasma membrane recruitment element.

53. The lipid delivery particle of claim 46, wherein the nuclear export signal is linked between the payload and the plasma membrane recruitment element.

54. The lipid delivery particle of claim 46, wherein the nuclear export signal is linked to the particle budding motif, the membrane penetrating particle, and / or the post -translational modification motif.

55. The lipid delivery particle of claim 46, wherein the nuclear export signal is linked to a cleavage site.

56. The lipid delivery particle of any one of claims 46 and 52-55, wherein the nuclear export signal comprises a sequence having at least about 80% sequence identity to the sequence set forth in any one of SEQ ID NOs: 87-267.

57. The lipid delivery particle of any one of claims 46 and 52-55, wherein the nuclear export signal comprises a sequence having at least one but not more than 8 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 87-267.

58. The lipid delivery particle of claim 56 or 57, wherein the nuclear export signal comprises a sequence as set forth in any one of SEQ ID NOs: 87-267.

59. The lipid delivery particle of claim 46, 52-58, wherein the lipid delivery particle comprises 2, 3, 4, 5, or more NES sequences.

60. The lipid delivery particle of any one of claims 1-59, wherein the chimeric protein comprises a linker sequence.

61. The lipid delivery particle of claim 60, wherein the linker sequence comprises a cleavable linker.

62. The lipid delivery particle of any one of claims 1 and 3-61, wherein the post-translational modification motif comprises myristoylation motif, acetylation motif, isoprenylation motif, palmitoylation motif, farnesylation motif, or any combination thereof.

63. The lipid delivery particle of any one of claims 1-62, wherein the particle budding motif comprises a sequence having at least about 80% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 1-32.

64. The lipid delivery particle of any one of claims 1-63, wherein the particle budding motif comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 1-32.

65. The lipid delivery particle of claim 63 or 64, wherein the particle budding motif comprises a sequence as set forth in any one of SEQ ID NOs: 1-32.

66. The lipid delivery particle of any one of claims 1-65, wherein the membrane penetrating peptide comprises a sequence having at least about 80% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 33-56.

67. The lipid delivery particle of any one of claims 1-66, wherein the membrane penetrating peptide comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 33-56.

68. The lipid delivery particle of claim 66 or 67, wherein the membrane penetrating peptide comprises a sequence as set forth in any one of SEQ ID NOs: 33-56.

69. The lipid delivery particle of any one of claims 1-68, wherein the post-translational modification motif comprises a sequence having at least about 80% sequence identity to SEQ ID NOs: 57-86.

70. The lipid delivery particle of any one of claims 1-69, wherein the post-translational modification motif comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 57-86.71 . The lipid delivery particle of claim 69 or 70, wherein the post-translational modification motif comprises a sequence as set forth in SEQ ID NOs: 57-86.

72. The lipid delivery particle of any one of claims 1-71, wherein the multimerization motif comprises a sequence having at least about 80% sequence identity to SEQ ID NOs: 281- 285.

73. The lipid delivery particle of any one of claims 1-72, wherein the multimerization motif comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 281-285.

74. The lipid delivery particle of claim 71 or 72, wherein the multimerization motif comprises a sequence as set forth in SEQ ID NOs: 281-285.

75. The lipid delivery particle of any one of claims 1-74, wherein the particle budding motif interacts with an endosomal sorting complex required for transport (ESCRT).

76. The lipid delivery particle of any one of claims 1-75, wherein the lipid delivery particle further comprises an envelope protein.

