WO2024138033A2

WO2024138033A2 - Compositions and methods for delivery of nucleic acid editors

Info

Publication number: WO2024138033A2
Application number: PCT/US2023/085507
Authority: WO
Inventors: Peter CABECEIRAS
Original assignee: Nvelop Therapeutics, Inc.
Priority date: 2022-12-21
Filing date: 2023-12-21
Publication date: 2024-06-27

Abstract

Disclosed herein, in aspects, are compositions, methods, kits, and systems relating to delivery of molecules (e.g., a prime editor and a recombinase) for targeted integration of nucleic acid molecules into cells, for instance, for in vivo delivery via lipid delivery particles.

Description

COMPOSITIONS AND METHODS FOR DELIVERY OF NUCLEIC ACID EDITORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to: US Provisional Application No. 63/476,534, filed December 21, 2022, which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Integration of exogenous DNA into genomic DNA has incredible therapeutic potential, not limited to gene replacement therapy. In some cases, retroviruses, such as lentiviruses, are capable of semirandomly integrating DNA sequences into the genome of host cells. They can serve as a functional delivery vector that can be administered intravenously and achieve efficient levels of tissue transduction in vivo. One major drawback of retroviral vectors and other modes of semi-random integration is that semi-random integration can lead to cell transformation. There remains a need to develop targeted integration strategies, especially when the sequences to be integrated are 100 base pairs or much longer. [0003] Targeted integration is a desirable type of nucleic acid editing that can be challenging to install with current gene editing modalities. Prime editing can result in a targeted integration, but the insertions that are installed by prime editing are, in some cases, limited to 100 base pairs of DNA or less before low efficiency of editing becomes preclusive. CRISPR, TALENs, and ZFNs can utilize a repair template which can be integrated at a target site through homology directed repair (HDR). However, the process of HDR in vivo can be inefficient and double -stranded DNA cleavage, which can be required for HDR, can result in semi-random insertion/deletion (indel) mutations and translocations.

SUMMARY

[0004] Disclosed herein, in some aspects, is a lipid delivery particle, comprising: (a) a lipid containing membrane; (b) a recombinase; and (c) a ribonucleoprotein complex that comprises: a prime editor comprising a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; and a guide nucleic acid molecule. In some cases, the recombinase and the ribonucleoprotein complex are within an inside cavity encapsulated by the lipid containing membrane. In some cases, the lipid delivery particle has a diameter that is less than 5 pm, less than 4 pm, less than 3 pm, less than 2 pm, or less than 1 pm. In some cases, the lipid containing membrane encapsulates a protein core. In some cases, the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule. In some cases, the recombinase mediates recombination between the first recombinase recognition sequence and a second recombinase recognition sequence. In some cases, the lipid delivery particle further comprises either (1) a donor nucleic acid molecule that comprises the second recombinase recognition sequence; or (2) a template RNA that encodes the donor nucleic acid molecule. In some cases, the donor nucleic acid molecule or the template RNA is within the inside cavity encapsulated by the lipid containing membrane. In some cases, the recombinase, the ribonucleoprotein complex, the donor nucleic acid molecule, and/or the template RNA is within the inside cavity of the protein core. [0005] Disclosed herein, in some aspects, is a lipid delivery particle, comprising: (a) a recombinase or a nucleic acid sequence encoding the recombinase; (b) (i) a ribonucleoprotein complex comprising: (1) a prime editor comprising a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; and (2) a guide nucleic acid molecule, or (ii) (1) a nucleic acid sequence encoding the prime editor; and (2) the guide nucleic acid molecule or a nucleic acid sequence encoding the guide nucleic acid molecule; and (c) a template RNA that encodes a donor nucleic acid molecule. In some cases, the donor nucleic acid molecule comprises a donor nucleic acid sequence and a second recombinase recognition sequence, and wherein the recombinase mediates recombination between a first recombinase recognition sequence and the second recombinase recognition sequence. In some cases, the lipid delivery particle has a diameter that is less than 5 pm, less than 4 pm, less than 3 pm, less than 2 pm, or less than 1 pm. In some cases, the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule. In some cases, the lipid delivery particle comprises a lipid containing membrane encapsulating a protein core.

[0006] Disclosed herein, in some aspects, is a lipid delivery particle comprising: a first nucleic acid sequence encoding a prime editor. In some cases, the prime editor comprises a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; a guide nucleic acid molecule or a second nucleic acid sequence encoding the guide nucleic acid molecule; a third nucleic acid sequence encoding a recombinase; and a donor nucleic acid sequence that comprises a second recombinase recognition sequence, or a template RNA encoding the donor nucleic acid sequence, and wherein the recombinase mediates recombination between a first recombinase recognition sequence and the second recombinase recognition sequence. In some cases, the lipid delivery particle has a diameter that is less than 5 pm, less than 4 pm, less than 3 pm, less than 2 pm, or less than 1 pm. In some cases, the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule. In some cases, the lipid delivery particle comprises a lipid containing membrane encapsulating a protein core.

[0007] Disclosed herein, in some aspects, is a system, comprising: (1) a lipid delivery particle comprising a lipid containing membrane; and a ribonucleoprotein complex comprising: (A) a prime editor comprising a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; and (B) a guide nucleic acid molecule, wherein the ribonucleoprotein complex is within an inside cavity encapsulated by the lipid containing membrane; and (2) a recombinase or a nucleic acid sequence encoding the recombinase. In some cases, the lipid delivery particle has a diameter that is less than 5 pm, less than 4 pm, less than 3 pm, less than 2 pm, or less than 1 pm. In some cases, the lipid delivery particle comprises a lipid containing membrane encapsulating a protein core. In some cases, the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule. In some cases, the recombinase mediates recombination between the first recombinase recognition sequence and a second recombinase recognition sequence.

[0008] Disclosed herein, in some aspects, is a system, comprising: (1) a lipid delivery particle comprising a lipid containing membrane; and a recombinase, wherein the recombinase is within an inside cavity encapsulated by the lipid containing membrane; and (2) (i) a ribonucleoprotein complex comprising: (A) a prime editor comprising a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; and (B) a guide nucleic acid molecule, or (ii) (A) a nucleic acid sequence encoding the prime editor; and (B) the guide nucleic acid molecule or a nucleic acid sequence encoding the guide nucleic acid molecule. In some cases, the lipid delivery particle has a diameter that is less than 5 pm, less than 4 pm, less than 3 pm, less than 2 pm, or less than 1 pm. In some cases, the lipid delivery particle comprises a lipid containing membrane encapsulating a protein core. In some cases, the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule. In some cases, the recombinase mediates recombination between the first recombinase recognition sequence and a second recombinase recognition sequence.

[0009] In any one of the foregoing or related aspects, the system further comprises either (3) a donor nucleic acid molecule comprising a donor nucleic acid sequence and the second recombinase recognition sequence, or (4) a template RNA that encodes the donor nucleic acid molecule.

[0010] In any one of the foregoing or related aspects, the lipid containing membrane comprises a phospholipid bilayer.

[0011] Disclosed herein, in some aspects, is a composition comprising: a first nucleic acid sequence encoding a first chimeric protein comprising a first plasma membrane recruitment element coupled to a prime editor, wherein the prime editor comprises a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; a guide nucleic acid molecule or a second nucleic acid sequence encoding the guide nucleic acid molecule; and a third nucleic acid sequence encoding a second chimeric protein comprising a second plasma membrane recruitment element coupled to a recombinase. In some cases, the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule. In some cases, the recombinase mediates recombination between the first recombinase recognition sequence and a second recombinase recognition sequence. In some cases, the composition further comprises either (1) a donor nucleic acid molecule that comprises the second recombinase recognition sequence; or (2) a template RNA that encodes the donor nucleic acid molecule. In some cases, the composition further comprises a fourth nucleic acid sequence encoding an envelope protein.

[0012] Disclosed herein, in some aspects, is a composition comprising: a first nucleic acid sequence encoding a prime editor, wherein the prime editor comprises a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; a guide nucleic acid molecule or a second nucleic acid sequence encoding the guide nucleic acid molecule; a third nucleic acid sequence encoding a recombinase; a donor nucleic acid sequence that comprises a second recombinase recognition sequence, or a template RNA encoding the donor nucleic acid sequence ; and a nucleic acid sequence encoding an envelope protein; wherein the recombinase mediates recombination between a first recombinase recognition sequence and the second recombinase recognition sequence. In some cases, the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule. In some cases, the first nucleic acid sequence encodes a first chimeric protein comprising a first plasma membrane recruitment element coupled to the prime editor. In some cases, the third nucleic acid sequence encodes a second chimeric protein comprising a second plasma membrane recruitment element coupled to the recombinase.

[0013] In any one of the foregoing or related aspects, the template RNA comprises a long terminal repeat (LTR) sequence. In some cases, the template RNA comprises at least two LTR sequences flanking a nucleic acid sequence encoding the donor nucleic acid molecule. In some cases, the at least two LTR sequences is capable of self-circularizing. In some cases, the LTR sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 345-352.

[0014] In any one of the foregoing or related aspects, the lipid delivery particle further comprises an envelope protein attached to the lipid containing membrane.

[0015] In any one of the foregoing or related aspects, the envelope protein is a viral envelope protein. In some cases, the viral envelope protein is selected from the group consisting of: a VSV-G protein, a FuG- B2 envelope protein, a FuG-E envelope protein, an HIV-1 envelope, a baboon retroviral envelope protein, and an ecotropic murine leukemia virus (MLV) envelope protein, and functional mutants thereof. In some cases, the viral envelope protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 83-104.

[0016] In any one of the foregoing or related aspects, the envelope protein is a human endogenous retroviral envelope protein. In some cases, the human endogenous retroviral envelope protein is selected from the group consisting of hENVHl, hENVH2, hENVH3, hENVKl, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)l, and hENVKcon, and functional mutants thereof. In some cases, the human endogenous retroviral envelope protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 49-82.

[0017] In any one of the foregoing or related aspects, the lipid delivery particle comprises a plasma membrane recruitment element.

[0018] In any one of the foregoing or related aspects, the plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof. In some cases, the plasma membrane recruitment element is part of a structural protein that forms the protein core. In some cases, the structural protein further comprises a retroviral protease (pro) protein.

[0019] In any one of the foregoing or related aspects, the plasma membrane recruitment element is a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof. In some cases, the plasma membrane recruitment element is a pleckstrin homology (PH) domain of a protein selected from the group consisting of: phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, and MAPK associated protein 1 (MAPKAP1), and functional mutants thereof. The lipid delivery particle of embodiment 24. In some cases, the plasma membrane recruitment element is selected from the group consisting of: a pleckstrin homology (PH) domain of human phospholipase C81, a pleckstrin homology (PH) domain of human Aktl, a pleckstrin homology (PH) domain of human Arc, human endogenous retroviral gag protein, a pleckstrin homology (PH) domain of human 3- phosphoinositide-dependent protein kinase 1 (hPDPKl), a transmembrane domain of human CD9, a transmembrane domain of human CD47, a transmembrane domain of human CD63, a transmembrane domain ofhuman CD81, a transmembrane domain ofhuman Daapl, a transmembrane domain ofmouse Grpl, a transmembrane domain ofhuman Grpl, a transmembrane domain ofhuman OSBP, a transmembrane domain ofhuman Btkl, a transmembrane domain ofhuman FAPP1, a transmembrane domain ofhuman CERT, a transmembrane domain ofhuman PKD, a transmembrane domain ofhuman PHLPP1, a transmembrane domain ofhuman SWAP70, and a transmembrane domain ofhuman MAPKAP1, and functional mutants thereof. In some cases, the plasma membrane recruitment element comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-48.

[0020] In any one of the foregoing or related aspects, the first plasma membrane recruitment element or the second plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof, a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof. In some cases, the first plasma membrane recruitment element or the second plasma membrane recruitment element is a pleckstrin homology (PH) domain of a protein selected from the group consisting of: phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3 -phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin- Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, and MAPK associated protein 1 (MAPKAP1), and functional mutants thereof. In some cases, the first plasma membrane recruitment element or the second plasma membrane recruitment element is selected from the group consisting of: a pleckstrin homology (PH) domain ofhuman phospholipase C81, a pleckstrin homology (PH) domain of human Aktl, a pleckstrin homology (PH) domain ofhuman Arc, human endogenous retroviral gag protein, a pleckstrin homology (PH) domain ofhuman 3 -phosphoinositide-dependent protein kinase 1 (hPDPKl), a transmembrane domain ofhuman CD9, a transmembrane domain ofhuman CD47, a transmembrane domain ofhuman CD63, a transmembrane domain ofhuman CD81, a transmembrane domain ofhuman Daapl, a transmembrane domain of mouse Grpl, a transmembrane domain ofhuman Grpl, a transmembrane domain ofhuman OSBP, a transmembrane domain ofhuman Btkl, a transmembrane domain ofhuman FAPP1, a transmembrane domain of human CERT, a transmembrane domain ofhuman PKD, a transmembrane domain ofhuman PHLPP1, a transmembrane domain ofhuman SWAP70, and a transmembrane domain of human MAPKAP1, and functional mutants thereof. In some cases, the first plasma membrane recruitment element or the second plasma membrane recruitment element comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-48. [0021] In any one of the foregoing or related aspects, the lipid delivery particle further comprises a first chimeric protein that comprises a second prime editor and a second plasma membrane recruitment element. In some cases, the second prime editor has the same sequence as the prime editor.

[0022] In any one of the foregoing or related aspects, the lipid delivery particle further comprises a second chimeric protein that comprises a second recombinase and a third plasma membrane recruitment element. In some cases, the second recombinase has the same sequence as the recombinase. In some cases, the second plasma membrane recruitment element or the third plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof. In some cases, the first chimeric protein or the second chimeric protein forms part of the protein core. In some cases, the second plasma membrane recruitment element or the third plasma membrane recruitment element is a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof. In some cases, the second plasma membrane recruitment element or the third plasma membrane recruitment element is a pleckstrin homology (PH) domain of a protein selected from the group consisting of: phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, and MAPK associated protein 1 (MAPKAP1), and functional mutants thereof. In some cases, the second plasma membrane recruitment element or the third plasma membrane recruitment element is selected from the group consisting of: a pleckstrin homology (PH) domain ofhuman phospholipase C81, a pleckstrin homology (PH) domain ofhuman Aktl, a pleckstrin homology (PH) domain ofhuman Arc, human endogenous retroviral gag protein, a pleckstrin homology (PH) domain ofhuman 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), a transmembrane domain ofhuman CD9, a transmembrane domain ofhuman CD47, a transmembrane domain ofhuman CD63, a transmembrane domain ofhuman CD81, a transmembrane domain ofhuman Daapl, a transmembrane domain of mouse Grpl, a transmembrane domain ofhuman Grpl, a transmembrane domain ofhuman OSBP, a transmembrane domain ofhuman Btkl, a transmembrane domain ofhuman FAPP1, a transmembrane domain ofhuman CERT, a transmembrane domain ofhuman PKD, a transmembrane domain ofhuman PHLPP1, a transmembrane domain ofhuman SWAP70, and a transmembrane domain of human MAPKAP1, and functional mutants thereof. In some cases, the second plasma membrane recruitment element or the third plasma membrane recruitment element comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-48.

[0023] In any one of the foregoing or related aspects, the composition further comprises a fifth nucleic acid sequence encoding a structural protein comprising a third plasma membrane recruitment element. In some cases, the third plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof. In some cases, the structural protein further comprises a retroviral protease (pro) protein. [0024] In any one of the foregoing or related aspects, the nucleic acid-guided polypeptide is a Cas protein. In some cases, the Cas protein is a type I, type II, type III, type IV, type V, or type VI Cas protein. The lipid delivery particle of embodiment 40. In some cases, the Cas protein is selected from the group consisting of: c2cl, Casl3a, Casl3b, Casl3c, Casl3d, c2c3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, Cas 14, Cas 10, CaslOd, CasF, CasG, CasH, Cas 12a, 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, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CasX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs and modified versions thereof. In some cases, the nucleic acid-guided polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 318-338.

[0025] In any one of the foregoing or related aspects, the nucleic acid polymerase is a reverse transcriptase. In some cases, the reverse transcriptase comprises an RNase H domain. In some cases, the reverse transcriptase lacks an RNase H domain. In some cases, the reverse transcriptase is selected from the group consisting of: murine leukemia virus reverse transcriptase (M-MLV RT) (optionally D200N, T306K, W313F, T330P, and L603W), friend murine leukemia virus reverse transcriptase (FMLV RT), human endogenous retrovirus Kcon reverse transcriptase (HERV Kcon RT), a AMV-RT, a MarathonRT, a transcription xenopolymerase (RTX), and a small reverse transcriptase (Tfl), and functional mutants thereof. In some cases, the reverse transcriptase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 339-344.

[0026] In any one of the foregoing or related aspects, the recombinase is selected from the group consisting of: Hin, Gin, Tn3, -six, CinH, ParA, y8, Bxbl, 4>C31, TP901, TGI, cpBTl, R4, cpRVl, cpFCl, MR11, Al 18, U153, gp29, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2 recombinase. In some cases, the recombinase comprises one or more recombinases independently selected from the group consisting of: Cre, Bxbl, FLP, Al 18, Abrogate, Airmid, Anglerfish, B2, B3, Benedict, BL3, Bob3, Bred, BxZ2, Cin, Conceptll, CreALSHG, Cre-R3M3, Doom, Dre, Fre, Gin, Hin, Hinder, HK022, ICleared, IntlO, Inti 1, Intl2, Intl3, Int3, Int4, Int8, Int9, Inti, K38, Kd, KSSJEB, LI, L5, LI, Lockley, Mariner (Himarl), Mariner (mosl), Min, Minos, MH (phiFCl), MR11, Mundrea, Museum, Nigri, P22, Panto, PattyP, Peaches, phi370.1, phiBTl, phiC31, phiJoe, phiK38, phiRVl, R Rl, R2, R3, R4, R5, RDF, Rebeuca, retrotransposases encoded by R2, Sarfire, Scowl, Sere, Severus, Sheen, Sin, SkiPole, SPBc, SprA, SV1, Switzer, Tc3, TD1-40, TGI, Theia, Tol2Tcl, TP901-1, Tre, Troube, U153, Vcre, Veracruz, Vika, WB, W , <p370.1 , cpBTl, cpCl, cpC31, cpFCl, and <pRV. In some cases, the recombinase recognizes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538.

[0027] In any one of the foregoing or related aspects, the lipid delivery particle is a retroviral particle or a lentiviral particle.

[0028] In any one of the foregoing or related aspects, the donor nucleic acid sequence encodes a therapeutic molecule. In some cases, the therapeutic molecule comprises at least a functional portion of a viral envelope protein, a hormone, a cytokine, a ligand, a receptor, an antibody, an enzyme, a transcription factor, a chimeric antigen receptor, a T cell receptor, an antigen, a secreted protein, or any combination thereof.

[0029] In any one of the foregoing or related aspects, the guide nucleic acid molecule comprises one or more guide RNA. In some cases, each of the one or more guide RNA comprises (A) a primer binding site, (B) a clamp segment, (C) a sequence encoding at least a portion of a first recombinase recognition sequence, (D) an aptamer, (E) spacer, or (F) scaffold, or any combinations thereof. In some cases, the guide nucleic acid molecule comprises a first guide RNA encoding at least a first portion of a first recombinase recognition sequence and a second guide RNA encoding at least a second portion of the first recombinase recognition sequence. In some cases, the first guide RNA and the second guide RNA work in a pair and collectively encode the first recombinase recognition sequence, optionally wherein the first and the second portion of the first recombinase recognition sequence have at least 6bp overlap. In some cases, the guide nucleic acid molecule comprises (A) a nicking guide RNA and (B) a guide RNA encoding a first recombinase recognition sequence. In some cases, the first recombinase recognition sequence or the second recombinase recognition sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538, and wherein the first recombinase recognition sequence and the second recombinase recognition sequence are a pair of recombinase recognition sequences for a cognate recombinase.

[0030] In any one of the foregoing or related aspects, the lipid delivery particle further comprises an additional recombinase or a fourth nucleic acid sequence encoding the additional recombinase.

[0031] In any one of the foregoing or related aspects, the system further comprises an additional recombinase or a nucleic acid encoding the additional recombinase.

[0032] In any one of the foregoing or related aspects, the composition further comprises a fourth nucleic acid sequence encoding an additional recombinase.

[0033] In any one of the foregoing or related aspects, the additional recombinase recognizes an additional pair of recombinase recognition sequences. In some cases, the additional recombinase and the recombinase are different. In some cases, the donor nucleic acid molecule further comprises the additional pair of recombinase recognition sequences. In some cases, the additional pair of recombinase recognition sequences comprises a third recombinase recognition sequence located at a 3’ end of the donor nucleic acid molecule and a fourth recombinase recognition sequence located at a 5’ end of the donor nucleic acid molecule. In some cases, the additional pair of recombinase recognition is capable of self-circularizing when contacted with the additional recombinase, and wherein the additional pair of recombinase recognition sequences has a faster integration rate than the first recombinase recognition sequence and the second recombinase recognition sequence, thereby the additional pair of recombinase recognition sequences recombines prior to recombination of the first recombinase recognition sequence and the second recombinase recognition sequence in the presence of the recombinase and the additional recombinase.

[0034] In any one of the foregoing or related aspects, the lipid delivery particle comprises a nucleic acid molecule that comprises the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, the fourth nucleic acid sequence, and the donor nucleic acid sequence.

[0035] In any one of the foregoing or related aspects, the composition comprises a nucleic acid molecule that comprises the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, the fourth nucleic acid sequence, and the donor nucleic acid sequence.

[0036] In any one of the foregoing or related aspects, the lipid delivery particle further comprises a MLHldn protein. In some cases, the MLHldn protein is a part of a third chimeric protein comprising a fourth plasma membrane recruitment element. In some cases, the fourth plasma membrane recruitment element is the same as the plasma membrane recruitment element, the second plasma membrane recruitment element or the third plasma membrane recruitment element.

[0037] In any one of the foregoing or related aspects, the system further comprising comprises a MLHldn protein or a nucleic acid sequence encoding the MLHldn protein. In some cases, the MLHldn protein is a part of a third chimeric protein comprising a fourth plasma membrane recruitment element. In some cases, the fourth plasma membrane recruitment element is the same as the plasma membrane recruitment element, the second plasma membrane recruitment element or the third plasma membrane recruitment element.

[0038] In any one of the foregoing or related aspects, the composition comprises a sixth nucleic acid sequence encoding a MLHldn protein. In some cases, the sixth nucleic acid sequence encodes a third chimeric protein comprising a fourth plasma membrane recruitment element and the MLHldn protein. In some cases, the fourth plasma membrane recruitment element is the same as the first plasma membrane recruitment element, the second plasma membrane recruitment element, or the third plasma membrane recruitment element.

[0039] Disclosed herein, in some aspects, is a pharmaceutical composition comprising (a) a lipid delivery particle described herein or a system of described herein; and (b) a pharmaceutically acceptable excipient.

[0040] Disclosed herein, in some aspects, is a kit comprising (a) a lipid delivery particle described herein, a system described herein, or a pharmaceutical composition described herein; and (b) an information material containing instructions for administering a dosage of the lipid delivery particle or the system, or a dosage form of the pharmaceutical composition to a subject.

[0041] Disclosed herein, in some aspects, is a method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject a lipid delivery particle described herein, a system described herein, or a pharmaceutical composition described herein.

[0042] Disclosed herein, in some aspects, is a lipid delivery particle described herein, a system described herein, or a pharmaceutical composition described herein for use as a medicament.

[0043] Disclosed herein, in some aspects, is use of a lipid delivery particle described herein, a system described herein, or a pharmaceutical composition described herein for the treatment of a disease or a condition described herein.

[0044] Disclosed herein, in some aspects, is use of a lipid delivery particle described herein, a system described herein, or a composition described herein for the manufacture of a medicament for the treatment of a disease or a condition described herein.

[0045] Disclosed herein, in some aspects, is a method comprising contacting a cell with a lipid delivery particle described herein. In some cases, the method comprising generating a template DNA in the cell using at least a portion of the template RNA as a template, wherein the template DNA encodes a therapeutic molecule, and optionally circularizing the template DNA in the cell; and expressing the therapeutic molecule from the template DNA in the cell.

[0046] Disclosed herein, in some aspects, is a method comprising contacting a cell with a system described herein.

[0047] Disclosed herein, in some aspects, is a method comprising administering a lipid delivery particle described herein to a subject in need thereof.

[0048] Disclosed herein, in some aspects, is a method of producing a lipid delivery particle described herein. In some cases, the method comprising contacting a producer cell described herein with a composition described herein.

[0049] Disclosed herein, in some aspects, is a lipid containing particle comprising: (a) a lipid containing membrane encapsulating a protein core; (b) a recombinase; and (c) a ribonucleoprotein complex that comprises (i) a prime editor comprising a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; and (ii) a guide nucleic acid molecule, wherein the recombinase and the ribonucleoprotein complex are within an inside cavity of the protein core. In some embodiments, the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule. In some embodiments, the recombinase mediates recombination between the first recombinase recognition sequence and a second recombinase recognition sequence. In some embodiments, the lipid containing particle further comprises either (1) a donor nucleic acid molecule that comprises the second recombinase recognition sequence; or (2) a template RNA that encodes the donor nucleic acid molecule. In some embodiments, the donor nucleic acid molecule or the template RNA is within the inside cavity of the protein core. [0050] Disclosed herein, in some aspects, is a lipid containing particle, comprising: (a) a recombinase or a nucleic acid sequence encoding the recombinase; (b) (i) a ribonucleoprotein complex comprising: (1) a prime editor comprising a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; and (2) a guide nucleic acid molecule, or (ii) (1) a nucleic acid sequence encoding the prime editor; and (2) the guide nucleic acid molecule or a nucleic acid sequence encoding the guide nucleic acid molecule; and (c) a template RNA that encodes a donor nucleic acid molecule, wherein the donor nucleic acid molecule comprises a donor nucleic acid sequence and a second recombinase recognition sequence, and wherein the recombinase mediates recombination between a first recombinase recognition sequence and the second recombinase recognition sequence. In some embodiments, the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule. In some embodiments, the lipid containing particle comprises a lipid containing membrane encapsulating a protein core. In some embodiments, the lipid containing membrane comprises a phospholipid bilayer. In some embodiments, the template RNA comprises a long terminal repeat (LTR) sequence. In some embodiments, the template RNA comprises at least two LTR sequences flanking a nucleic acid sequence encoding the donor nucleic acid molecule. In some embodiments, the LTR sequence has at least 80% identity to any one of the sequences listed in Table 6-A. In some embodiments, the lipid containing particle further comprises a membrane-fusion protein attached to the lipid containing membrane. In some embodiments, the membrane-fusion protein is a viral envelope protein. In some embodiments, the viral envelope protein is derived from VSV-G protein. In some embodiments, the viral envelope protein comprises an amino acid sequence having at least 80% identity to any one of the sequences in Table 1-C. In some embodiments, the membrane-fusion protein is a human endogenous retroviral envelope protein. In some embodiments, the human endogenous retroviral envelope protein is derived from hENVHl, hENVH2, hENVH3, hENVKl, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)l, hENVK_con. In some embodiments, the human endogenous retroviral envelope protein comprises an amino acid sequence having at least 80% identity to any one of the sequences in Table 2-B. In some embodiments, the membrane -fusion protein is a non-immunogenic membrane -fusion protein. In some embodiments, the protein core comprises a structural protein comprising a plasma membrane localization domain. In some embodiments, the plasma membrane localization domain is derived from a retroviral gag protein. In some embodiments, the structural protein further comprises a retroviral protease (pro) protein. In some embodiments, the plasma membrane localization domain is derived from a human endogenous retroviral structural protein. In some embodiments, the plasma membrane localization domain is derived from a humanized viral structural protein. In some embodiments, the plasma membrane localization domain is derived from a mammalian protein. In some embodiments, the plasma membrane localization domain is a pleckstrin homology (PH) domain. In some embodiments, the plasma membrane localization domain is a pleckstrin homology (PH) domain derived from phospholipase C81 (PLC31), Aktl, Arc, endogenous retroviral gag protein, 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxy sterol -binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate- adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, or MAPK associated protein 1 (MAPKAP1), or a mutant thereof. In some embodiments, the plasma membrane localization domain is a pleckstrin homology (PH) domain derived from human phospholipase C31, human Aktl, human Arc, human endogenous retroviral gag protein, human 3-phosphoinositide- dependent protein kinase 1 (hPDPKl), human CD9, human CD47, human CD63, human CD81, human Daapl, mouse Grpl, human Grpl, human OSBP, human Btkl, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, or human MAPKAP1, or a mutant thereof. In some embodiments, the plasma membrane localization domain comprises an amino acid sequence having at least 80% identity to any of the sequences listed in Table 3. In some embodiments, the lipid containing particle further comprises a first combinatorial protein that comprises a second prime editor and a second plasma membrane localization domain. In some embodiments, the second prime editor has the same sequence as the prime editor. In some embodiments, the lipid containing particle further comprises a second combinatorial protein that comprises a second recombinase and a third plasma membrane localization domain. In some embodiments, the second recombinase has the same sequence as the recombinase. In some embodiments, the first combinatorial protein or the second combinatorial protein forms part of the protein core. In some embodiments, the second plasma membrane localization domain or the third plasma membrane localization domain is derived from a human endogenous retroviral structural protein. In some embodiments, the second plasma membrane localization domain or the third plasma membrane localization domain is derived from a humanized viral structural protein. In some embodiments, the second plasma membrane localization domain or the third plasma membrane localization domain is derived from a mammalian protein. In some embodiments, the second plasma membrane localization domain or the third plasma membrane localization domain is a pleckstrin homology (PH) domain. In some embodiments, the second plasma membrane localization domain or the third plasma membrane localization domain is a pleckstrin homology (PH) domain derived from phospholipase C81 (PLC31), Aktl, Arc, endogenous retroviral gag protein, 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, or MAPK associated protein 1 (MAPKAP1), or a mutant thereof. In some embodiments, the second plasma membrane localization domain or the third plasma membrane localization domain is a pleckstrin homology (PH) domain derived from human phospholipase C81, human Aktl, human Arc, human endogenous retroviral gag protein, human 3-phosphoinositide- dependent protein kinase 1 (hPDPKl), human CD9, human CD47, human CD63, human CD81, human Daapl, mouse Grpl, human Grpl, human OSBP, human Btkl, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, or human MAPKAP1, or a mutant thereof. In some embodiments, the second plasma membrane localization domain or the third plasma membrane localization domain comprises an amino acid sequence having at least 80% identity to any of the sequences listed in Table 3. In some embodiments, the nucleic acid-guided polypeptide is derived from a Cas protein. In some embodiments, the Cas protein is a type I, type II, type III, type IV, type V, or type VI Cas protein. In some embodiments, the Cas protein is selected from the group consisting of: c2cl, Casl3a, Casl3b, Casl3c, Casl3d, c2c3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, Casl4, CaslO, CaslOd, CasF, CasG, CasH, Casl2a, 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, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CasX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs and modified versions thereof. In some embodiments, the nucleic acid- guided polypeptide comprises an amino acid sequence having at least 80% identity to any one of the sequences listed in Table 4-A. In some embodiments, the nucleic acid polymerase is a reverse transcriptase. In some embodiments, the reverse transcriptase comprises an RNase H domain. In some embodiments, the reverse transcriptase lacks an RNase H domain. In some embodiments, the reverse transcriptase is derived from murine leukemia virus reverse transcriptase (M-MLV RT), friend murine leukemia virus reverse transcriptase (FMLV RT), or human endogenous retrovirus Kcon reverse transcriptase (Kcon RT). In some embodiments, the reverse transcriptase comprises an amino acid sequence having at least 80% identity to any one of the sequences listed in Table 4-B. In some embodiments, the recombinase is selected from the group consisting of: Hin, Gin, Tn3, -six, CinH, ParA, y8, Bxbl, C31, TP901, TGI, cpBTl, R4, cpRVl, cpFCl, MR11, A118, U153, gp29, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2 recombinase. In some embodiments, the recombinase comprises an amino acid sequence having at least 80% identity to any one of the sequences listed in Table 5A-5D. In some embodiments, the lipid containing particle is a retroviral particle. In some embodiments, the lipid containing particle is a lentiviral particle. In some embodiments, the donor nucleic acid sequence encodes a therapeutic protein. In some embodiments, the therapeutic protein comprises at least a functional portion of a viral envelope protein, a hormone, a cytokine, a ligand, a receptor, an antibody, an enzyme, a transcription factor, a chimeric antigen receptor, a T cell receptor, an antigen, a secreted protein, or any combination thereof.

[0051] Disclosed herein, in some aspects, is a composition comprising: a first nucleic acid sequence encoding a first combinatorial protein comprising a first plasma membrane localization domain coupled to a prime editor, wherein the prime editor comprises a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; a guide nucleic acid molecule or a second nucleic acid sequence encoding the guide nucleic acid molecule; and a third nucleic acid sequence encoding a second combinatorial protein comprising a second plasma membrane localization domain coupled to a recombinase. In some embodiments, the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule. In some embodiments, the recombinase mediates recombination between the first recombinase recognition sequence and a second recombinase recognition sequence. In some embodiments, the lipid containing particle further comprises either (1) a donor nucleic acid molecule that comprises the second recombinase recognition sequence; or (2) a template RNA that encodes the donor nucleic acid molecule.

[0052] Disclosed herein, in some aspects, is a composition comprising: a first nucleic acid sequence encoding a prime editor, wherein the prime editor comprises a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; a guide nucleic acid molecule or a second nucleic acid sequence encoding the guide nucleic acid molecule; a third nucleic acid sequence encoding a recombinase; and a template RNA, wherein the template RNA encodes a donor nucleic acid molecule comprising a second recombinase recognition sequence, and wherein the recombinase mediates recombination between a first recombinase recognition sequence and the second recombinase recognition sequence. In some embodiments, the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule. In some embodiments, the first nucleic acid sequence encodes a first combinatorial protein comprising a first plasma membrane localization domain coupled to the prime editor. In some embodiments, the third nucleic acid sequence encodes a second combinatorial protein comprising a second plasma membrane localization domain coupled to the recombinase. In some embodiments, the template RNA comprises a long terminal repeat (LTR) sequence. In some embodiments, the template RNA comprises at least two LTR sequences flanking a nucleic acid sequence encoding the donor nucleic acid molecule. In some embodiments, the LTR sequence has at least 80% identity to any one of the sequences listed in Table 6-A. In some embodiments, the composition further comprises a fourth nucleic acid sequence encoding a membrane-fusion protein. In some embodiments, the membrane -fusion protein is a viral envelope protein. In some embodiments, the viral envelope protein is derived from VSV-G protein. In some embodiments, the viral envelope protein comprises an amino acid sequence having at least 80% identity to any one of the sequences in Table 1-C. In some embodiments, the membrane-fusion protein is a human endogenous retroviral envelope protein. In some embodiments, the human endogenous retroviral envelope protein is derived from hENVHl, hENVH2, hENVH3, hENVKl, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)l, hENVK_COn. In some embodiments, the human endogenous retroviral envelope protein comprises an amino acid sequence having at least 80% identity to any one of the sequences in Table 2-B. In some embodiments, the membrane -fusion protein is a non-immunogenic membrane -fusion protein. In some embodiments, the composition further comprises a fifth nucleic acid sequence encoding a structural protein comprising a third plasma membrane localization domain. In some embodiments, the third plasma membrane localization domain is derived from a retroviral gag protein. In some embodiments, the structural protein further comprises a retroviral protease (pro) protein. In some embodiments, the third plasma membrane localization domain is derived from a human endogenous retroviral structural protein. In some embodiments, the third plasma membrane localization domain is derived from a humanized viral structural protein. In some embodiments, the third plasma membrane localization domain is derived from a mammalian protein. In some embodiments, the third plasma membrane localization domain is a pleckstrin homology (PH) domain. In some embodiments, the third plasma membrane localization domain is a pleckstrin homology (PH) domain derived from phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3 -phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, or MAPK associated protein 1 (MAPKAP1), or a mutant thereof. In some embodiments, the third plasma membrane localization domain is a pleckstrin homology (PH) domain derived from human phospholipase C81, human Aktl, human Arc, human endogenous retroviral gag protein, human 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), human CD9, human CD47, human CD63, human CD81, human Daapl, mouse Grpl, human Grpl, human OSBP, human Btkl, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, or human MAPKAP1, or a mutant thereof. In some embodiments, the third plasma membrane localization domain comprises an amino acid sequence having at least 80% identity to any of the sequences listed in Table 3. In some embodiments, the first plasma membrane localization domain or the second plasma membrane localization domain is derived from a human endogenous retroviral structural protein. In some embodiments, the first plasma membrane localization domain or the second plasma membrane localization domain is derived from a humanized viral structural protein. In some embodiments, the first plasma membrane localization domain or the second plasma membrane localization domain is derived from a mammalian protein. In some embodiments, the first plasma membrane localization domain or the second plasma membrane localization domain is a pleckstrin homology (PH) domain. In some embodiments, the first plasma membrane localization domain or the second plasma membrane localization domain is a pleckstrin homology (PH) domain derived from phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, or MAPK associated protein 1 (MAPKAP1), or a mutant thereof. In some embodiments, the first plasma membrane localization domain or the second plasma membrane localization domain is a pleckstrin homology (PH) domain derived from human phospholipase C81, human Aktl, human Arc, human endogenous retroviral gag protein, human 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), human CD9, human CD47, human CD63, human CD81, human Daapl, mouse Grpl, human Grpl, human OSBP, human Btkl, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, or human MAPKAP1, or a mutant thereof. In some embodiments, the first plasma membrane localization domain or the second plasma membrane localization domain comprises an amino acid sequence having at least 80% identity to any of the sequences listed in Table 3. In some embodiments, the nucleic acid-guided polypeptide is derived from a Cas protein. In some embodiments, the Cas protein is a type I, type II, type III, type IV, type V, or type VI Cas protein. In some embodiments, the Cas protein is selected from the group consisting of: c2cl, Cas 13a, Cas 13b, Casl3c, Casl3d, c2c3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, Casl4, CaslO, CaslOd, CasF, CasG, CasH, Casl2a, 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, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CasX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs and modified versions thereof. In some embodiments, the nucleic acid-guided polypeptide comprises an amino acid sequence having at least 80% identity to any one of the sequences listed in Table 4-A. In some embodiments, the nucleic acid polymerase is a reverse transcriptase. In some embodiments, the reverse transcriptase comprises an RNase H domain. In some embodiments, the reverse transcriptase lacks an RNase H domain. In some embodiments, the reverse transcriptase is derived from murine leukemia virus reverse transcriptase (M-MLV RT), friend murine leukemia virus reverse transcriptase (FMLV RT), or HERV Kcon RT. In some embodiments, the reverse transcriptase comprises an amino acid sequence having at least 80% identity to any one of the sequences listed in Table 4-B. In some embodiments, the recombinase is selected from the group consisting of: Hin, Gin, Tn3, -six, CinH, ParA, y8, Bxbl, C31, TP901, TGI, cpBTl, R4, cpRVl, cpFCl, MR11, A118, U153, gp29, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2 recombinase. In some embodiments, the recombinase comprises an amino acid sequence having at least 80% identity to any one of the sequences listed in Table 5A-5D. In some embodiments, the donor nucleic acid sequence encodes a therapeutic protein. In some embodiments, the therapeutic protein comprises at least a functional portion of a viral envelope protein, a hormone, a cytokine, a ligand, a receptor, an antibody, an enzyme, a transcription factor, a chimeric antigen receptor, a T cell receptor, an antigen, a secreted protein, or any combination thereof.