77. The lipid delivery particle of claim 76, wherein the envelope protein comprises a VSV glycoprotein (VSV-G), a human immunodeficiency virus (HIV) GP160 glycoprotein, a Baboon Endogenous Retrovirus (BaEVTR) glycoprotein, a fusion protein of Vesicular stomatitis Indiana virus and Rabies virus glycoprotein (FuG-E), an ecotropic Murine Leukemia Virus envelope protein (MLV ENV ecotropic), a human T-cell lymphotropic virus type 1 (HTLV-1) glycoprotein, an amphotrophic Murine Leukemia Virus envelope protein (MLV ENV amphotropic), a moloney murine leukemia virus 10A1 strain glycoprotein (MLV 10A1), a Baculovirus envelope glycoprotein (GP64), a pantropic MLV envelope protein, a xenotropic MLV envelope protein, a xenotropic murine leukemia virus (XMLV) envelope protein, a Moloney murine leukemia virus (MMLV) envelope protein, a moloney murine sarcoma virus (MoMSVg) envelope protein, a simian endogenous type D retrovirus protein (RD-114), a gibbon ape leukemia virus (GALV) envelope protein, a feline leukemia virus (FLV) envelope protein, a mouse mammary tumor virus (MMTV) envelope protein, an avian leukosis virus envelope protein, a Rous Sarcoma virus envelope protein, or an endogenous feline virus envelope protein (RD114 ENV), or a mutant thereof.

78. The lipid delivery particle of claim 76, wherein the envelope protein comprises a HIV-1 envelope protein, a respiratory syncytial virus glycoprotein, a SARS-CoV glycoprotein, a rabies glycoprotein, a Mokola virus glycoprotein, a Semliki Forest virus glycoprotein, a Venezuelan equine encephalitis virus glycoprotein, an Ebola virus glycoprotein, a Marburg virus glycoprotein, or a mutant thereof.

79. The lipid delivery particle of claim 76, wherein the envelope protein comprises a glycoprotein of a mammalian endogenous retrovirus or mutant thereof.

80. The lipid delivery particle of claim 79, wherein the glycoprotein of the mammalian endogenous retrovirus is a glycoprotein of a human endogenous retrovirus (hERV).

81. The lipid delivery particle of claim 80, wherein the glycoprotein of a hERV comprises a hENVHl, a hENVH2, a hENVH3, a hENVKl, a hENVK2, a hENVK3, a hENVK4, a hENVK5, a hENVK6, a hENVT, a hENVW, a hENVFRD, a hENVR, a hENVR(b), a hENVR(c)l, a hENVR(c)2 or a hENVKcon, or a biologically active mutant thereof.

82. The lipid delivery particle of any one of claims 76-81, wherein the envelope protein is fused to a targeting moiety, optionally an scFv.

83. The lipid delivery particle of claim 76, wherein the envelope protein comprises a non- viral envelope protein.

84. A chimeric protein comprising:(a) a plasma membrane recruitment element;(b) a payload; and(c) one, two, three, or all of:(i) a particle budding motif;(ii) a membrane penetrating peptide;(iii) a post-translational modification motif that promotes assembly of a lipid delivery particle and / or promotes loading of the payload into the lipid delivery particle; or(iv) a multimerization motif.

85. A chimeric protein comprising:(a) a plasma membrane recruitment element;(b) a payload; and(c) one, two, three, or all of :(i) a particle budding motif;(ii) a membrane penetrating peptide;(iii) a post-translational modification motif selected from the group consisting of: myristoylation motif, acetylation motif, isoprenylation motif, palmitoylation motif, and famesylation motif; or(iv) a multimerization motif.

86. The chimeric protein of claim 84 or 85, wherein the plasma membrane recruitment element comprises a pleckstrin homology (PH) domain.

87. The chimeric protein of any one of claims 84-86, wherein the plasma membrane recruitment element comprises a mutated PH domain or an engineered PH domain.

88. The chimeric protein on any one of claims 84-87, wherein the PH domain is from a protein selected from a group consisting of human phospholipase C51, human Aktl, human Aktl with E17K substitution, human 3 -phosphoinositide-dependent protein kinase 1, human Daapl, mouse Grpl, human Grpl, human OSBP, human Btkl, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, and human MAPKAP1 .

89. The chimeric protein of any one of claims 86-88, wherein the plasma membrane recruitment element further comprises a retroviral gag protein.

90. The chimeric protein of claim 89, wherein the PH domain is linked to an N-terminus of the retroviral gag protein.

91. The chimeric protein of any one of claims 84-90, wherein the PH domain is linked to a gag-payload fusion construct.

92. The chimeric protein of any one of claims 84-91, wherein the plasma membrane recruitment element is linked via its C-terminus to an N-terminus of the payload.

93. The chimeric protein of any one of claims 84-92, wherein the plasma membrane recruitment element is linked via its N-terminus to a C-terminus of the payload.