[0053] Disclosed herein, in some aspects, is a system, comprising: (1) a lipid containing particle comprising (a) a lipid containing membrane encapsulating a protein core; and (b) a ribonucleoprotein complex comprising: (A) a prime editor comprising a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; and (B) a guide nucleic acid molecule, wherein the ribonucleoprotein complex is within an inside cavity of the protein core; and (2) a recombinase or a nucleic acid sequence encoding the recombinase.

[0054] Disclosed herein, in some aspects, is a system, comprising: (1) a lipid containing particle comprising (a) a lipid containing membrane encapsulating a protein core; and (b) a recombinase, wherein the recombinase is within an inside cavity of the protein core; and (2) (i) a ribonucleoprotein complex comprising: (A) a prime editor comprising a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; and (B) a guide nucleic acid molecule, or (ii) (A) a nucleic acid sequence encoding the prime editor; and (B) the guide nucleic acid molecule or a nucleic acid sequence encoding the guide nucleic acid molecule.

[0055] In some embodiments, the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule; and wherein the recombinase mediates recombination between the first recombinase recognition sequence and a second recombinase recognition sequence. In some embodiments, the system further comprises either (3) a donor nucleic acid molecule comprising a donor nucleic acid sequence and the second recombinase recognition sequence, or (4) a template RNA that encodes the donor nucleic acid molecule. In some embodiments, the lipid containing membrane comprises a phospholipid bilayer. In some embodiments, the template RNA comprises a long terminal repeat (LTR) sequence. In some embodiments, the template RNA comprises at least two LTR sequences flanking a nucleic acid sequence encoding the donor nucleic acid molecule. In some embodiments, the LTR sequence has at least 80% identity to any one of the sequences listed in Table 6-A. In some embodiments, the lipid containing particle further comprises a membrane-fusion protein attached to the lipid containing membrane. In some embodiments, the membrane -fusion protein is a viral envelope protein. In some embodiments, the viral envelope protein is derived from VSV-G protein. In some embodiments, the viral envelope protein comprises an amino acid sequence having at least 80% identity to any one of the sequences in Table 1-C. In some embodiments, the membrane -fusion protein is a human endogenous retroviral envelope protein. In some embodiments, the human endogenous retroviral envelope protein is derived from hENVHl, hENVH2, hENVH3, hENVKl, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)l, hENVK_COn. In some embodiments, the human endogenous retroviral envelope protein comprises an amino acid sequence having at least 80% identity to any one of the sequences in Table 2-B. In some embodiments, the membrane-fusion protein is a non-immunogenic membrane -fusion protein. In some embodiments, the protein core comprises a structural protein comprising a plasma membrane localization domain. In some embodiments, the plasma membrane localization domain is derived from a retroviral gag protein. In some embodiments, the structural protein further comprises a retroviral protease (pro) protein. In some embodiments, the plasma membrane localization domain is derived from a human endogenous retroviral structural protein. In some embodiments, the plasma membrane localization domain is derived from a humanized viral structural protein. In some embodiments, the plasma membrane localization domain is derived from a mammalian protein. In some embodiments, the plasma membrane localization domain is a pleckstrin homology (PH) domain. In some embodiments, the plasma membrane localization domain is a pleckstrin homology (PH) domain derived from phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3-phosphoinositide- dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, or MAPK associated protein 1 (MAPKAP1), or a mutant thereof. In some embodiments, the plasma membrane localization domain is a pleckstrin homology (PH) domain derived from human phospholipase C81, human Aktl, human Arc, human endogenous retroviral gag protein, human 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), human CD9, human CD47, human CD63, human CD81, human Daapl, mouse Grpl, human Grpl, human OSBP, human Btkl, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, or human MAPKAP1, or a mutant thereof. In some embodiments, the plasma membrane localization domain comprises an amino acid sequence having at least 80% identity to any of the sequences listed in Table 3. In some embodiments, the lipid containing particle further comprises a first combinatorial protein that comprises a second prime editor and a second plasma membrane localization domain. In some embodiments, the second prime editor has the same sequence as the prime editor. In some embodiments, the lipid containing particle further comprises a second combinatorial protein that comprises a second recombinase and a third plasma membrane localization domain. In some embodiments, the second recombinase has the same sequence as the recombinase. In some embodiments, the first combinatorial protein or the second combinatorial protein forms part of the protein core. In some embodiments, the second plasma membrane localization domain or the third plasma membrane localization domain is derived from a human endogenous retroviral structural protein. In some embodiments, the second plasma membrane localization domain or the third plasma membrane localization domain is derived from a humanized viral structural protein. In some embodiments, the second plasma membrane localization domain or the third plasma membrane localization domain is derived from a mammalian protein. In some embodiments, the second plasma membrane localization domain or the third plasma membrane localization domain is a pleckstrin homology (PH) domain. In some embodiments, the second plasma membrane localization domain or the third plasma membrane localization domain is a pleckstrin homology (PH) domain derived from phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, or MAPK associated protein 1 (MAPKAP1), or a mutant thereof. In some embodiments, the second plasma membrane localization domain or the third plasma membrane localization domain is a pleckstrin homology (PH) domain derived from human phospholipase C81, human Aktl, human Arc, human endogenous retroviral gag protein, human 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), human CD9, human CD47, human CD63, human CD81, human Daapl, mouse Grpl, human Grpl, human OSBP, human Btkl, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, or human MAPKAP1, or a mutant thereof. In some embodiments, the second plasma membrane localization domain or the third plasma membrane localization domain comprises an amino acid sequence having at least 80% identity to any of the sequences listed in Table 3. In some embodiments, the nucleic acid-guided polypeptide is derived from a Cas protein. In some embodiments, the Cas protein is a type I, type II, type III, type IV, type V, or type VI Cas protein. In some embodiments, the Cas protein is selected from the group consisting of: c2cl, Cas 13a, Cas 13b, Casl3c, Casl3d, c2c3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, Casl4, CaslO, CaslOd, CasF, CasG, CasH, Casl2a, 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, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CasX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs and modified versions thereof. In some embodiments, the nucleic acid-guided polypeptide comprises an amino acid sequence having at least 80% identity to any one of the sequences listed in Table 4-A. In some embodiments, the nucleic acid polymerase is a reverse transcriptase. In some embodiments, the reverse transcriptase comprises an RNase H domain. In some embodiments, the reverse transcriptase lacks an RNase H domain. In some embodiments, the reverse transcriptase is derived from murine leukemia virus reverse transcriptase (M-MLV RT), friend murine leukemia virus reverse transcriptase (FMLV RT), or HERV Kcon RT. In some embodiments, the reverse transcriptase comprises an amino acid sequence having at least 80% identity to any one of the sequences listed in Table 4-B. In some embodiments, the recombinase is selected from the group consisting of: Hin, Gin, Tn3, 0-six, CinH, ParA, y8, Bxbl, C31, TP901, TGI, cpBTl, R4, cpRVl, cpFCl, MR11, A118, U153, gp29, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2 recombinase. In some embodiments, the recombinase comprises an amino acid sequence having at least 80% identity to any one of the sequences listed in Table 5A-5D. In some embodiments, the lipid containing particle is a retroviral particle. In some embodiments, the lipid containing particle is a lentiviral particle. In some embodiments, the donor nucleic acid sequence encodes a therapeutic protein. In some embodiments, the therapeutic protein comprises at least a functional portion of a viral envelope protein, a hormone, a cytokine, a ligand, a receptor, an antibody, an enzyme, a transcription factor, a chimeric antigen receptor, a T cell receptor, an antigen, a secreted protein, or any combination thereof.

[0056] Disclosed herein, in some aspects, is a method comprising contacting a cell with the lipid containing particle described herein.

[0057] Disclosed herein, in some aspects, is a method comprising contacting a cell with the system described herein.

[0058] Disclosed herein, in some aspects, is a method comprising administering the lipid containing particle described herein to a subject in need thereof.

[0059] Disclosed herein, in some aspects, is a method of producing the lipid containing particle described herein.

[0060] Disclosed herein, in some aspects, is a method of producing a lipid containing particle, comprising contacting a producer cell with the composition described herein.

INCORPORATION BY REFERENCE

[0061] 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 [0062] 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:

[0063] FIG. 1 shows a schematic of the architecture of prime edit recombinase mediated cassette exchange virus-like particles (PERMCEVLP) according to some embodiments of the present disclosure. As shown in the figure, inside a lipid delivery particle, a prime editor ribonucleoprotein complex, which includes prime editor and prime editor guide RNA (PEgRNA). The primer editor is fused to the C- terminus of a gag polyprotein via a linker that can be cleaved by a protease upon particle maturation. Additionally, the lipid delivery particle can have a recombinase that is fused to the C-terminus of a gag polyprotein via a linker that can be cleaved by a protease upon particle maturation. An LTR-flanked recombination template RNA (e.g., up to 10 kb) can be reverse transcribed into an LTR-flanked recombination template DNA which can serve as a donor sequence for the integration mediated by the prime editor ribonucleoprotein complex and the recombinase.

[0064] FIG. 2 depicts the two-step targeted integration of a template nucleic acid molecule (e.g. , up to 10 kb) mediated by PERMCEVLP according to some embodiments of the present disclosure. The first step involves insertion of a recombinase recognition sequence at a specific target site in a DNA sequence via prime-editing technology. The second step involves using the recombinase recognition sequence inserted from the first step as a landing pad to introduce an LTR-flanked target sequence through a recombinase mediated cassette exchange.

DETAILED DESCRIPTION

[0065] The practice of some methods disclosed herein employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

[0066] One way of creating a targeted integration is to use recombinases (such as tyrosine or serine recombinases and integrases) and a template nucleic acid molecule (e.g., DNA) to perform recombinase mediated cassette exchange (RMCE). Without wishing to be bound by certain theories, prime editing and RMCE can be relatively scarless modes of nucleic acid editing compared to HDR because the indel frequencies of prime editing and RMCE can be relatively low. Prime editing can be used to insert a 20-40 base pair recombinase recognition sequence. The recombinase recognition sequence can serve as a landing pad for subsequent RMCE.

[0067] One challenge of targeted integration editing strategy is the simultaneous delivery of multiple components that are used to install sequential edits. For example, in order to edit genome of a cell, prime editor, one or two prime editing guide RNAs (PEgRNAs), optional nicking guide RNA, template nucleic acid molecule, and recombinase all can be delivered to the nucleus of the cell in order for sequential edits to take place. This can be a challenge for ex vivo delivery, let alone in vivo delivery, and can be a hurdle in the development of synthetic biological therapeutic interventions that can stably install customizable DNA sequence(s) in a targeted fashion.

[0068] In some aspects, the present disclosure relates to compositions, methods, kits, and systems that can facilitate targeted integration of large DNA fragments into the genome of cells (e.g., mammalian cells), or targeted manipulation (e.g, deletion, reversion, translocation, or other cassette exchange) of the genome of cells. In some aspects, provided herein are unique lipid delivery particles that are capable of in vivo delivery of payloads (used herein interchangeably with “cargoes” or “freights”), which are unique for editing large DNA fragment(s) in the genome of cells.

[0069] In some cases, these payloads comprise two gene editors that will install at least two edits, one after the other. In some cases, a first edit comprises a targeted insertion that can serve as a target site or a landing pad for a second edit and/or one or more further edits. In some cases, the second edit is also a targeted insertion. In some cases, the first edit comprises prime editing. In some cases, the prime editing inserts a recombinase recognition sequence into a targeted site of a DNA sequence within a cell. In some cases, the second edit comprises a RMCE. The RMCE can comprise recombinase recognition sequences and cognate recombinases/bacterial integrases, for example, AttP/B sites and Bxbl recombinase, and loxP sites and CRE recombinase. In some cases, these payloads comprise an exogenous nucleic acid molecule or a protein encoded by the exogenous nucleic acid molecule. The RMCE can also comprise the exogenous nucleic acid molecule, for example, a template nucleic acid molecule. In some cases, the template nucleic acid molecule comprises a recombinase recognition sequence that can be recognized by the same recombinase as the recombinase recognition sequence inserted in the target DNA sequence in the cell (e.g., AttP/B sites or loxP sites), and a donor sequence to be inserted into the target site of a DNA sequence. In some cases, the donor sequence encodes a therapeutic molecule.

[0070] In some cases, the payloads (e.g., any components of prime editor and RMCE) are packaged into a delivery vehicle, e.g., a lipid-containing particle disclosed herein, including lentivirus (LV), retrovirus (RV), adeno-associated virus (AAV), virus-like particle (VLP), anellovirus (ANV), adenovirus (AV), a viral-like particle (VLP), or combination thereof. In some cases, the payloads (e.g., one or more components of prime editor and RMCE) are packaged into a lipid delivery particle, including non-viral human endogenous viral-like particles, pleckstrin homology ectosome-like particle, or completely humanized ectosome-like particle. The system or method provided herein can involve simultaneous delivery of payloads in an all-in-one delivery vehicle in order to install desired targeted insertions. Alternatively, payloads of the systems or methods provided herein can be sequentially delivered in multiple delivery vehicles in order to install desired targeted insertions. The targeted integration system using prime editor and recombinase described in the present disclosure include publicly disclosed methods such as those described in Yarncill et a/., Nat Biotechnol. 2023 April ; 41(4): 500-512 and U.S. Patent Nos. 11,572,556, 11,827,881, and 11,834,658, each of which is incorporated herein by reference in its entirety. DEFINITIONS

[0071] As used in the present disclosure, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a chimeric protein” includes a plurality of chimeric proteins.

[0072] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2-fold, of a value. Where particular values are described in the application, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0073] As used herein, a “cell” can generally refer to a biological cell. A cell can be the basic structural, functional and/or biological unit of a living organism. A cell can originate from any organism having one or more cells. Some examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, com, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, fems, clubmosses, homworts, liverworts, mosses), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens C. Agardh, and the like), seaweeds (e.g. , kelp), a fungal cell (e.g. , a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g, fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g. , fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g. , a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.), and etcetera. Sometimes a cell is not originating from a natural organism (e.g., a cell can be a synthetically made, sometimes termed an artificial cell).

[0074] The term “antibody,” as used herein, refers to a proteinaceous binding molecule with immunoglobulin-like functions. The term antibody includes antibodies (e.g., monoclonal and polyclonal antibodies), as well as derivatives, variants, and fragments thereof. Antibodies include immunoglobulins (Ig’s) of different classes (i.e., IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgGl, IgG2, etc.). A derivative, variant or fragment thereof can refer to a functional derivative or fragment which retains the binding specificity (e.g., complete and/or partial) of the corresponding antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, variable fragment (Fv), single chain variable fragment (scFv), minibodies, diabodies, and single-domain antibodies (“sdAb” or “nanobodies” or “camelids”). The term antibody includes antibodies and antigen-binding fragments of antibodies that have been optimized, engineered or chemically conjugated. Examples of antibodies that have been optimized include affinity- matured antibodies. Examples of antibodies that have been engineered include Fc optimized antibodies (e.g., antibodies optimized in the fragment crystallizable region) and multispecific antibodies (e.g., bispecific antibodies).

[0075] The term “nucleotide,” as used herein, generally refers to a base-sugar-phosphate combination. A nucleotide can comprise a synthetic nucleotide. A nucleotide can comprise a synthetic nucleotide analog. Nucleotides can be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP) and deoxyribonucleoside triphosphates such as dATP, dCTP, diTP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives can include, for example, [aS]dATP, 7-deaza-dGTP and 7-deaza- dATP, and nucleotide derivatives that confer nuclease resistance on the nucleic acid molecule containing them. The term nucleotide as used herein can refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Illustrative examples of dideoxyribonucleoside triphosphates can include ddATP, ddCTP, ddGTP, ddITP, and ddTTP. A nucleotide can be unlabeled or detectab ly labeled by well-known techniques. Labeling can also be carried out with quantum dots. Detectable labels can include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Fluorescent labels of nucleotides can include fluorescein, 5 -carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5-dichloro-6-carboxyfluorescein (JOE), rhodamine, 6-carboxyrhodamine (R6G), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 4-

(4 'dimethylaminophenylazo) benzoic acid (DABCYL), Cascade Blue, Oregon Green, Texas Red, Cyanine and 5-(2'-aminoethyl)aminonaphthalene-l-sulfonic acid (EDANS). Specific examples of fluorescently labeled nucleotides can include [R6G]dUTP, [TAMRA]dUTP, [R110]dCTP, [R6G]dCTP, [TAMRA]dCTP, [JOE]ddATP, [R6G]ddATP, [FAM]ddCTP, [R110]ddCTP, [TAMRA]ddGTP, [ROX]ddTTP, [dR6G]ddATP, [dR110]ddCTP, [dTAMRA] ddGTP, and [dROX]ddTTP available from Perkin Elmer, Foster City, Calif; FluoroLink DeoxyNucleotides, FluoroLink Cy3-dCTP, FluoroLink Cy5- dCTP, FluoroLink Fluor X-dCTP, FluoroLink Cy3-dUTP, and FluoroLink Cy5-dUTP available from Amersham, Arlington Heights, Ill.; Fluorescein- 15 -dATP, Fluorescein- 12-dUTP, Tetramethyl-rodamine- 6-dUTP, IR770-9-dATP, Fluorescein- 12-ddUTP, Fluorescein- 12-UTP, and Fluorescein- 15-2 '-dATP available from Boehringer Mannheim, Indianapolis, Ind.; and Chromosome Labeled Nucleotides, BODIPY-FL-14-UTP, BODIPY-FL-4-UTP, BODIPY-TMR-14-UTP, BODIPY-TMR-14-dUTP, BODIPY-TR-14-UTP, BODIPY-TR-14-dUTP, Cascade Blue-7-UTP, Cascade Blue-7-dUTP, fluorescein-12-UTP, fluorescein- 12-dUTP, Oregon Green 488-5-dUTP, Rhodamine Green-5-UTP, Rhodamine Green-5 -dUTP, tetramethylrhodamine-6-UTP, tetramethylrhodamine-6-dUTP, Texas Red-5 - UTP, Texas Red-5-dUTP, and Texas Red-12-dUTP available from Molecular Probes, Eugene, Oreg. Nucleotides can also be labeled or marked by chemical modification. A chemically modified single nucleotide can be biotin-dNTP. Some examples of biotinylated dNTPs can include, biotin-dATP (e.g., bio-N6-ddATP, biotin- 14-dATP), biotin-dCTP (e.g., biotin-11-dCTP, biotin- 14-dCTP), and biotin-dUTP (e.g., biotin- 11 -dUTP, biotin- 16-dUTP, biotin-20-dUTP). [0076] The terms “polynucleotide,” “oligonucleotide,” “nucleic acid”, and “nucleic acid molecule” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof, either in single-, double-, or multi-stranded form. A polynucleotide can be exogenous or endogenous to a cell. A polynucleotide can exist in a cell- free environment. A polynucleotide can be a gene or fragment thereof. A polynucleotide can be DNA. A polynucleotide can be RNA. A polynucleotide can have any three-dimensional structure, and can perform any function, known or unknown. A polynucleotide can comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. Some examples of analogs include: 5 -bromouracil, peptide nucleic acid, xeno nucleic acid, morpholines, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine. Examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell- free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers. The sequence of nucleotides can be interrupted by non-nucleotide components.