94. The chimeric protein of any one of claims 84-93, wherein the payload comprises a therapeutic agent selected from the group consisting of an antibody, a cytokine, a therapeutic protein, a small molecule therapeutic, a therapeutic DNA, and a therapeutic RNA.

95. The chimeric protein of any one of claims 84-94, wherein the payload comprises a ribonucleoprotein, a zinc-finger, a transcription activator-like effector (TALE), or a CRISPR-based genome editing or modulating protein.

96. The chimeric protein of any one of claims 84-95, wherein the chimeric protein comprises the particle budding motif.

97. The chimeric protein of any one of claims 84-96, wherein the particle budding motif is PTAP, PPXY, LPYX, PPEE, LYPL, LYPSL, PPPY, PPEY, PSAP, LYPAL, PPAP, PPPE, YMYL, YRKL, YQCL, YCYL, LYRTL, YPXnL, or any combination thereof, and wherein n is an integer from 1 to 5.

98. The chimeric protein of claim 96 or 97, wherein the particle budding motif is linked to the payload or the plasma membrane recruitment element.

99. The chimeric protein of claim 96 or 97, wherein the particle budding motif is linked between the payload and the plasma membrane recruitment element.

100. The chimeric protein of claim 96 or 97, wherein the particle budding motif is linked to the chimeric protein based on a construct arrangement listed in Table 12 or Table 15.

101. The chimeric protein of any one of claims 84-100, wherein the chimeric protein comprises the membrane penetrating peptide.

102. The chimeric protein of claim 101, wherein the membrane penetrating peptide is linked to the payload or the plasma membrane recruitment element.

103. The chimeric protein of claim 101, wherein the membrane penetrating peptide is linked between the payload and the plasma membrane recruitment element.

104. The chimeric protein of claim 101, wherein the membrane penetrating peptide is linked to the chimeric protein based on a construct arrangement listed in Table 13.

105. The chimeric protein of any one of claims 84-104, wherein the chimeric protein comprises the post-translational modification motif.

106. The chimeric protein of claim 105, wherein the chimeric protein comprises the post- translational modification motif to one or more amino acids of the payload or one or more amino acids of the plasma membrane recruitment element.

107. The chimeric protein of claim 105, wherein the chimeric protein comprises the post- translational modification motif to one or more amino acids of a region in the chimeric protein outside of the payload and the plasma membrane recruitment element.

108. The chimeric protein of claim 105, wherein the chimeric protein comprises the post- translational modification motif to one or more amino acids of a region in the chimeric protein between the payload and the plasma membrane recruitment element.

109. The chimeric protein of claim 105 wherein the post-translational modification motif is linked to the chimeric protein based on a construct arrangement listed in Table 14 or Table 15.

110. The chimeric protein of any one of claims 84-109, wherein the chimeric protein comprises the multimerization motif.

111. The chimeric protein of any one of claims 84-110, wherein the multimerization motif comprises a leucine zipper oligomerization motif.

112. The chimeric protein of claim 110 or 111, wherein the multimerization motif is linked to the payload or the plasma membrane recruitment element.

113. The chimeric protein of claim 110 or 111, wherein the multimerization motif is linked between the payload and the plasma membrane recruitment element.

114. The chimeric protein of claim 110 or 111, wherein the multimerization motif is linked to the chimeric protein based on a construct arrangement listed in Table 16.

115. The chimeric protein of any one of claims 84-114, wherein the chimeric protein comprises the particle budding motif and the membrane penetrating peptide.

116. The chimeric protein of any one of claims 84-114, wherein the chimeric protein comprises the particle budding motif and the post -translational modification motif.

117. The chimeric protein of any one of claims 84-114, wherein the chimeric protein comprises the particle budding motif and the multimerization motif.

118. The chimeric protein of any one of claims 84-114, wherein the chimeric protein comprises the membrane penetrating peptide and the post -translation al modification motif.

119. The chimeric protein of any one of claims 84-114, wherein the chimeric protein comprises the membrane penetrating peptide and the multimerization motif.

120. The chimeric protein of any one of claims 84-114, wherein the chimeric protein comprises the post-translational modification motif and the multimerization motif.