[0077] The term “gene,” as used herein, refers to a nucleic acid (e.g., DNA such as genomic DNA and cDNA) and its corresponding nucleotide sequence that is involved in encoding an RNA transcript. The term as used herein with reference to genomic DNA includes intervening, non-coding regions as well as regulatory regions and can include 5’ and 3’ ends. In some uses, the term encompasses the transcribed sequences, including 5’ and 3’ untranslated regions (5’-UTR and 3’-UTR), exons and introns. In some genes, the transcribed region will contain “open reading frames” that encode polypeptides. In some uses of the term, a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region”) necessary for encoding a polypeptide. In some cases, genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters. A gene can refer to an “endogenous gene” or a native gene in its natural location in the genome of an organism. A gene can refer to an “exogenous gene” or a non-native gene. A non-native gene can refer to a gene not normally found in the host organism, but which is introduced into the host organism by gene transfer. A non-native gene can also refer to a gene not in its natural location in the genome of an organism. A non-native gene can also refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and/or deletions (e.g., non-native sequence). [0078] The terms “target polynucleotide,” “target nucleic acid,” and "target sequence," as used herein, refer to a nucleic acid or polynucleotide which is targeted by a payload of the present disclosure. A target polynucleotide can be DNA (e.g., endogenous or exogenous). DNA can refer to template to generate mRNA transcripts and/or the various regulatory regions which regulate transcription of mRNA from a DNA template. A target polynucleotide can be a portion of a larger polynucleotide, for example a chromosome or a region of a chromosome. A target polynucleotide can refer to an extrachromosomal sequence (e.g., an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) or a region of an extrachromosomal sequence. A target polynucleotide can be RNA. RNA can be, for example, mRNA which can serve as template encoding for proteins. A target polynucleotide comprising RNA can include the various regulatory regions which regulate translation of protein from an mRNA template. A target polynucleotide can encode for a gene product (e.g., DNA encoding for an RNA transcript or RNA encoding for a protein product) or comprise a regulatory sequence which regulates expression of a gene product. In general, the term “target sequence” refers to a nucleic acid sequence on a single strand of a target nucleic acid. The target sequence can be a portion of a gene, a regulatory sequence, genomic DNA, cell free nucleic acid including cfDNA and/or cfRNA, cDNA, a chimeric gene, and RNA including mRNA, miRNA, rRNA, and others. A target polynucleotide, when targeted by a payload, can result in altered gene expression and/or activity. A target polynucleotide, when targeted by a payload, can result in an edited nucleic acid sequence. A target nucleic acid can comprise a nucleic acid sequence that may not be related to any other sequence in a nucleic acid sample by a single nucleotide substitution. A target nucleic acid can comprise a nucleic acid sequence that may not be related to any other sequence in a nucleic acid sample by a 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions. In some embodiments, the substitution does not occur within 5, 10, 15, 20, 25, 30, or 35 nucleotides of the 5’ end of a target nucleic acid. In some embodiments, the substitution does not occur within 5, 10, 15, 20, 25, 30, 35 nucleotides of the 3’ end of a target nucleic acid.

[0079] The term “expression” refers to one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell. “Up-regulated,” with reference to expression, generally refers to an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and/or polypeptide sequence relative to its expression level in a wild-type state while “down-regulated” generally refers to a decreased expression level of a polynucleotide (e.g. , RNA such as mRNA) and/or polypeptide sequence relative to its expression in a wild-type state.

[0080] The terms “complement,” “complements,” “complementary,” and “complementarity,” as used herein, generally refer to a sequence that is fully complementary to and hybridizable to the given sequence. In some cases, a sequence hybridized with a given nucleic acid is referred to as the “complement” or “reverse-complement” of the given molecule if its sequence of bases over a given region is capable of complementarily binding those of its binding partner, such that, for example, A-T, A- U, G-C, and G-U base pairs are formed. In general, a first sequence that is hybridizable to a second sequence is specifically or selectively hybridizable to the second sequence, such that hybridization to the second sequence or set of second sequences is preferred (e.g., thermodynamically more stable under a given set of conditions, such as stringent conditions commonly used in the art) to hybridization with nontarget sequences during a hybridization reaction. Hybridizable sequences can share a degree of sequence complementarity over all or a portion of their respective lengths, such as between 25%-100% complementarity, including at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence complementarity. Sequence identity, such as for the purpose of assessing percent complementarity, can be measured by any suitable alignment algorithm, including the Needleman-Wunsch algorithm (see e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html, optionally with default settings), the BLAST algorithm (see e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (see e.g., the EMBOSS Water aligner available at www.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html, optionally with default settings). Optimal alignment can be assessed using any suitable parameters of a chosen algorithm, including default parameters.

[0081] Complementarity can be perfect or substantial/sufficient. Perfect complementarity between two nucleic acids can mean that the two nucleic acids can form a duplex in which every base in the duplex is bonded to a complementary base by Watson-Crick pairing. Substantial or sufficient complementary can mean that a sequence in one strand is not completely and/or perfectly complementary to a sequence in an opposing strand, but that sufficient bonding occurs between bases on the two strands to form a stable hybrid complex in set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted by using the sequences and standard mathematical calculations to predict the Tm of hybridized strands, or by empirical determination of Tm by using routine methods.

[0082] The term “mutant,” as used herein in the context of a protein, a polypeptide, or a nucleic acid, can refer to a protein, a polypeptide, or a nucleic acid, whose sequence is similar to (e.g. , at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%) but has one or more amino acid or nucleotide differences from the sequence of a reference protein, a polypeptide, or a nucleic acid. Mutant can include a functional mutant and a non-functional mutant of a reference molecule (protein, polypeptide, or nucleic acid).

[0083] The term “functional mutant,” as used herein in the context of a protein or polypeptide, can refer to a protein or polypeptide, whose amino acid sequence is substantially similar to (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to) but has one or more amino acid differences from the amino acid sequence of a reference protein or polypeptide, and retains at least one function of the reference protein or polypeptide.

[0084] The term “regulating” with reference to expression or activity, as used herein, refers to altering the level of expression or activity. Regulation can occur at the transcriptional level, post-transcriptional level, translational level, and/or post-translational level. [0085] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to a polymer of at least two amino acid residues joined by peptide bond(s). This term does not connote a specific length of polymer, nor is it intended to imply or distinguish whether the peptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers comprising at least one modified amino acid. In some cases, the polymer can be interrupted by non-amino acids. The terms include amino acid chains of any length, including full length proteins, and proteins with or without secondary and/or tertiary structure (e.g, domains). The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, oxidation, and any other manipulation such as conjugation with a labeling component. The terms “amino acid” and “amino acids,” as used herein, generally refer to natural and non-natural amino acids, including modified amino acids and amino acid analogues. Modified amino acids can include natural amino acids and non-natural amino acids, which have been chemically modified to include a group or a chemical moiety not naturally present on the amino acid. Amino acid analogues can refer to amino acid derivatives. The term “amino acid” includes both D-amino acids and L-amino acids. [0086] The term “variant,” when used herein with reference to a polypeptide, refers to a polypeptide related, but not identical, to a wild type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., enzymatic activity) and/or function. Variants include polypeptides comprising one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wild type polypeptide. Variants also include derivatives of the wild type polypeptide and fragments of the wild type polypeptide.

[0087] The term “percent (%) identity,” as used herein, refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence that are identical to the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). Alignment, for purposes of determining percent identity, can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Percent identity of two sequences can be calculated by aligning a test sequence with a comparison sequence using BLAST, determining the number of amino acids or nucleotides in the aligned test sequence that are identical to amino acids or nucleotides in the same position of the comparison sequence, and dividing the number of identical amino acids or nucleotides by the number of amino acids or nucleotides in the comparison sequence.

[0088] A Cas protein referred to herein can be a type of protein or polypeptide. A Cas protein can refer to a nuclease. A Cas protein can refer to an endoribonuclease. A Cas protein can refer to any modified (e.g., shortened, mutated, lengthened) polypeptide sequence or homologue of the Cas protein. A Cas protein can be codon optimized. A Cas protein can be a codon-optimized homologue of a Cas protein. A Cas protein can be enzymatically inactive, partially active, constitutively active, fully active, inducible active and/or more active, (e.g., more than the wild type homologue of the protein or polypeptide.). A Cas protein can be a Type II Cas protein. A Cas protein can be Cas9. A Cas protein can be a Type V Cas protein. A Cas protein can be Cpfl or Cas 12a. A Cas protein can be C2cl. A Cas protein can be C2c3. A Cas protein can be a Type VI Cas protein. A Cas protein can be C2c2 or Cas 13a. A Cas protein can be Casl3b. A Cas protein can be Casl3c. A Cas protein can be Casl3d. A Cas protein can be Casl4. A Cas protein (e.g., variant, mutated, enzymatically inactive and/or conditionally enzymatically inactive site- directed polypeptide) can bind to a target nucleic acid. A Cas protein (e.g., variant, mutated, enzymatically inactive and/or conditionally enzymatically inactive endoribonuclease) can bind to a target RNA or DNA.

[0089] The term “crRNA,” as used herein, can generally refer to a nucleic acid with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity to a wild type exemplary crRNA (e.g. , a crRNA from .S', pyogenes). crRNA can generally refer to a nucleic acid with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity to a wild type exemplary crRNA (e.g., a crRNA from .S'. pyogenes, S. aureus, etc). crRNA can refer to a modified form of a crRNA that can comprise a nucleotide change such as a deletion, insertion, or substitution, variant, mutation, or chimera. A crRNA can be a nucleic acid having at least about 60% sequence identity to a wild type exemplary crRNA (e.g., a crRNA from .S', pyogenes, S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides. For example, a crRNA sequence can be at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100 % identical to a wild type exemplary crRNA sequence (e.g., a crRNA from .S'. pyogenes, S. aureus, etc) over a stretch of at least 6 contiguous nucleotides.

[0090] The term “tracrRNA,” as used herein, can generally refer to a nucleic acid with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity to a wild type exemplary tracrRNA sequence (e.g. , a tracrRNA from .S', pyogenes). tracrRNA can refer to a nucleic acid with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and/or sequence similarity to a wild type exemplary tracrRNA sequence (e.g., a tracrRNA from .S', pyogenes, S. aureus, etc). tracrRNA can refer to a modified form of a tracrRNA that can comprise a nucleotide change such as a deletion, insertion, or substitution, variant, mutation, or chimera. A tracrRNA can refer to a nucleic acid that can be at least about 60% identical to a wild type exemplary tracrRNA (e.g., a tracrRNA from .S', pyogenes, S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides. For example, a tracrRNA sequence can be at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100 % identical to a wild type exemplary tracrRNA (e.g., a tracrRNA from .S', pyogenes, S. aureus, etc) sequence over a stretch of at least 6 contiguous nucleotides. [0091] As used herein, a “guide nucleic acid” can refer to a nucleic acid that can hybridize to another nucleic acid. A guide nucleic acid can be RNA, which is referred to as “guide RNA” or “gRNA.” A guide nucleic acid can be DNA. The guide nucleic acid can be programmed to bind to a sequence of nucleic acid site -specifically. The nucleic acid to be targeted, or the target nucleic acid, can comprise nucleotides. The guide nucleic acid can comprise nucleotides. A portion of the target nucleic acid can be complementary to a portion of the guide nucleic acid. The strand of a double-stranded target polynucleotide that is complementary to and hybridizes with the guide nucleic acid can be called the complementary strand. The strand of the double-stranded target polynucleotide that is complementary to the complementary strand, and therefore may not be complementary to the guide nucleic acid can be called noncomplementary strand. A guide nucleic acid can comprise a polynucleotide chain and can be called a “single guide nucleic acid.” A single guide nucleic acid can comprise a crRNA. A single guide nucleic acid can comprise a crRNA and a tracrRNA. A guide nucleic acid can comprise two polynucleotide chains and can be called a “double guide nucleic acid.” A double guide nucleic acid can comprise a crRNA and a tracrRNA. If not otherwise specified, the term “guide nucleic acid” can be inclusive, referring to both single guide nucleic acids and double guide nucleic acids.

[0092] A guide nucleic acid can comprise a segment that can be referred to as a “nucleic acid-targeting segment” or a “nucleic acid-targeting sequence.” A nucleic acid-targeting segment can comprise a subsegment that can be referred to as a “protein binding segment” or “protein binding sequence” or “Cas protein binding segment”.

[0093] The term “targeting sequence,” as used herein, refers to a nucleotide sequence and the corresponding amino acid sequence which encodes a targeting polypeptide which mediates the localization (or retention) of a protein to a sub -cellular location, e.g. , plasma membrane or membrane of a given organelle, nucleus, cytosol, mitochondria, endoplasmic reticulum (ER), Golgi, chloroplast, apoplast, peroxisome or another organelle. For example, a targeting sequence can direct a protein (e.g. , a receptor polypeptide or an adaptor polypeptide) to a nucleus utilizing a nuclear localization signal (NLS); outside of a nucleus of a cell, for example to the cytoplasm, utilizing a nuclear export signal (NES); mitochondria utilizing a mitochondrial targeting signal; the endoplasmic reticulum (ER) utilizing an ER- retention signal; a peroxisome utilizing a peroxisomal targeting signal; plasma membrane utilizing a membrane localization signal; or combinations thereof.

[0094] As used herein, “nuclear localization domain” can refer to a nuclear localization signal or other sequence or domain capable of traversing a nuclear membrane, thereby entering the nucleus. A nuclear localization domain can be fused in-frame with a polypeptide, in which case the nuclear localization domain can be referred to as a “heterologous nuclear localization domain.”

[0095] As used herein, “nuclear export domain” can refer to a nuclear export signal or other sequence or domain that is present in a protein and capable of targeting the protein for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport. A nuclear export domain can be fused in-frame with a polypeptide, in which case the nuclear export domain can be referred to as a “heterologous nuclear export domain.” [0096] As used herein, “fusion” or “chimera” can refer to a protein and/or nucleic acid comprising one or more non-native sequences (e.g., moieties). A chimera or fusion can comprise one or more of the same non-native sequences. A chimera or fusion can comprise one or more of different non-native sequences. A chimera or fusion can be a chimeric protein. The terms “chimeric protein” and “combinatorial protein” as used herein are interchangeable unless otherwise specified. A chimera or fusion can comprise a nucleic acid affinity tag. A chimera or fusion can comprise a barcode. A fusion can comprise a peptide affinity tag. A chimera or fusion can provide for subcellular localization of the site-directed polypeptide (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an endoplasmic reticulum (ER) retention signal, and the like). A chimera or fusion can provide a non-native sequence (e.g. , affinity tag) that can be used to track or purify.

[0097] As used herein, “non-native” can refer to a nucleic acid or polypeptide sequence that is not found in a native nucleic acid or protein. Non-native can refer to affinity tags. Non-native can refer to chimeras or fusions, e.g., chimeric proteins or chimeric nucleic acids. Non-native can refer to a naturally occurring nucleic acid or polypeptide sequence that comprises mutations, insertions and/or deletions. A non-native sequence can exhibit and/or encode for an activity (e.g., enzymatic activity, methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitinating activity, etc.) that can also be exhibited by the nucleic acid and/or polypeptide sequence to which the non-native sequence is fused. A non-native nucleic acid or polypeptide sequence can be linked to a naturally occurring nucleic acid or polypeptide sequence (or a variant thereof) by genetic engineering to generate a chimeric nucleic acid and/or polypeptide sequence encoding a chimeric nucleic acid and/or polypeptide.

[0098] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal such as a human. Mammals include murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0099] The terms “treatment” and “treating,” as used herein, refer to an approach for obtaining beneficial or desired results including a therapeutic benefit and/or a prophylactic benefit. For example, a treatment can comprise administering a system or cell population disclosed herein. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, a composition can be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.

[0100] The term “effective amount” or “therapeutically effective amount” refers to the quantity of a composition, for example a composition comprising immune cells such as lymphocytes (e.g., T lymphocytes and/or NK cells) comprising a system of the present disclosure, which is sufficient to result in a desired activity upon administration to a subject in need thereof. Within the context of the present disclosure, the term “therapeutically effective” refers to that quantity of a composition that is sufficient to delay the manifestation, arrest the progression, relieve or alleviate at least one symptom of a disorder treated by the methods of the present disclosure.

[0101] The term "recombine," or "recombination," is used herein in the context of a nucleic acid modification (e.g. , a genomic modification) to refer to the process by which two or more nucleic acid molecules, or two or more regions of a single nucleic acid molecule, are modified by the action of a protein, e.g., a recombinase. Recombination can result in insertion, inversion, excision, or translocation of nucleic acids, e.g., in or between one or more nucleic acid molecules.