121. The chimeric protein of any one of claims 84-114, wherein the chimeric protein comprises the particle budding motif, the membrane penetrating peptide, and the post- translational modification motif.

122. The chimeric protein of any one of claims 84-114, wherein the chimeric protein comprises the particle budding motif, the membrane penetrating peptide, and the multimerization motif.

123. The chimeric protein of any one of claims 84-114, wherein the chimeric protein comprises the particle budding motif, the post-translational modification motif, and the multimerization motif.

124. The chimeric protein of any one of claims 84-114, wherein the chimeric protein comprises the post-translational modification motif, the membrane penetrating peptide, and the multimerization motif.

125. The chimeric protein of any one of claims 84-114, wherein the chimeric protein comprises the particle budding motif, the membrane penetrating peptide, the post- translational modification motif, and the multimerization motif.

126. The chimeric protein of any one of claims 84-114, wherein the chimeric protein comprises a tandem repeat of the particle budding motif, the membrane penetrating peptide, the post-translational modification motif, or any combination thereof.

127. The chimeric protein of claim 126, wherein the tandem repeat of the particle budding motif further comprises a linker sequence.

128. The chimeric protein of any one of claims 84-127, wherein the chimeric protein further comprises a nuclear localization signal (NLS) or a nuclear export signal (NES).

129. The chimeric protein of claim 128, wherein the nuclear localization signal is linked to the payload or the plasma membrane recruitment element.

130. The chimeric protein of claim 128 or 129, wherein the nuclear localization signal comprises a sequence having at least about 80% sequence identity to SEQ ID NOs: 268- 280.

131. The chimeric protein of any one of claims 128-130, wherein the nuclear localization signal comprises a sequence having at least one but not more than 8 modifications of an amino acid sequence as set forth in SEQ ID NOs: 268-280.

132. The chimeric protein of claim 130 or 131, wherein the nuclear localization signal comprises a sequence as set forth in SEQ ID NOs: 268-280.

133. The lipid delivery particle of any one of claims 128-132, wherein the chimeric protein comprises 2, 3, 4, 5, or more NLS sequences.

134. The chimeric protein of claim 128, wherein the nuclear export signal is linked to the payload or the plasma membrane recruitment element.

135. The chimeric protein of claim 128, wherein the nuclear export signal is linked between the payload and the plasma membrane recruitment element.

136. The chimeric protein of claim 128, wherein the nuclear export signal is linked to the particle budding motif, the membrane penetrating particle, the post -translational modification motif, and / or the multimerization motif.

137. The chimeric protein of claim 128, wherein the nuclear export signal is linked to a cleavage site.

138. The chimeric protein of any one of claims 128 and 134-137, wherein the nuclear export signal comprises a sequence having at least about 80% sequence identity to SEQ ID NOs: 87-267.

139. The chimeric protein of claim 128 and 134-138, wherein the nuclear export signal comprises a sequence having at least one but not more than 8 modifications of an amino acid sequence as set forth in SEQ ID NOs: 87-267.

140. The chimeric protein of claim 138 or 139, wherein the nuclear export signal comprises a sequence as set forth in SEQ ID NOs: 87-267.

141. The chimeric protein of any one of claims 128, and 134-140, wherein the chimeric protein comprises 2, 3, 4, 5, or more NES sequences.

142. The chimeric protein of any one of claims 84-141, wherein the chimeric protein further comprises a linker sequence.

143. The chimeric protein of claim 142, wherein the linker sequence comprises a cleavable linker.

144. The chimeric protein of any one of claims 84-143, wherein the post-translational modification comprises myristoylation motif, acetylation motif, isoprenylation motif, palmitoylation motif, famesylation motif, or any combination thereof.

145. The chimeric protein of any one of claims 84-144, wherein the particle budding motif comprises a sequence having at least about 80% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 1-32.

146. The chimeric protein of any one of claims 84-145, wherein the particle budding motif comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 1-32.

147. The chimeric protein of claim 145 or 146, wherein the particle budding motif comprises a sequence as set forth in any one of SEQ ID NOs: 1-32.