LIPID DELIVERY PARTICLES

[0102] In some aspects, the present disclosure relates to lipid delivery particles for delivery of a payload (e.g., directly packaged into the lipid delivery particles or encoded by a template nucleic acid molecule that is packaged into the lipid delivery particles) into a cell in vitro, ex vivo, or in vivo. The term “lipid delivery particle” is used herein interchangeably with “lipid containing particle.” In some cases, the lipid delivery particles of the present disclosure have high efficiency for in vivo delivery of a payload (e.g., a component of prime editor, a recombinase, or a template nucleic acid molecule encoding payloads for genomic integration) into a cell of a subject. In some cases, the lipid delivery particles of the present disclosure deliver template nucleic acid molecules into a cell of a subject for genomic integration of at least a portion of the template nucleic acid molecules. In some cases, the lipid delivery particles of the present disclosure deliver components of prime editor and RMCE to complete the genomic integration of the at least a portion of the template nucleic acid molecules in a two-step process. In some cases, the lipid delivery particles of the present disclosure include viral-like particles. The lipid delivery particles disclosed herein can be highly efficient for in vivo delivery of payload upon administration into a subject, e.g., a high percentage of payload loaded in the lipid delivery particle is delivered to the cells of the subject, is delivered to the desired subcellular location (e.g., cell nucleus or cell cytoplasm) of the cells of the subject. In some cases, the lipid delivery particles of the present disclosure have high efficiency for in vivo delivery of a prime editor, recombinase, and a template nucleic acid molecule encoding a therapeutic molecule to allow for genomic integration of the payload at a target site of interest. In some cases, the lipid delivery particle has a diameter that is less than 5 pm. In some cases, the lipid delivery particle comprises a lipid containing membrane encapsulating the payloads, and optionally a protein core encapsulating the payloads. In some cases, the lipid delivery particle of the present disclosure is an engineered particle. In some cases, the lipid delivery particle of the present disclosure is a viral-like particle. The lipid delivery particles can be minimally immunogenic and are partially or fully humanized. The lipid delivery particles can be resistant to inactivation by human serum complement. The at least a portion of the template nucleic acid molecule integrated into the genome of the cell of the subject can stably express a therapeutic molecule. In some cases, the lipid delivery particle of the present disclosure is a humanized viral -like particle. In some cases, the lipid delivery particle of the present disclosure is not a cell. [0103] In some cases, the lipid delivery particles are used to deliver genome editing components into cells of a subject and can have a high efficiency of in vivo gene editing carried out by the delivered genome editing components. In some cases, the genome editing components include components for prime-editing (e.g., forming a "prime-editing system" or a “prime editor”). In some cases, the primeediting components insert a sequence at a target site in the genome of the cells of a subject. In some cases, the sequence inserted by prime editing is a recombinase recognition sequence. In some cases, the primeediting components insert two recombinase recognition sequences at a target site in the genome of the cells of a subject. In some cases, the lipid delivery particles are used to deliver components for RMCE (recombinase mediated cassette exchange) (e.g., forming a "RMCE system"). In some cases, the RMCE components include a recombinase and a template nucleic acid molecule. In some cases, the lipid delivery particles are used to deliver components for both prime editing and RMCE to complete a two-step gene insertion process at a target site in the genome of the cells of a subject. The RMCE components can mediate cassette exchange between a template nucleic acid molecule and a target sequence as guided by the recombinase recognition sequences present in the template nucleic acid molecule and the target sequence (e.g., the recombinase recognition sequence inserted via prime editing), respectively, thereby inserting a donor sequence in the template nucleic acid molecule into target sequence adjacent to the recombinase recognition sequence. In some cases, the template nucleic acid molecule comprises a second recombinase recognition sequence and a donor sequence. In some embodiments, the donor sequence is inserted into the target site in the genome of the cell. In some cases, the donor sequence encodes a therapeutic molecule (e.g. , an antibody, a transcription factor, or a chimeric antigen receptor (CAR)). In other cases, the lipid delivery particles are used to deliver components for both prime editing and RMCE to complete a two-step gene deletion process at a target site in the genome of the cells of a subject. The RMCE components (e.g., a recombinase, optionally recombination directionality factors) can mediate deletion of a sequence at the target site as guided by the two recombinase recognition sequences present in the target sequence (e.g., the recombinase recognition sequences inserted via prime editing), thereby deleting a sequence in the target sequence flanked by the recombinase recognition sequences. The two recombinase recognition sequences inserted to the target sequence can be the same sequence. The two recombinase recognition sequences inserted to the target sequence can be different sequences that work together as a pair with the recombinase. The two recombinase recognition sequences inserted to the target sequence can be positioned in the same direction, flanking a sequence to be deleted at the target site. In another case, the lipid delivery particles are used to deliver components for both prime editing and RMCE to complete a two-step gene inversion process at a target site in the genome of the cells of a subject. The RMCE components (e.g., a recombinase, optionally recombination directionality factors) can mediate inversion of a sequence at the target site as guided by the two recombinase recognition sequences present in the target sequence (e.g., the recombinase recognition sequences inserted via prime editing), thereby inverting a sequence in the target sequence flanked by the recombinase recognition sequences. The two recombinase recognition sequences inserted to the target sequence can be the same sequence. The two recombinase recognition sequences inserted to the target sequence can be different sequences that work together as a pair with the recombinase. The two recombinase recognition sequences inserted to the target sequence can be positioned in an opposite direction, flanking a sequence to be inverted at the target site. [0104] In some cases, the lipid delivery particles provided herein comprise a lipid-based external layer (e.g., a lipid membrane) encapsulating the payloads. In some cases, the lipid delivery particle provided herein comprise a lipid containing membrane enclosing a protein core that encapsulates the payloads. In some cases, the lipid membrane contains phospholipid. In some cases, the lipid membrane is a phospholipid bilayer membrane that comprises proteins (e.g., proteins that are anchored in the membrane via transmembrane domain or attached to the membrane via covalent binding or non-covalent interactions) and other biomolecules. In some cases, the protein core comprises a structural protein. The structural protein can comprise a plasma membrane recruitment element. The terms “plasma membrane localization domain” and “plasma membrane recruitment element” as used herein are interchangeable unless otherwise specified. The plasma membrane recruitment element can be part of a chimeric protein. The chimeric protein comprising the plasma membrane recruitment element (e.g., a gag protein) can form at least part of the structural protein, which can form at least part of the protein core. In some cases, the structural protein can further comprise a retroviral protease (pro) protein. In some cases, the structural protein can further comprise a retroviral polymerase protein (e.g., a retroviral reverse transcriptase). In some cases, the lipid delivery particle provided herein does not comprise a protein core.

[0105] The chimeric protein can further comprise a payload. A payload (e.g., one or more components of a prime-editing system and a RMCE system) can be loaded in the lipid delivery particles. In some cases, the payloads are loaded inside the protein core formed by a plasma membrane recruitment element (e.g. , a gag protein). In some cases, a payload is loaded in the lipid delivery particle by attaching to the external lipid-based layer. The external lipid containing membrane (lipid-based layer) can be a single lipid layer or lipid bilayer made of two layers of lipid molecules. In some cases, the external lipid-based layer contains phospholipid. In some cases, the lipid delivery particle has one or more envelope proteins inserted in or attached to the outside of the external lipid layer. The envelope protein can help fusion of the lipid delivery particle with membrane of a target cell, thus delivering the payload loaded in the lipid delivery particle to the target cell. In some cases, the term “envelope protein” is a membrane fusion protein as described herein.

[0106] A dimension (e g. , diameter) of the lipid delivery particle can be less than 5 pm, less than 4 pm, less than 3 pm, less than 2 pm, or less than 1 pm. A dimension (e.g. , diameter) of the lipid delivery particle can be about 10 nm to about 1000 nm, such as about 10 nm to 50 nm, 10 nm to 100 nm, 10 nm to 200 nm, 10 nm to 300 nm, 10 nm to 400 nm, 10 nm to 500 nm, 10 nm to 600 nm, 10 nm to 800 nm, 20 nm to 50 nm, 20 nm to 100 nm, 20 nm to 200 nm, 20 nm to 300 nm, 20 nm to 400 nm, 20 nm to 500 nm, 20 nm to 600 nm, 20 nm to 800 nm, 50 nm to 100 nm, 50 nm to 200 nm, 50 nm to 300 nm, 50 nm to 400 nm, 50 nm to 500 nm, 50 nm to 600 nm, 50 nm to 800 nm, 100 nm to 200 nm, 100 nm to 300 nm, 100 nm to 400 nm, 100 nm to 500 nm, 100 nm to 600 nm, 100 nm to 800 nm, 200 nm to 300 nm, 200 nm to 400 nm, 200 nm to 500 nm, 200 nm to 600 nm, 200 nm to 800 nm, 400 nm to 600 nm, 400 nm to 800 nm, or 600 nm to 800 nm. In some cases, the lipid delivery particle comprise viral-like particles and have a dimension (e.g., diameter) of about 10 nm to about 100 nm, such as about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 40 nm, about 10 nm to about 50 nm, about 10 nm to about 60 nm, about 10 nm to about 80 nm, about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm to about 50 nm, about 20 nm to about 60 nm, about 20 nm to 80 nm, about 40 nm to about 50 nm, about 40 nm to about 60 nm, or about 40 nm to about 80 nm. In some cases, the lipid delivery particle comprise exosomes, and have a size of about 50 nm to about 200 nm, such as about 50 nm to about 80 nm, about 50 nm to about 100 nm, about 50 nm to about 120 nm, about 50 nm to about 150 nm, about 50 nm to about 160 nm, about 50 to about 180 nm, about 60 nm to about 80 nm, about 60 nm to about 100 nm, about 60 nm to about 120 nm, about 60 nm to about 160 nm, about 60 nm to about 160 nm, about 60 nm to about 180 nm, about 80 nm to about 100 nm, about 80 nm to about 120 nm, about 80 nm to about 160 nm, about 80 nm to about 180 nm, about 80 nm to about 180 nm, about 100 nm to about 120 nm, about 100 nm to about 150 nm, about 100 nm to about 180 nm, about 120 nm to about 150 nm, about 120 nm to about 180 nm, about 150 nm to about 180 nm, or about 150 nm to about 200 nm.

[0107] The lipid delivery particles (e.g., viral -like particles) described herein can package payloads (e.g., any components of prime editor and RMCE) by integrating all production DNA into the genomic DNA of production cell lines. Once cell lines are created, payload delivering particles can be produced in a constitutive or inducible fashion. Protein payloads can be packaged into lipid delivery particle by fusing plasma membrane recruitment elements to protein-based payload. Protein payloads can be packaged into lipid delivery particle by fusing plasma membrane recruitment elements to an RBP that binds an RBP binding sequence located on an mRNA encoding the protein payload.

[0108] In some aspects, provided herein is a lipid delivery particle (e.g., a viral-like particle) comprising a lipid containing membrane, a recombinase, and a ribonucleoprotein complex. In some cases, the ribonucleoprotein complex that comprises: a prime editor comprising a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; and a guide nucleic acid molecule. In some cases, the recombinase and the ribonucleoprotein complex are within an inside cavity encapsulated by the lipid containing membrane. In some cases, the lipid delivery particle is engineered. In some cases, the lipid delivery particle is not a cell. In some cases, the lipid delivery particle has a diameter that is less than 5 pm.

[0109] In some aspects, provided herein is a lipid delivery particle (e.g., a viral-like particle) comprising (a) a lipid containing membrane, (b) a recombinase or a nucleic acid sequence encoding the recombinase; (c) (i) a ribonucleoprotein complex comprising: (1) a prime editor comprising a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; and (2) a guide nucleic acid molecule, or (ii) (1) a nucleic acid sequence encoding the prime editor; and (2) the guide nucleic acid molecule or a nucleic acid sequence encoding the guide nucleic acid molecule; and a template RNA that encodes a donor nucleic acid molecule, wherein the donor nucleic acid molecule comprises a donor nucleic acid sequence and a second recombinase recognition sequence. In some cases, the recombinase mediates recombination between a first recombinase recognition sequence and the second recombinase recognition sequence.

[0110] In some aspects, provided herein is a lipid delivery particle comprising: a first nucleic acid sequence encoding a prime editor, wherein the prime editor comprises a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; a guide nucleic acid molecule or a second nucleic acid sequence encoding the guide nucleic acid molecule; a third nucleic acid sequence encoding a recombinase; and a donor nucleic acid sequence that comprises a second recombinase recognition sequence, or a template RNA encoding the donor nucleic acid sequence. In some cases, the recombinase mediates recombination between a first recombinase recognition sequence and the second recombinase recognition sequence, [oni] In some aspects, provided herein is a lipid delivery particle (e.g., a viral-like particle) that includes an envelope protein (e.g., a human endogenous retroviral (HERV) envelope protein); a chimeric protein comprising a plasma membrane recruitment element (e.g., coupled to a nuclear export sequence (NES)); and a payload (e.g., one or more components of a prime-editing system and a RMCE system, such as a recombinase and a template nucleic acid molecule). In some cases, the template nucleic acid molecule encodes a therapeutic molecule into the genome of a recipient cell.

[0112] In some aspects, provided herein is a lipid delivery particle (e.g., a viral-like particle) that includes an envelope protein (e.g., a human endogenous retroviral (HERV) envelope protein, or a humanized viral envelope protein); and a chimeric protein comprising a plasma membrane recruitment element (e.g. , coupled to a cleavable linker); and a payload (e.g. , one or more components of a primeediting system and a RMCE system, such as a recombinase and a template nucleic acid molecule). In some cases, the template nucleic acid molecule encodes a therapeutic molecule into the genome of a recipient cell.

[0113] In some aspects, provided herein is a lipid delivery particle (e.g., a viral-like particle) that includes a plasma membrane recruitment molecule (e.g. , a human endogenous retroviral (HERV) structural protein, e.g., HERV gag, a membrane protein, a pleckstrin homology (PH) domain) and a nuclear export sequence (NES).

[0114] In some aspects, provided herein is a lipid delivery particle (e.g., a viral-like particle) that includes a chimeric protein comprising i) a plasma membrane recruitment molecule (e.g., a human endogenous retroviral (HERV) structural protein, e.g., HERV gag, a membrane protein, or a transmembrane domain thereof, or a pleckstrin homology (PH) domain), ii) a cleavable linker, and iii) a payload (e.g., one or more components of a prime -editing system and a RMCE system comprising a recombinase and a template nucleic acid molecule). In some cases, the template nucleic acid molecule encodes a therapeutic molecule into the genome of a recipient cell.

Viral-like Particles

[0115] In some aspects, disclosed herein are compositions, methods, and systems related to viral-like particles that can be utilized to deliver payload into a cell.

[0116] A viral-like particle (VLP) disclosed herein can comprise one or more virus-derived proteins, such as a structural protein of VLPs and an envelope protein. In some cases, the virus-derived protein is present as part of a chimeric protein that forms the VLP. In some cases, the VLPs do not comprise a protein core but comprises plasma membrane recruitment elements that are not gag proteins (e.g., are pH domain or a membrane protein described herein). [0117] In some cases, the loading capacity of the VLPs disclosed herein has a loading capacity that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18- fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 80-fold, 100-fold higher than a conventional VLP. [0118] In some cases, the VLPs comprise structural proteins (e.g., gag protein). In some cases, the structural protein described herein forms basic structure of the viral-like particle, e.g. , at least part of the capsid that encapsulate the protein core of the VLP. Structural proteins of viral-like particle can include a plasma membrane recruitment element. In some cases, the plasma membrane recruitment element described herein also facilitates self-assembly of the VLP, e.g., facilitates localization of plasma membrane and packaging of the viral-like particle by forming the membrane enclosure. In some cases, the structural protein described herein facilitates releases of the VLP from the producer cell from which the VLP is produced. In some cases, the structural protein of VLP (e.g., plasma membrane recruitment element) is a viral protein, e.g., derived from a virus. In some cases, the structural protein of VLP is a mammalian protein, e.g., derived from a mammal, e.g., human. In some cases, the structural protein of VLP is a human endogenous protein. In some cases, the structural protein of VLP (e.g., plasma membrane recruitment element) is a polyprotein derived from a virus, a homologue thereof, a fragment thereof, a variant thereof, or any combination thereof. For instance, the structural protein of VLP (e.g. , 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. In some cases, the gag protein is derived from Friend murine leukemia virus (FMLV). In some cases, the retroviral gag polyprotein is a gag polyprotein of an alpha retrovirus, a beta retrovirus, a gamma retrovirus, a delta retrovirus, an epsilon retrovirus, or a spumavirus. In some cases, the retroviral gag polyprotein is a gag polyprotein of a human immunodeficiency virus.

[0119] Examples of the structural protein of VLP (e.g. , plasma membrane recruitment element) comprises Human Papillomavirus (HPV) LI protein, HPV L2 protein, Hepatitis B virus (HBV) core protein, Chikungunya virus (CHIKV) C-E3-E2-6k-El, human immunodeficiency virus (HIV) gag-pol, HIV gag, Respiratory syncytial virus (RSV) M, RSV NP, Human metapneumovirus (HMPV) M, Influenza Ml, Zika virus (ZIKV) C, ZIKV prM/M, Dengaue virus (DENV) C-prM, West Nile Virus (WNV) prME protein, WNV CprME protein, Filovirus VP40 or Z protein, Baculovirus P 1 protein, Rotavirus VP7, Rotavirus VP2 protein, Rotavirus VP6 protein, SARS M protein, SARS E protein, SARS N protein, Porcine Circovirus Type 2 (PCV2) capsid, baculovirus VP2 protein, baculovirus VP5 protein, baculovirus VP3 protein, or baculovirus VP7 protein, Hepatitis C virus (HCV) core protein, Ebola nucleocapsid, Parovirus VP1 protein, Parovirus VP2 protein, Newcastle disease virus (NDV) M protein, hepatitis E virus (HeV) M protein, Nipah virus (NIV) M protein, Human polyomavirus 2 (JCPyV) VP1 protein, Human parainfluenza virus type 3 (HPIV3) M protein, HPIV3N protein, or Mumps virus (MuV) M proteins, a homologue thereof, a fragment thereof, a variant thereof, or any combination thereof. Human Endogenous VLP

[0120] In some aspects, provided herein are viral-like particles that have reduced or no immunogenicity to human subjects, e.g., non-viral human endogenous viral-like particles (heVLPs), that comprise human endogenous viral components.

[0121] Different from viral-like particles according to some embodiments of the present disclosure, heVLPs described herein can package protein payload by integrating all production DNA into the genomic DNA of production cell lines. Once cell lines are created, protein delivery heVLPs can be produced in a constitutive or inducible fashion. Protein payloads are packaged into he VLP by fusing select human-endogenous GAG proteins or other plasma membrane recruitment elements to proteinbased payload.

[0122] The he VLP systems described herein can have the potential to be simpler, more efficient and safer than conventional, artificially derived lipid/gold nanoparticles and viral particle-based delivery systems because heVLPs can be comprised of human-derived components. The payload inside the particles may or may not be human derived, but the he VLP is derived from human or comprises human endogenous components or synthetic non-immunogenic components. “Synthetic” components include surface scFv/nanobody/darpin peptides that have been demonstrated to not be immunostimulatory and can be used to enhance targeting and cellular uptake of lipid delivery particles. This means that the exterior surface of the particle lacks components that can be significantly immunostimulatory, which can minimize immunogenicity and antibody neutralization of these particles.

[0123] In some cases, excluding payload, the heVLPs provided herein do not contain exogenous viral components inherent to other VLPs and this represents a significant and novel advancement in technology. In some cases, the heVLPs can utilize chemical -based dimerizers, and heVLPs can have the ability to package and deliver payload molecules including therapeutic or diagnostic agents, including biomolecules and chemicals, e.g., specialty single and/or double-stranded DNA molecules (e.g., plasmid, mini circle, closed-ended linear DNA, AAV DNA, episomes, bacteriophage DNA, homology directed repair templates, etc.), single and/or double-stranded RNA molecules (e.g., single guide RNA, prime editing guide RNA, messenger RNA, transfer RNA, long non-coding RNA, circular RNA, RNA replicon, circular or linear splicing RNA, micro RNA, small interfering RNA, short hairpin RNA, piwi-interacting RNA, toehold switch RNA, RNAs that can be bound by RNA binding proteins, bacteriophage RNA, internal ribosomal entry site containing RNA, etc.), proteins, chemical compounds and/or molecules (e.g., small molecules), and combinations of the above listed payloads (e.g, AAV particles).

[0124] The heVLPs described herein are different from conventional retroviral particles, virus-like particles (VLPs), exosomes and other previously described extracellular vesicles that can be loaded with payload, at least because heVLPs can be produced by a strategic overexpression of human-derived components in human cells, heVLPs have a vast diversity of possible payloads and loading strategies, heVLPs lack a limiting DNA/RNA length constraint, heVLPs lack proteins derived from pol and exogenous gag, and heVLPs have unique mechanisms of cellular entry. [0125] Described herein are compositions and methods for payload delivery that can be used with a diverse array of protein and nucleic acid molecules, such as genome editing (e.g., delivering a therapeutic molecule that encodes a therapeutic protein) that are applicable to many disease therapies.

[0126] In some aspects, provided herein are engineered heVLPs, comprising a membrane comprising a phospholipid bilayer with one or more HERV-derived ENV/glycoprotein(s) (e.g., overexpressed from exogenous sources, such as plasmids or stably integrated transgenes, in heVLP production cells) (e.g., as shown in Table 2- A or Table 2-B) or other human endogenous envelope protein on the external side; and a human endogenous GAG protein, other plasma membrane recruitment element (e.g., as shown in Table 3), and/or biomolecule/chemical payload disposed in the core of the heVLP on the inside of the membrane (e.g. , in the protein core enclosed by the phospholipid bilayer).

[0127] In some cases, the lipid delivery particles (e.g., VLPs) provided herein comprise a plasma membrane recruitment element that is a PH domain derived from phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3 -phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, or MAPK associated protein 1 (MAPKAP1), or a mutant thereof (e.g., a functional mutant thereof). In some cases, the plasma membrane recruitment element comprises a PH domain derived from a human protein. In some cases, the plasma membrane recruitment element comprises a PH domain derived from human phospholipase C81, human Aktl, human Arc, human endogenous retroviral gag protein, human 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), human CD9, human CD47, human CD63, human CD81, human Daapl, mouse Grpl, human Grpl, human OSBP, human Btkl, human FAPP1, human CERT, human PKD, human PHLPP1, human SWAP70, or human MAPKAP1, or a mutant thereof. In some cases, the plasma membrane recruitment element comprises any one of the sequences in Table 3.

[0128] In some aspects, provided herein are human endogenous VLPs, comprising a membrane comprising a phospholipid bilayer with one or more HERV-derived ENVZglycoprotein(s) (e.g. , overexpressed from exogenous sources, such as plasmids or stably integrated transgenes, in heVLP production cells) (e.g., as shown in Table 2- A or Table 2-B) or other human endogenous envelope protein on the external side; and a viral structural protein (e.g., a retroviral gag protein) on the inside of the membrane (e.g., in the protein core enclosed by the phospholipid bilayer).

[0129] In some aspects, provided herein are human endogenous VLPs, comprising a membrane comprising a phospholipid bilayer with one or more viral envelope proteins disclosed herein; and a human endogenous GAG protein, other plasma membrane recruitment element, and/or biomolecule/chemical payload disposed in the core of the heVLP on the inside of the membrane (e.g. , in the protein core enclosed by the phospholipid bilayer). [0130] The payload may or may not be fused to a human-endogenous GAG or other plasma membrane recruitment element. In some cases, the heVLP does not comprise a non-human gag and/or pol protein. In some cases, the heVLP does not express gag and/or pol proteins except for gag proteins that are encoded in the human genome or gag proteins that are encoded by a consensus sequence that is derived from gag proteins found in the human genome. Human-derived GAG or other plasma membrane recruitment elements fused to payload can be overexpressed from exogenous sources, such as plasmids or stably integrated transgenes, in heVLP production cells.

[0131] Human-endogenous GAG proteins and human pleckstrin homology (PH) domains can localize to biological membranes. PH domains can interact with phosphatidylinositol lipids and proteins within biological membranes, such as PIP2, PIP3, bg-subunits of GPCRs, and PKC. However, in addition to localizing to phospholipid bilayers, human-endogenous GAG proteins can also drive budding and particle formation. This dual functionality of human-endogenous GAG can enable packaging of payload and budding/formation of particles. One such human-endogenous GAG protein used for this purpose is the human Arc protein that can be fused to protein-based payload to recruit payload to the cytosolic side of the phospholipid bilayer. These human-endogenous GAG phospholipid bilayer recruitment domains can be fused to the N-terminus or C-terminus of protein-based payload via polypeptide linkers of variable length regardless of the location or locations of one or more nuclear localization sequence(s) (NLS) within the payload. In some cases, the linker between protein-based payload and the human-endogenous GAG phospholipid bilayer recruitment domain is a polypeptide linker 5-20, e.g., 8-12, e.g., 10, amino acids in length primarily composed of glycines and serines.

[0132] The human-endogenous GAG or other phospholipid bilayer recruitment domain can localize the payload to the phospholipid bilayer and this protein payload is packaged within heVLPs that bud off from the producer cell into extracellular space. The use of these human-endogenous GAG and other phospholipid bilayer recruitment domains is novel and unique in that these human-endogenous GAG and other proteins can facilitate for localization of payload to the cytosolic face of the plasma membrane within the heVLP production cells. The use of these human-endogenous GAG and other phospholipid bilayer recruitment domains can allow for payload to localize to the nucleus of the transduced cells without the utilization of exogenous retroviral GAG or chemical and/or light-based dimerization systems. [0133] heVLPs can also package and deliver a combination of DNA and RNA if heVLPs are produced via transient transfection of a production cell line. DNA that is transfected into cells will possess sizedependent mobility such that a fraction of the transfected DNA will remain in the cytosol while another fraction of the transfected DNA will localize to the nucleus. One fraction of the transfected DNA in the nucleus can express components that create heVLPs and the other fraction in the cytosol/near the plasma membrane will be encapsulated and delivered in heVLPs.

[0134] In some cases, the payload is limited by the diameter of the particles, which e.g., in some embodiments range from 150nm to 500nm. [0135] Other examples of heVLPs, human endogenous viral structural proteins, and plasma membrane recruitment elements include those described in international publication no. WO 2020/252455, which is incorporated herein by reference in its entirety.

[0136] In some embodiments, in order for efficient recruitment of payload into heVLPs, the payload comprises a covalent or non-covalent connection to a human-endogenous GAG or other Plasma membrane recruitment element, such as those shown in Table 3. Covalent connections, for example, can include direct protein-protein chimeras generated from a single reading frame, inteins that can form peptide bonds, other proteins that can form covalent connections at R-groups and/or RNA splicing. Non- covalent connections, for example, can include DNA/DNA, DNA/RNA, and/or RNA/RNA hybrids (nucleic acids base pairing to other nucleic acids via hydrogen bonding interactions), protein domains that dimerize or multimerize with or without the need for a chemical compound/molecule to induce the protein-protein binding (such as DmrA/DmrB/DmrC (Takara Bio), FKBP/FRB, dDZFs, and Leucine zippers), single chain variable fragments, nanobodies, affibodies, proteins that bind to DNA and/or RNA, proteins with quaternary structural interactions, optogenetic protein domains that can dimerize or multimerize in the presence of certain light wavelengths, and/or naturally reconstituting split proteins.

[0137] In some embodiments, the payload comprises a fusion to a dimerization domain or proteinprotein binding domain that may or may not require a molecule to trigger dimerization or protein-protein binding.

[0138] In some cases, excluding payloads (e.g., prime editor, recombinase, and the template nucleic acid molecule), the lipid delivery particles (e.g., viral -like particles) provided herein do not contain exogenous viral components inherent to other VLPs and this represents a significant and novel advancement in technology. The lipid delivery particles (e.g., viral-like particles) can utilize chemical-based dimerizers, and lipid delivery particles (e.g., viral -like particles) can have the ability to package and deliver payload molecules including therapeutic or diagnostic agents, including biomolecules and chemicals, e.g., specialty single and/or double-stranded DNA molecules (e.g., plasmid, mini circle, closed-ended linear DNA, AAV DNA, extrachromosomal genetic materials, bacteriophage DNA, homology directed repair templates, etc.), single and/or double-stranded RNA molecules (e.g., single guide RNA, prime editing guide RNA, messenger RNA, transfer RNA, long non-coding RNA, circular RNA, RNA replicon, circular or linear splicing RNA, micro RNA, small interfering RNA, short hairpin RNA, piwi-interacting RNA, toehold switch RNA, RNAs that can be bound by RNA binding proteins, bacteriophage RNA, internal ribosomal entry site containing RNA, etc.), proteins, chemical compounds and/or molecules (e.g., small molecules), and combinations of the above listed payloads (e.g, AAV particles).

ENVELOPE PROTEIN

[0139] 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.

[0140] 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.

[0141] The envelope protein can be 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 can be embedded or inserted in the membrane, spanning through the membrane, with certain portions located at the outside of the membrane, or certain portions extending to the inside of the particle, or both. The envelope protein within the lipid delivery particle described herein can be overexpressed from an exogenous source, such as plasmids or stably integrated transgenes, in the production cells. [0142] 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 surface of a target cell and participate in the fusion of the 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 mechanisms is 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-mediated proteolysis, or (ii) low pH/acidification. The cathepsin-mediated proteolysis can be pH dependent or pH independent. Other fusion triggering mechanisms 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.

[0143] 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 the 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.

[0144] 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 Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), nanobodies, or camelid VHH 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 non-covalently 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. [0145] In some cases, the targeting moiety linked to the membrane -fusion protein binds a cell surface marker on the target cell, e.g, a protein, glycoprotein, receptor, cell surface ligand, agonist, lipid, sugar, class I transmembrane protein, class II transmembrane protein, or class III transmembrane protein.

[0146] 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

[0147] In some cases, an envelope protein has a viral origin. For example, a suitable envelope protein is from a DNA virus, an RNA 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 different virus. 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. [0148] In some cases, the envelope protein is a vesicular stomatitis virus glycoprotein (VSVG) or a mutant thereof. In some cases, the envelope protein is a Human immunodeficiency virus GP160 or a mutant thereof. In some cases, the envelope protein is a Baboon Endogenous Retrovirus (BaEVTR) glycoprotein or a mutant thereof. In some cases, the envelope protein is a modified Baboon Endogenous Retrovirus (BaEVTRless) glycoprotein or a 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 mutant thereof. In some cases, the envelope protein pantropic murine leukemia virus envelope protein (MLV) or a mutant thereof. In some cases, the envelope protein is a modified Fusion protein of Vesicular stomatitis Indiana virus and Rabies vims Glycoproteins (FuG-E P440E) or a mutant thereof. In some cases, the envelope protein is a FuG-B2 envelope glycoprotein or a mutant thereof. In some cases, the envelope protein is an ecotropic Murine Leukemia Vims envelope protein (MLV ENV ecotropic) or a mutant thereof. In some cases, the envelope protein is an amphotrophic Murine Leukemia Vims envelope protein (MLV ENV amphotropic) or a mutant thereof. In some cases, the envelope protein is a Moloney murine leukemia vims envelope protein (MMLV) or a mutant thereof. In some cases, the envelope protein is a Moloney murine sarcoma vims envelope protein (MoMSVg) or a mutant thereof. In some cases, the envelope protein is a moloney murine leukemia vims 10A1 strain Glycoprotein (MLV 10A1) or a mutant thereof. In some cases, the envelope protein is a xenotropic murine leukemia vims envelope protein (MLV ENV xenotropic) or a mutant thereof. In some cases, the envelope protein is a xenotropic murine leukemia vims-related envelope protein (XMRV) or a mutant thereof. In some cases, the envelope protein is a Baculovims envelope glycoprotein (GP64) or a mutant thereof. In some cases, the envelope protein is an endogenous feline vims envelope protein (RD114 ENV) or a mutant thereof. In some cases, the envelope protein is a mammalian endogenous retrovims protein. The mammalian endogenous retrovims protein can be a koala retrovims protein (KoRV) or a Jaagsiekte sheep retrovims protein (enJSRV), or a mutant thereof.

[0149] In some cases, the envelope protein is a simian endogenous type D retrovims protein (RD-114) or a mutant thereof. In some cases, the envelope protein is a gibbon ape leukemia vims envelope protein (GALV) or a mutant thereof. In some cases, the envelope protein is a feline leukemia vims envelope protein (FLV) or a mutant thereof. In some cases, the envelope protein is a mouse mammary tumor vims envelope protein (MMTV) or a mutant thereof. In some cases, the envelope protein is an avian leukosis vims envelope protein or a mutant thereof. In some cases, the envelope protein is a rous sarcoma vims envelope protein or a mutant thereof.

[0150] 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 vimses or a mutant thereof, e.g. , Hepatitis B vims (HBV) or hepatitis C vims (HCV), VSV-G glycoprotein or a mutant thereof, a Marburg vims glycoprotein or a mutant thereof, an Ebola vims glycoprotein or a mutant thereof. To fuse with a target muscle cell, for example, a skeletal muscle cell, the envelope protein can be a Ross River vims glycoprotein or a mutant thereof, or a VSV-G or a mutant thereof. To fuse with a target ocular cell, for example, a photoreceptor cell or a retinal cell, the envelope protein can be an Ebola vims glycoprotein or a mutant thereof, a Marburg vims glycoprotein or a mutant thereof, or a VSV-G or a mutant thereof. To fuse with a target immune cell, for example, CD8+ T cell, an HTLV-1 glycoprotein or a mutant thereof, or a VSV- G glycoprotein or a mutant thereof. To fuse with a target immune cell, for example, CD4+ T cell, the envelope protein can be a HIV-1 envelope or a mutant thereof, a HTLV-1 glycoprotein or a mutant thereof, or a VSV-G glycoprotein or a mutant thereof. To fuse with a target lung cells, the envelope protein can be a respiratory syncytial virus glycoprotein or a mutant thereof, or a SARS-CoV glycoprotein or a 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 mutant thereof, a Mokola virus glycoprotein or a mutant thereof, a Semliki Forest virus glycoprotein or a mutant thereof, a Venezuelan equine encephalitis virus glycoprotein or a mutant thereof, a FuG-E or a mutant thereof, a FuG-B2 or a mutant thereof, or a VSV-G or a mutant thereof. To fuse with a target sensory cell, such as an auditory cell, including hair cells, cochlear cells, etc., the envelope protein can be an Ebola virus glycoprotein or a mutant thereof, a Marburg virus glycoprotein or a mutant thereof, or a VSV-G or a mutant thereof.

[0151] In some cases, the envelope protein includes those described in Table 2-C 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 includes those described in Table 2-C and a heterologous peptide sequence fused to the N-terminal or C-terminal.

[0152] 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 2-C. 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: 83-104. 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: 83-104. 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: 83-104 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: 83-104. 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: 83-104. 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: 83-104. 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: 83-104 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: 83-104. 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: 83-104. 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: 83-104 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: 83-104. 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: 83-104. 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: 83-104. 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: 83-104.

Table 2-C. Exemplary envelope proteins from virus origin

Envelope protein of human origin

[0153] 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%, at least 95%, at least 98%, or at least 99%. Using an envelope protein of a human 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). [0154] In some cases, the envelope protein is a HERV envelope protein such as any one of those listed in Table 2- A. 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 mutant thereof. 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 mutant thereof. In some cases, the envelope protein is a hENVR(b) or a mutant thereof. In some cases, the envelope protein is a hENVR(c)2 or a mutant thereof. In some cases, the envelope protein is a hENVR(c)l or a mutant thereof. 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-A. Exemplary HERV envelope proteins

[0155] In some cases, the envelope protein includes those described in Table 2-B with at least one amino acid substitution, deletion, or insertion. For example, for those amino acid sequences start with a N- terminal methionine, the N-terminal methionine can be absent. In some cases, the envelope protein includes those described in Table 2-B and a heterologous peptide sequence fused to the N-terminal or C- terminal. [0156] 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: 49-82. 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: 49-82. 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: 49-82. 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: 49-82. 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: 49-82. 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: 49-82. 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: 49-82. 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: 49-82. 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: 49-82. 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: 49-82. 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: 49-82. 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: 49-82.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: 49-82. 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: 49-82.

Table 2-B. Exemplary sequences for human HERV envelope proteins

PLASMA MEMBRANE RECRUITMENT ELEMENT

[0157] 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 template nucleic acid molecule and a plasma membrane recruitment element. In some cases, the membrane encapsulates a protein core. In some cases, the template nucleic acid is within an inside of the protein core. 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, 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. [0158] 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 or together with an envelope protein disclosed herein in a producer cell can lead to formation of the lipid delivery particle.

[0159] 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. The plasma 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 mutant thereof. The plasma membrane recruitment element can comprise a gag from Moloney murine leukemia virus (MMLV) or a mutant thereof. In some cases, the plasma membrane recruitment element forms structural protein that forms the protein core of the lipid delivery particles described herein. The plasma membrane recruitment element can comprise Respiratory syncytial virus (RSV) M or a mutant thereof. The plasma membrane recruitment element can comprise Human Papillomavirus (HPV) LI protein or a mutant thereof. The plasma membrane recruitment element can comprise HPV L2 protein or a mutant thereof. The plasma membrane recruitment element can comprise Hepatitis B virus (HBV) core protein or a mutant thereof. The plasma membrane recruitment element can comprise Hepatitis C virus (HCV) core protein or a mutant thereof. The plasma membrane recruitment element can comprise hepatitis E virus (HeV) M protein or a mutant thereof. The plasma membrane recruitment element can comprise Chikungunya virus (CHIKV) C-E3-E2-6k-El or a mutant thereof. The plasma membrane recruitment element can comprise RSV NP or a mutant thereof. The plasma membrane recruitment element can comprise Human metapneumovirus (HMPV) M or a mutant thereof. The plasma membrane can comprise a glycoprotein from a flavivirus. 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 mutant thereof. The plasma membrane recruitment element can comprise ZIKV prM/M or a mutant thereof. The plasma membrane recruitment element can comprise Dengaue virus (DENV) C-prM or a mutant thereof. The plasma membrane recruitment element can comprise West Nile Virus (WNV) prME protein or a mutant thereof. The plasma membrane recruitment element can comprise WNV CprME protein or a mutant thereof. The plasma membrane recruitment element can comprise Filovirus VP40 or Z protein or a mutant thereof. The plasma membrane recruitment element can comprise Baculovirus Pl protein or a mutant thereof. The plasma membrane recruitment element can comprise Rotavirus VP7 or a mutant thereof. The plasma membrane recruitment element can comprise Rotavirus VP2 protein or a mutant thereof. The plasma membrane recruitment element can comprise Rotavirus VP6 protein or a mutant thereof. The plasma membrane recruitment element can comprise Porcine Circovirus Type 2 (PCV2) capsid or a mutant thereof. The plasma membrane recruitment element can comprise baculovirus VP2 protein or a mutant thereof. The plasma membrane recruitment element can comprise baculovirus VP5 protein or a mutant thereof. The plasma membrane recruitment element can comprise baculovirus VP3 protein or a mutant thereof. The plasma membrane recruitment element can comprise or baculovirus VP7 protein or a mutant thereof. The plasma membrane recruitment element can comprise Ebola nucleocapsid or a mutant thereof. The plasma membrane recruitment element can comprise Parovirus VP 1 protein or a mutant thereof. The plasma membrane recruitment element can comprise Parovirus VP2 protein or a mutant thereof. The plasma membrane recruitment element can comprise Newcastle disease virus (NDV) M protein or a mutant thereof. The plasma membrane recruitment element can comprise Human polyomavirus 2 (JCPyV) VP1 protein or a mutant thereof. The plasma membrane recruitment element can comprise Human parainfluenza virus type 3 (HPIV3) M protein or a mutant thereof. The plasma membrane recruitment element can comprise HPIV3N protein or a mutant thereof. The plasma membrane recruitment element can comprise or Mumps virus (MuV) M proteins or a mutant thereof. The plasma membrane recruitment element can comprise SARS M protein or a mutant thereof. The plasma membrane recruitment element can comprise SARS E protein or a mutant thereof. The plasma membrane recruitment element can comprise SARS N protein or a mutant thereof.