148. The chimeric protein of any one of claims 84-147, wherein the membrane penetrating peptide comprises a sequence having at least about 80% sequence identity to the sequence as set forth in any one of SEQ ID NOs: 33-56.

149. The chimeric protein of any one of claims 84-148, wherein the membrane penetrating peptide comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 33-56.

150. The chimeric protein of claim 148 or 149, wherein the membrane penetrating peptide comprises a sequence as set forth in any one of SEQ ID NOs: 33-56.

151. The chimeric protein of any one of claims 84-150, wherein the post-translational modification motif comprises a sequence having at least about 80% sequence identity to SEQ ID NOs: 57-86.

152. The chimeric protein of any one of claims 84-151, wherein the post-translational modification motif comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 57-86.

153. The chimeric protein of claim 151 or 152, wherein the post-translational modification motif comprises a sequence as set forth in SEQ ID NOs: 57-86.

154. The chimeric protein of any one of claims 84-153, wherein the multimerization motif comprises a sequence having at least about 80% sequence identity to SEQ ID NOs: 281- 285.

155. The chimeric protein of any one of claims 84-154, wherein the multimerization motif comprises a sequence having at least one but not more than 10 modifications of an amino acid sequence as set forth in any one of SEQ ID NOs: 281-285.

156. The chimeric protein of claim 154 or 155, wherein the multimerization motif comprises a sequence as set forth in SEQ ID NOs: 281-285.

157. The chimeric protein of any one of claims 84-156, wherein the particle budding motif interacts with an endosomal sorting complex required for transport (ESCRT).

158. A method for preparing a lipid delivery particle comprising a payload, the method comprising:(a) providing a producer cell that produces the lipid delivery particle of any one of claims 1-83; and(b) collecting the lipid delivery particle from the producer cell.

159. The method of claim 158, wherein the producer cell comprises a nucleic acid sequence encoding the chimeric protein.

160. The method of claim 159, wherein the method further comprises expressing in the producer cell the nucleic acid molecule encoding the chimeric protein.

161. The method of any one of claims 158-160, wherein the producer cell comprises a nucleic acid sequence encoding the envelope protein.

162. The method of claim 161, wherein the method further comprises expressing in the producer cell the nucleic acid molecule encoding the envelope protein.

163. The method of any one of claims 158-162, wherein the method further comprises culturing the producer cell in a medium and maintaining the producer under conditions to produce the lipid delivery particle.

164. The method of any one of claims 158-163, wherein the method further comprises harvesting the medium and purifying the extracellular vesicle.

165. The method of claim 164, wherein the purifying retains the structural integrity of the extracellular vesicle.

166. A lipid delivery particle produced by the method of any one of claims 158-165.

167. A nucleic acid sequence encoding the chimeric protein of any one of claims 1 -157.

168. A composition comprising:(a) a first nucleic acid sequence encoding an envelope protein; and(b) a second nucleic acid sequence encoding a chimeric protein, wherein the chimeric protein comprises:(i) a plasma membrane recruitment element;(ii) a payload; and(iii) one, two, there, or all of :

1. a particle budding motif ;2. a membrane penetrating peptide;3. a post-translational modification motif; or4. a multimerization motif.

169. A vector comprising the nucleic acid sequence of claim 167 or 168.

170. The vector of claim 169, wherein the vector is a vector selected from a plasmid, a cosmid, a bacterial vector, a viral vector, or an artificial chromosome.

171. A cell comprising the lipid delivery particle of any one of claims 1-83.

172. A cell produced by the method of any one of claims 158-165.

173. A pharmaceutical formulation comprising the lipid delivery particle of any one of claims 1-83, and a pharmaceutically acceptable carrier, excipient, adjuvant or diluent.

174. A kit comprising:(a) the lipid delivery particle of any one of claims 1-83, or the pharmaceutical formulation of claim 173; and(b) instructions for use of the lipid delivery particle or the pharmaceutical formulation.

175. The lipid delivery particle of any one of claims 1-83, wherein the chimeric protein enhances an efficacy of the lipid delivery particle compared to an efficacy of an otherwise identical lipid delivery particle without the chimeric protein.

176. The lipid delivery particle of claim 175, wherein the efficacy of the lipid delivery particle is a base editing percentage.

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