[0160] 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 gag from 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 hGAGK_con 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.

[0161] The plasma membrane recruitment element can include a pleckstrin homology (PH) domain from a human protein or a 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 C81 or a mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human Aktl or a mutant thereof. The plasma membrane recruitment element can comprise a mutant PH domain of human Aktl with E17K substitution or a mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human 3- phosphoinositide-dependent protein kinase 1 or a mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human Daapl or a mutant thereof. The plasma membrane recruitment element can comprise a PH domain of mouse Grp 1 or a mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human Grpl or a mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human OSBP or a mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human Btkl or a mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human FAPP1 or a mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human CERT or a mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human PKD or a mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human PHLPP1 or a mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human SWAP70 or a mutant thereof. The plasma membrane recruitment element can comprise a PH domain of human MAPKAP1 or a mutant thereof.

[0162] The plasma membrane recruitment element can also include a membrane protein (e.g. , a human membrane protein), a transmembrane domain thereof, or a mutant thereof. For example, the transmembrane domain of a human protein can be a tetraspanin. In some cases, the plasma membrane recruitment element comprises a transmembrane domain of human CD9 or a mutant thereof. In some cases, the plasma membrane recruitment element comprises a transmembrane domain of human CD47 or a mutant thereof. In some cases, the plasma membrane recruitment element comprises a transmembrane domain of human CD63 or a mutant thereof. In some cases, the plasma membrane recruitment element comprises a transmembrane domain of human CD81.

[0163] The plasma membrane recruitment element can comprise a retroviral gag or a 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 virus (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 mutant thereof. The plasma membrane recruitment element can include a gag of MLV or a mutant thereof. The plasma membrane recruitment element can include a gag of RSV or a mutant thereof. The plasma membrane recruitment element can include a gag of Friend murine leukemia virus (FMLV) or mutant thereof.

[0164] In some cases, the plasma membrane recruitment element includes those described in Table 3 with a further truncation on the N-terminus. For example, for those 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 3 with a further truncation on the C-terminus. In some cases, the plasma membrane recruitment element includes those described in Table 3 with one amino acid substitution. In some cases, the plasma membrane recruitment element includes those described in Table 3 with two or more amino acid substitutions. In some cases, the plasma membrane recruitment element includes those described in Table 3 and a heterologous peptide sequence fused to the N-terminal or C-terminal.

[0165] 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 3. 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: 1-48. 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: 1-48. 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: 1-48 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: 1-48. 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: 1-48. 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 any one of SEQ ID NOs: 1-48. 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: 1-48. 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: 1-48. 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: 1-48. 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: 1-48. 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: 1-48. 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: 1-48. 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: 1-48 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: 1-48.

Table 3. Exemplary plasma membrane recruitment elements and their sequences

[0166] *hGAGK con is a consensus sequence derived 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, HERVK108, HERV-K109, HERV-K115, HERV- KI lp22, and HERV-K12ql3.

CHIMERIC PROTEIN

[0167] In aspects, disclosed herein are chimeric proteins that are suitable for assembly of a payload into a lipid delivery particle, e.g., lipid delivery particle, and delivery of the payload into a cell. 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 payload 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. In aspects, disclosed herein are chimeric proteins that are suitable for assembly of a payload into a lipid delivery particle described herein (e.g., heVLPs), and delivery of the payload into a cell.

[0168] 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 include a structural protein. The structural protein can comprise a plasma membrane recruitment element or polypeptide (e.g. , retroviral gag protein). The plasma membrane recruitment element can be fused to a payload (e.g., a prime editor or a recombinase). 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 prime editor. In some cases, the payload further comprises a guide nucleic acid molecule that forms a ribonucleoprotein complex with the prime editor. In some cases, the chimeric protein comprises a payload that comprises a recombinase. In some cases, the chimeric protein is suitable for delivery by a lipid delivery particle disclosed herein.

[0169] 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. 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 payload is present as a "free" entity separate from the plasma membrane recruitment element. For instance, the payload can be free and present within an inside of the protein core of the lipid delivery 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.

[0170] 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 protein-protein chimeras generated from a single reading frame. In some cases, nucleic acids base pairing to 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, aflfibodies, DmrA/DmrB/DmrC, FKBP/FRB, dDZFs, Leucine zippers, proteins that bind to DNA and/or RNA, optogenetic 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

7. 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: 478-513.

Table 7. Exemplary RNA binding proteins (RBP) and corresponding RBP binding sequences

[0171] In some cases, a chimeric protein can comprise a recombinase. In some cases, a chimeric protein can comprise a recombinase and a plasma membrane recruitment element or polypeptide (e.g. , retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain) described herein. In some embodiments, a chimeric protein comprises a recombinase linked to the plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain) via a cleavable linker. In some embodiments, the recombinase is directly coupled to the plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain).

[0172] In some cases, a chimeric protein can comprise a prime editor. In some cases, a chimeric protein can comprise a prime editor and a plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain) described herein. In some embodiments, a chimeric protein comprises a prime editor linked to the plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain) via a linker. The linker can be a cleavable linker or a non- cleavable linker. In some embodiments, the prime editor is directly coupled to the plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain).

[0173] In other embodiments, the chimeric protein comprises a recombinase, a plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain), and a prime editor. In some cases, the recombinase is directly fused to the prime editor. In some cases, the recombinase is linked to the prime editor via a linker. In some cases, the recombinase is linked to the prime editor via a non-cleavable linker. In some cases, the recombinase is linked to the prime editor via a cleavable linker. In some cases, the recombinase, the prime editor, and the plasma membrane recruitment element or polypeptide (e.g. , retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain) are linked from N-terminus to C-terminus directly or operatively via a linker. In some cases, the prime editor, the recombinase, and the plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain) are linked from N-terminus to C-terminus directly or operatively via a linker. In some cases, the plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain), the prime editor, and the recombinase are linked from N-terminus to C-terminus directly or operatively via a linker. In some cases, the plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain), the recombinase and the prime editor are linked from N-terminus to C-terminus directly or operatively via a linker. In some cases, the recombinase, the plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain) and the prime editor are linked from N-terminus to C-terminus directly or operatively via a linker. In some cases, the prime editor, the plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain) and the recombinase are linked from N-terminus to C-terminus directly or operatively via a linker. The linker can be cleavable linker or non-cleavable linker.

[0174] In other embodiments, the chimeric protein comprises a second payload (e.g., a recombinase), a plasma membrane recruitment element or polypeptide (e.g. , retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain), and a first payload (e.g., a prime editor). In some cases, the second payload (e.g. , a recombinase) is directly fused to the first payload (e.g. , a prime editor). In some cases, the second payload (e.g. , a recombinase) is linked to the first payload (e.g. , a prime editor) via a linker. In some cases, the second payload (e.g., a recombinase) is linked to the first payload (e.g. , a prime editor) via a non-cleavable linker. In some cases, the second payload (e.g. , a recombinase) is linked to the first payload (e.g., a prime editor) via a cleavable linker. In some cases, the second payload (e.g. , a recombinase), the first payload (e.g. , a prime editor), and the plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain) are linked from N-terminus to C-terminus directly or operatively via a linker. In some cases, the first payload (e.g., a prime editor), the second payload (e.g., a recombinase), and the plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain) are linked from N-terminus to C- terminus directly or operatively via a linker. In some cases, the plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain), the first payload (e.g., a prime editor), and the second payload (e.g., a recombinase) are linked from N-terminus to C-terminus directly or operatively via a linker. In some cases, the plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain), the second payload (e.g., a recombinase) and the first payload (e.g., a prime editor) are linked from N-terminus to C-terminus directly or operatively via a linker. In some cases, the second payload (e.g., a recombinase), the plasma membrane recruitment element or polypeptide (e.g., retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain) and the first payload (e.g., a prime editor) are linked from N-terminus to C- terminus directly or operatively via a linker. In some cases, the first payload (e.g., a prime editor), the plasma membrane recruitment element or polypeptide (e.g. , retroviral gag protein, human endogenous retroviral gag protein, or a pleckstrin homology domain) and the second payload (e.g., a recombinase) are linked from N-terminus to C-terminus directly or operatively via a linker. The linker can be cleavable linker or non-cleavable linker.

[0175] In some cases, a second payload (e.g., a recombinase) or a chimeric protein comprising a second payload (e.g., a recombinase) can be delivered by a lipid delivery particle that also contains a first payload (e.g., a prime editor) or a chimeric protein comprising a first payload (e.g., a prime editor). In other cases, a second payload (e.g., a recombinase) or a chimeric protein comprising a second payload (e.g., a recombinase) can be delivered by a lipid delivery particle that does not contain a first payload (e.g. , a prime editor) or a chimeric protein comprising a first payload (e.g. , a prime editor).

[0176] In some cases, a recombinase or a chimeric protein comprising a recombinase can be delivered by a lipid delivery particle that also contains a prime editor or a chimeric protein comprising a prime editor. In other cases, a recombinase or a chimeric protein comprising a recombinase can be delivered by a lipid delivery particle that does not contain a prime editor or a chimeric protein comprising a prime editor.

Nuclear Export Signal

[0177] Direction of nuclear transport within the cell can be governed by nuclear targeting signals within payload proteins. As used herein, the term “nuclear export signal” refers to a sequence of amino acids that targets a payload 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.

[0178] 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 Table 1-A. 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 Table 1-A. 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 set forth in SEQ ID NOs: 353-453. In some cases, the NES sequence described herein comprises a sequence with greater than 80% sequence identity to any sequence listed in Table 1- A. 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;31(l):393-396; and Xu D, et a . Mol Biol Cell. 2012 Sep;23(18):3673-6, each of which is incorporated herein by reference in its entirety. Any of the NES sequences described in the NES sequence 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.

[0179] In some cases, a chimeric protein disclosed herein include a nuclear export sequence (NES). In some cases, the NES facilitates localization of the chimeric protein in the cytosol of a target cell relative to the nucleus.

[0180] 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 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.

[0181] In some cases, a payload is a protein (e.g. , a recombinase, a prime editor) 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 near the one or both 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 positioned at 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 payload protein sequence. In some cases, an NES sequence is positioned at the N-terminus and an NES sequence is positioned at the C-terminus of the payload protein 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 payload protein.

[0182] In some cases, the NES sequence that can be 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 disclosed herein comprises a sequence such as those described in T la Cour, et al., Nucleic Acids Res. 2003;31 ( 1 ) : 393 - 396; and Xu D, et al. Mol Biol Cell. 2012 Sep;23(18):3673-6, each of which is incorporated herein by reference in its entirety. Any of the NES sequences described in the NES sequence database (NESdb°; prodata.swmed.edu/LRNes) or (NESbase; services.healthtech.dtu.dk/datasets/NESbase-1.0) can be used in a fusion protein disclosed herein, e.g., for the purpose of packaging a payload protein into the molecular assembly, e.g., the viral -like particle.

[0183] In eukaryotic cells, transport of proteins between the nucleus and the cytoplasm can be mediated by transport factors in the karyopherin- 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.

Table 1-A Exemplary NES sequences

Nuclear Localization Signal

[0184] 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 that targets a payload (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, 6 NLSs, 7 NLSs, 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, a NLS 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 1-B 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 1-B. In some cases, the NLS sequence described herein can comprise a sequence with greater than 80% sequence identity to any sequence listed in Table 1-B. 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 set forth in SEQ ID NOs: 454-477

[0185] 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.

[0186] 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; the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK; the c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence RMRIZFKNKGKDT AELRRRRVE V S VELRK AKKDEQILKRRN V of the IBB domain from importin-alpha; the sequences VSRKRPRP and PPKKARED of the myoma T protein; the sequence PQPKKKPL of human p53; the sequence SALIKKKKKMAP of mouse c-abl IV; the sequences DRLRR and PKQKKRK of the influenza virus NS 1; the sequence RKLKKKIKKL of the Hepatitis virus delta antigen; the sequence REKKKFLKRR of the mouse Mxl protein; the sequence KRKGDE VDGVDEV AKKKS KK of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK 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 .

[0187] Other examples of an NLS sequence include KRTADGSEFESPKKKRKV, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, and MDSLLMNRRKFLY QFKNVRWAKGRRETYLC, SPKKKRKVEAS, AGCCCCAAGAAgAAGAGaAAGGTGGAGGCCAGC, GPKKKRKVAAA, 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.

[0188] 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.

Table 1-B Exemplary NLS sequences

[0189] 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 payload 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. The cleavable linker sequence provided herein can be a cleavable sequence that is recognized and cleaved by 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, e.g., pro derived from Moloney murine leukemia virus (MMLV) or Friend murine leukemia virus (FMLV)). Examples of cleavable linker sequences that can be used in the chimeric protein include TSTLLMENSS, PRSSLYPALTP, VQALVLTQ, and PLQVLTLNIERR, and sequences having at least 80% identity to the foregoing.

[0190] 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, (GGS)x, (GGGGS)x, (GGSG)x, and (SGGG)x, and wherein x is an integer from 1 to 50.

[0191] In some cases, the linker is a P2A linker, PAPAP linker, a (EAAK)s linker, or a XTEN linker (comprising a sequence of SGSETPGTSESATPES). In some cases, the linker is EAAAK. In some cases, the linker is GGASPAGG. In some cases, the linker is A(EAAK)4ALEA(EAAK)4A. In some cases, the linker is GGASPAAPAPAG. In some cases, the linker is AHHSEDPGGGGSGGGGSGGGGS. In some cases, the linker is EAAAKGGGSEAAAK. In some cases, the linker is GGGSEAAKGGGS. In some cases, the linker is SGGSSGGSSGSETPGTSEATPESSGGSSGGSST. In some cases, the linker comprises an NLS. In some cases, the linker is SGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGS. In some cases, the linker is a flexible linker. Additional suitable linkers are described in International Publication No. WO2023205744, which is incorporated herein by reference in its entirety.

[0192] In some cases, the chimeric protein has one of the following configurations of components positioned in an order from N-terminus to C-terminus:

[plasma membrane recruitment element]-[n * NES] -[cleavable linker]-[mi * NLS]-[payload protein]-[m2 * NLS];

[plasma membrane recruitment element]-[cleavable linker]-[mi * NLS]-[payload protein]-[m2 * NLS]-[n * NES]; [plasma membrane recruitment element]-[cleavable linker l]-[mi * NLS]-[payload protein]-] -[m2 * NLS]-[cleavable linker 2]-[n * NES]; and

[plasma membrane recruitment element]-[cleavable linker l]-[mi * NLS]-[payload protein]-[m2 * NLS]; [mi * NLS]-[payload protein]-[m2 * NLS] -[cleavable linker]-[n * NES] -[plasma membrane recruitment element];

[n * NES]-[mi * NLS]-[payload protein]-[m2 * NLS] -[cleavable linker] -[plasma membrane recruitment element];

[n * NES] -[cleavable linker l]-[mi * NLS]-[payload protein]-[m2 * NLS] -[cleavable linker 2]-[plasma membrane recruitment element]; and

[mi * NLS]-[payload protein]-[m2 * NLS] -[cleavable linker] -[plasma membrane recruitment element]; wherein n, mi, and m2 are integers in the range of from 0 to 10, respectively, and denote the number of repeats of the respective sequences they refer to. 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.

Cleavable Linker and Protease

[0193] 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 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 virus (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 PLQVLTLNIERR, as well as variant sequences having at least 80% identity to the foregoing sequences. [0194] In some cases, the cleavable linker is ASPRAGGK that can be recognized by granzyme A. In some cases, the cleavable linker is YEADSLEE that can be recognized by granzyme B. In some cases, the cleavable linker is Y QYRAL that can be recognized by granzyme K. In some cases, the cleavable linker is LGVLIV that can be recognized by Cathepsin D.

PRIME EDITING SYSTEM

[0195] In one aspect, 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. The prime editing system can be delivered to a cell by the lipid delivery particles disclosed herein and can perform the first step of a two-step targeted genome modification process described in the present disclosure. The first step can comprise introducing a first recombinase recognition sequence into a target nucleic acid molecule by the prime editing system. The two-step targeted genome modification process can include targeted genome insertion, targeted genome deletion, and targeted genome inversion. In some cases, the two-step targeted modification is free of double-strand DNA breaks.

[0196] 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 nucleic acid-guided polypeptide 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.

[0197] 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 nucleic acid- guided polypeptide 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 linked to 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. [0198] 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, e.g., inserting a recombinase recognition sequence. In some cases, the prime editing system can function through a multi-step process: 1) the Cas9 domain can bind and nick the 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 containing the edited DNA sequence (e.g., a recombinase recognition 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 heteroduplex using the edited strand as a template for repair, which completes this editing process.

[0199] 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 (e.g, insertion of a recombinase recognition sequence), 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 (edited DNA, e.g, a recombinase recognition sequence) into the locus.

[0200] In some cases, a lipid delivery particle described herein can comprise a chimeric protein that forms part of a protein core. The chimeric protein can comprise a prime editor. The chimeric protein can comprise a structural protein (e.g., Plasma membrane recruitment element). The chimeric protein can comprise a cleavable linker that connects the prime editor and the structural protein. In some cases, a lipid delivery particle described herein can comprise at least two chimeric proteins. In some cases, a lipid delivery particle described herein can comprise at a first chimeric protein and a second chimeric protein. In some cases, the first chimeric protein comprises a first prime editor and a first structural protein. In some cases, the second chimeric protein comprises a second prime editor and a second structural protein. In some cases, the second prime editor has the same sequence as the first prime editor. In some cases, the second structural protein has the same sequence as the first structural protein. In some cases, a lipid delivery particle described herein can comprise at least two prime editors. In some cases, the two prime editors have the same sequence. In some cases, the second prime editor is inside the protein core in its free from after cleaved from the second chimeric protein comprising a cleavable linker. In some cases, the second prime editor is inside the protein core in its free from after cleaved by a protease.

[0201] 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. In some cases, the prime editor comprises a reverse transcriptase (RT) fused with Cas9 H 840A nickase (Cas9n (H840A)) and a prime-editing guide RNA (pegRNA). 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(wt)] and (b) a PEgRNA encoding at least a portion of recombinase recognition sequence. 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)] and (b) a PEgRNA encoding at least a portion of recombinase recognition sequence. 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 encoding at least a portion of recombinase recognition sequence; 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 encoding at least a portion of a recombinase recognition sequence; 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 having the following N-terminus to C-terminus structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K) (W313F)]; (b) a PEgRNA encoding at least a portion of a recombinase recognition sequence; 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 having the following N-terminus to C-terminus structure: [NLS]-[Cas9(H840A)]-[linker]- [MMLV_RT(D200N)(T330P)(L603W)(T306K) (W313F)]; (b) a PEgRNA encoding at least a portion of a recombinase recognition sequence; (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: MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIVKEGGLKLIQIQDNGT GIRKEDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTADGKCAYRASYSD GKLKAPPKPCAGNQGTQITVEDLFYNIATRRKALKNPSEEYGKILEVVGRYSVHNAGISFSVKKQ GETVADVRTLPNASTVDNIRSIFGNAVSRELIEIGCEDKTLAFKMNGYISNANYSVKKCIFLLFINH RLVESTSLRKAIETVYAAYLPKNTHPFLYLSLEISPQNVDVNVHPTKHEVHFLHEESILERVQQHI ESKLLGSNSSRMYFTQTLLPGLAGPSGEMVKSTTSLTSSSTSGSSDKVYAHQMVRTDSREQKLD AFLQPLSKPLSSQPQAIVTEDKTDISSGRARQQDEEMLELPAPAEVAAKNQSLEGDTTKGTSEMS EKRGPTSSNPRKRHREDSDVEMVEDDSRKEMTAACTPRRRIINLTSVLSLQEEINEQGHEVLREM LHNHSFVGCVNPQWALAQHQTKLYLLNTTKLSEELFYQILIYDFANFGVLRLSEPAPLFDLAMLA LDSPESGWTEEDGPKEGLAEYIVEFLKKKAEMLADYFSLEIDEEGNLIGLPLLIDNYVPPLEGLPIF ILRLATEVNWDEEKECFESLSKECAMFYSIRKQYISEESTLSGQQSEVPGSIPNSWKWTVEHIVYK ALRSHILPPKHFTEDGNILQLANLPDLYKVF (SEQ ID NO: 514). In some cases, other strategies for evading MMR 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.

[0202] 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, including DNA replication. The flap endonuclease includes those described in Patel et al., Nucleic Acids Research, 2012, 40(10): 4507-4519 and Tsutakawa et al., Cell, 2011, 145(2): 198-211, each of which is incorporated herein by reference in its entirety.

[0203] 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.

Nucleic acid-guided polypeptide

[0204] A prime editor disclosed herein can comprise a nucleic acid-guided polypeptide. The nucleic acid-guided polypeptide can be any suitable nuclease. The nucleic acid-guided polypeptide can be an engineered nuclease that is functionally equivalent to any suitable nuclease. The term "functional equivalent" refers to a second biomolecule that is equivalent in function, but not necessarily equivalent in structure to a first biomolecule. A "functional equivalent" of a protein (e.g., a Cas nuclease) embraces any homolog, paralog, fragment, naturally occurring, engineered, mutated, or synthetic version of the protein (e.g., a Cas nuclease) which bears an equivalent function.

[0205] 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, type IV 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, Cas 13b, Cas 13c, Cas 13d).

[0206] In some embodiments, the nuclease is 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; Gameau, J. E., et al, “The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA,” Nature (2010) 468:67-71 ; Sapranauskas, R., et al. “The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli,” Nucleic Acids Res (2011) 39: 9275-9282).

[0207] 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-E2 86; linek, M., et al, “A programmable dual -RNA-guided DNA endonuclease in adaptive bacterial immunity,” Science (2012) 337:816-821; Sternberg, S. H., et al, “DNA interrogation by the CRISPR RNA-guided endonuclease Cas9,” Nature (2014) 507:62; Deltcheva, E., et al, “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III,” Nature (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 engineering using 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).

[0208] 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 encoded by 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.

[0209] 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 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), which is able to form a complex with a Cas protein.

[0210] 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. et al. “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, atype 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., II, 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.

[0211] A Cas protein can be a Class 1 or a Class 2 Cas protein. A Cas protein can be a type I, type II, type III, type IV, type V, or type VI Cas protein. 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, protein-protein 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.

[0212] Examples of Cas proteins that can be used as part of the prime editor 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, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, Casl4, CaslO, CaslOd, CasF, CasG, CasH, Casl2a (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, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CasX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof. Examples of mutant Cas9 proteins or Cas9 variants include SpG, SpRY, eSpCas9(l.l), SpCas9-HFl, nSpCas9, SpCas9(H840A), dSpCas9, SpCas9(N863A), SpCas9(D839A), SpCas9(H983A), as well as others described in Chuang CK et al., Int JMol Sci. 2021 Sep 13;22(18):9872, which is incorporated herein by reference in its entirety.

[0213] Another example of a Cas protein that can be used as part of the prime editor includes Cas 14. A Cas 14 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 Casl4 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 (e.g., the Casl4 protein is a variant Casl4 protein, a chimeric protein, and the like). A Casl4 protein includes 3 partial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains) that are not contiguous with respect to the primary amino acid sequence of the Casl4 protein but form a RuvC domain once the protein is produced and folds. A naturally occurring Casl4 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 Casl4 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 Casl4 protein. Examples of Casl4 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 Casl4 polypeptide or a nucleic acid molecule encoding Casl4 polypeptide. In some cases, the donor sequence disclosed herein encodes Casl4a polypeptide. In some cases, the donor sequence disclosed herein encodes Casl4b polypeptide. In some cases, the donor sequence disclosed herein encodes Casl4c polypeptide.

[0214] 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, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sihiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, Leptotrichia wadeii, Leptotrichia wadeii F0279, Rhodobacter capsulatus SB1003, Rhodobacter capsulatus R121, Rhodobacter capsulatus DE442, Lachnospiraceae bacterium NK4A179, Lachnospiraceae bacterium MA2020, Clostridium aminophilum DSM 10710, Paludibacter propionicigenes WB4, Carnobacterium gallinarum DMS4847, Camobacterium 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).

[0215] A Cas protein can be derived from a variety of bacterial species including Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Listeria seeligeri. Listeria weihenstephanensis FSL R90317, Listeria weihenstephanensis FSL M60635, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp. Torquens, Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp. Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobacter eiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella succinogenes, Campylobacter jejuni subsp. Jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.

[0216] 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. [0217] 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).

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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 acidcleaving 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. [0227] 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.

[0228] 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).

[0229] 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 domains is deleted or mutated, the resulting Cas protein, known as a nickase, can generate a single-strand break at a CRISPR RNA (crRNA) recognition sequence within a doublestranded DNA but not a double-strand break. Such a nickase can cleave the complementary strand or the non-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 Cpfl 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 D10A (aspartate to alanine at position 10 of Cas9) mutation in the RuvC domain and 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.

[0230] 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 wildtype Cas protein. The mutation can result in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the complementary strand of the target nucleic acid but reducing its ability to cleave the non-complementary strand of the target nucleic acid. The mutation can result in one or more of the plurality of nucleic acid-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 the target nucleic acid. The mutation 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 Asp 10, 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.

[0231] As examples, residues D10, 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.

[0232] 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 H840A mutation can be combined with one or more of D10A, N854A, or N856A mutations to produce a site- directed polypeptide substantially lacking DNA cleavage activity. A N854A mutation can be combined with one or more of H840A, D10A, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity. A N856A mutation can be combined with one or more of H840A, N854A, or D10A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity.

[0233] 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.

[0234] 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 from .S', 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 amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof.

[0235] In some embodiments, a nucleic acid-guided polypeptide is a Cas9 or variant thereof. In some embodiments, the Cas9 or variant thereof is a nuclease active Cas9 domain, a nuclease inactive Cas9 domain, or a Cas9 nickase domain or a variant thereof. In some embodiments, a nucleic acid-guided polypeptide is Cas9, Casl2e, Casl2d, Casl2a, Casl2bl, Casl3a, Casl2c, or Argonaute (Ago domain), any of which optionally has a nickase activity. In some embodiments, a nucleic acid-guided polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 99% identical to any one of sequences listed in Table 4-A below. In some embodiments, a nucleic acid-guided polypeptide 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: 318-338. In some cases, a nucleic acid-guided polypeptide is a Cas9 H840A nickase. In some cases, a nucleic acid-guided polypeptide is Cas9 D10A nickase. Cas9-H840A. In some cases, a nucleic acid-guided polypeptide is a Casl2a/b nickase.

Table 4-A. Exemplary nucleic acid-guided polypeptide sequences

[0236] In some cases, the priming editing system delivered by the lipid delivery particles of the present disclosure use a chimeric protein, comprising a nucleic acid-guided polypeptide and a nucleic acid polymerase (e.g., a reverse transcriptase or an RNA-dependent DNA polymerase), and a guide nucleic acid molecule. In some cases, the chimeric protein comprises a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme. In some cases, the guide nucleic acid molecule comprises a prime editing guide RNA (PEgRNA), capable of identifying the target site and providing the new genetic information to replace the target DNA nucleotides. The prime editing system disclosed herein can mediate targeted insertions, deletions, and/or base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates.

[0237] One or more components of a prime editing system that can be delivered by the lipid delivery particles of the present disclosure include those described in International Patent Publication Nos. WO2020191242, WO2020191234, W02020086908, WO2021072328, WO2021226558, and WO2020191248, and Anzalone AV, et al. Nature. 2019 Dec;576(7785): 149-157; Anzalone AV, et al. Nat Biotechnol. 2021 Dec 9; Hsu JY, et al. Nat Commun. 2021 Feb 15; 12(1): 1034; Nelson JW, et al. Nat Biotechnol. 2021 Oct 4; Chen PJ, et al. Cell. 2021 Oct 28;184(22):5635-5652.e29; Scholefield J, et al. Gene Ther. 2021 Aug;28(7-8):396-401; Newby GA, et al. Mol Ther. 2021 Nov 3;29(11):3107-3124, each of which is incorporated herein by reference in its entirety.

Nucleic acid polymerase

[0238] A prime editor can comprise a nucleic acid polymerase. In some cases, the nucleic acid polymerase is coupled to the nucleic acid-guided polypeptide. In some cases, the nucleic acid polymerase is directly linked to the nucleic acid-guided polypeptide. In some cases, the nucleic acid polymerase is operably linked to the nucleic acid-guided polypeptide via a linker. In some cases, the nucleic acid polymerase comprises a reverse transcriptase or a polymerase. In some cases, the nucleic acid polymerase comprises a DNA polymerase. The DNA polymerase can be any suitable DNA polymerase that can work with the prime editing system, such as a reverse transcriptase. While the term “reverse transcriptase” is used throughout the specification, a person having ordinary skill in the art should appreciate that any suitable DNA polymerase can be used in place of the reverse transcriptase. In various embodiments, an extension on guide nucleic acid molecule can provide template for polymerization of a replacement strand containing the edit (e.g., insertion of a recombinase recognition sequence). In some cases, such extension can be formed from RNA or DNA. In the case of an RNA extension, the polymerase of the prime editor can be an RNA-dependent DNA polymerase (such as, a reverse transcriptase). In the case of a DNA extension, the polymerase of the prime editor can be a DNA-dependent DNA polymerase.

[0239] In some cases, the nucleic acid polymerase is a reverse transcriptase. In some cases, the nucleic acid polymerase is a truncated version of a reverse transcriptase. In some cases, the truncated version of a reverse transcriptase retains at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the reverse transcriptase. In some cases, the truncation is at a C- terminal of the reverse transcriptase. In some cases, the truncation is at aN-terminal of the reverse transcriptase.

[0240] In some cases, the reverse transcriptase is moloney murine leukemia virus reverse transcriptase (M-MLV RT). In some cases, the reverse transcriptase is Friend murine leukemia virus reverse transcriptase (FMLV RT). In some cases, the reverse transcriptase is human endogenous retrovirus Kcon reverse transcriptase (HERV Kcon RT). In some cases, the reverse transcriptase is wild type M-MLV RT. M-MLV RT can comprise fingers/palm domain (residues 1-275), thumb domain (residues 276-361), connection domain (residues 362-496), and RNase H domain (residues 497-671). In some cases, the reverse transcriptase is a variant of M-MLV RT. In some cases, the reverse transcriptase is a wild type HERV Kcon RT. In some cases, the reverse transcriptase is a variant of HERV Kcon RT. Suitable reverse transcriptase can include variants with at least 80%, least 82%, at least 85%, least 88%, at least 90%, least 92%, at least 95%, least 97%, least 98%, or at least 99% sequence identity to the polymerase or fragments thereof (e.g, a truncated version of the wild-type polymerase) listed in Table 4-B. In some cases, the reverse transcriptase is a truncated version of M-MLV RT. In some cases, the truncated version of M- MLV RT lacks a portion of the RNase H domain. In some cases, the truncated version of M-MLV RT lacks at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the RNase H domain. In some cases, the truncated version of M-MLV RT lacks the RNase H domain in its entirety. In some cases, the reverse transcriptase comprises transcription xenopolymerase (RTX) or mutant thereof. In some cases, the reverse transcriptase comprises avian myeloblastosis virus reverse transcriptase (AMV-RT) or a mutant thereof. In some cases, the reverse transcriptase comprises Eubacterium rectale maturase RT (MarathonRT) or a mutant thereof. In some cases, the reverse transcriptase comprises a transcription xenopolymerase (RTX) or a mutant thereof. In some cases, the reverse transcriptase comprises a small reverse transcriptase (Tfl) or a mutant thereof.

[0241] In other cases, the nucleic acid polymerase can comprise a variant reverse transcriptase comprising at least one of the mutations selected from P5 IX, S67X, E69X, L139X, T197X, D200X, H204X, F209X, E302X, T306X, F309X, W313X, T330X, L345X, L435X, N454X, D524X, E562X, D583X, H594X, L603X, E607X, or D653X in the wild type M-MLV RT or at a corresponding amino acid position in another wild type reverse transcriptase polypeptide sequence, wherein "X" can be any amino acid. In some embodiments, the mutations comprise at least one of D200N, T300P, L603W, E69K, E607K, L139P, L435G, N454K, T306K, W313F, P51L, S67K, T197A, H204R, E302K, F309N, W313F, T330P, L435G, N454K, D524G, D583N, H594Q, and D653N. In some cases, the nucleic acid polymerase can comprise a mutant M-MLV reverse transcriptase comprising one or more of mutations selected from the group consisting of D200N, T306K, W313F, T330P, and L603W relative to the wild type.

[0242] In some cases, a reverse transcriptase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 339-344. In some cases, a reverse transcriptase comprises an amino acid sequence having at least 80% sequence identity to any one of the sequences set forth in SEQ ID NOs: 339-344. In some cases, a reverse transcriptase comprises an amino acid sequence having at least 85% sequence identity to any one of the sequences set forth in SEQ ID NOs: 339-344. In some cases, a reverse transcriptase comprises an amino acid sequence having at least 90% sequence identity to any one of the sequences set forth in SEQ ID NOs: 339-344. In some cases, a reverse transcriptase comprises an amino acid sequence having at least 95% sequence identity to any one of the sequences set forth in SEQ ID NOs: 339-344. In some cases, a reverse transcriptase comprises an amino acid sequence having 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 339-344.

Table 4-B. Exemplary nucleic acid polymerases

[0243] The prime editing system described here contemplates any publicly-available reverse transcriptase described or disclosed in any of the following U.S. patents (each of which are incorporated by reference in their entireties): U.S. Patent Nos: 10,202,658; 10,189,831; 10,150,955; 9,932,567; 9,783,791; 9,580,698; 9,534,201; and 9,458,484, and any variant thereof that can be made using known methods for installing mutations, or known methods for evolving proteins. The following references describe reverse transcriptase in art. Each of their disclosures are incorporated herein by reference in their entireties. Herzig, E., Voronin, N., Kucherenko, N. & Hizi, A. A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication. J. Virol. 89, 8119-8129 (2015); Mohr, G. et al. A Reverse Transcriptase-Casl Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition. Mol. Cell 72, 700- 714.e8 (2018); Zhao, C., Liu, F. & Pyle, A. M. An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron. RNA 24, 183-195 (2018); Zimmerly, S. & Wu, L. An Unexplored Diversity of Reverse Transcriptases in Bacteria. Microbiol Spectr 3, MDNA3-0058-2014 (2015);

Ostertag, E. M. & Kazazian Jr, H. H. Biology of Mammalian LI Retrotransposons; Annual Review of Genetics 35, 501-538 (2001); Perach, M. & Hizi, A. Catalytic Features of the Recombinant Reverse Transcriptase of Bovine Leukemia Virus Expressed in Bacteria. Virology 259, 176-189 (1999); Lim, D. et al. Crystal structure of the moloney murine leukemia virus RNase H domain. J. Virol. 80, 8379-8389 (2006); Zhao, C. & Pyle, A. M. Crystal structures of a group II intron maturase reveal a missing link in spliceosome evolution. Nature Structural & Molecular Biology 23, 558-565 (2016); Griffiths, D. J. Endogenous retroviruses in the human genome sequence. Genome Biol. 2, REVIEWS1017 (2001); Baranauskas, A. et al. Generation and characterization of new highly thermostable and processive M- MuLV reverse transcriptase variants. Protein Eng Des Sei 25, 657-668 (2012); Herschhom, A. & Hizi, A. Retroviral reverse transcriptases. Cell. Mol. Life Sci. 67, 2717^_,2747 (2010); Liu, M. et al. Reverse Transcriptase-Mediated Tropism Switching in Bordetella Bacteriophage. Science 295, 2091-2094 (2002); Nowak, E. et al. Structural analysis of monomeric retroviral reverse transcriptase in complex with an RNA/DNA hybrid; Nucleic Acids Res 41, 3874-3887 (2013); Monot, C. et al. The Specificity and Flexibility of LI Reverse Transcription Priming at Imperfect T-Tracts. PLOS Genetics 9, e 1003499 (2013); Chunwei Zheng et al. , Development of a flexible split prime editor using truncated reverse transcriptase, bioRxiv 2021.08.26.457801; Avidan, O., Meer, M. E., Oz, I. & Hizi, A. The processivity and fidelity of DNA synthesis exhibited by the reverse transcriptase of bovine leukemia virus. European Journal of Biochemistry 269, 859-867 (2002).

Guide nucleic acid molecule

[0244] In some cases, a guide nucleic acid molecule for a prime editing system is a guide RNA, 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 at least a portion of a first recombinase recognition sequence, (D) an aptamer, (E) spacer, or (F) scaffold, or any combinations thereof. In some cases, a guide RNA comprises a sequence encoding at least a portion of a first recombinase recognition 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.

[0245] 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 a primer 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 which recruits and binds to the MS2cp protein). In some cases, the PEgRNA comprises an aptamer and the prime editor further comprises an aptamer binding protein (e.g. , fused to 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 a recombinase recognition 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 recombinase recognition sequence and which is incorporated into the genomic DNA target locus through the prime editing process.

[0246] 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. In one embodiment, the new duplex sequences installed by the multi-flap prime editing systems can comprise a recombinase recognition sequence, e.g., a Bxbl recombinase attB/attP site or a Cre recombinase loxP site. In some cases, the recombinase recognition sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538.

[0247] 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 recombinase recognition 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.

[0248] In some cases, a guide RNA comprises a sequence encoding full sequence of a first recombinase recognition sequence. The guide RNA can complete the insertion of the first recombinase recognition 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 first recombinase recognition sequence with a second guide RNA that is a nicking guide RNA when delivered together with a prime editor described herein.

[0249] 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 a first recombinase recognition sequence. In some cases, the two or more guide RNAs comprise a second guide RNA encoding at least a second portion of the first recombinase recognition sequence. In some cases, the first guide RNA and the second guide RNA work in a pair and collectively encode the first recombinase recognition sequence, thereby inserting the first recombinase recognition 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 first recombinase recognition sequence have at least 6bp overlap. In some cases, the first recombinase recognition sequence is an attB site. In some cases, the first portion of the first recombinase recognition is 46 bp. In some cases, the first portion of the first recombinase recognition is 42 bp. In some cases, the first portion or the second portion of the first recombinase recognition 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 the first recombinase recognition sequence in inserted instead. The deletion can be mediated by the following steps: (a) reverse transcription of the sequence encoding the first portion of the first recombinase recognition sequence in the first guide RNA and the sequence encoding the second portion of the first recombinase recognition sequence in the second guide RNA, wherein the first and the second portion of the first recombinase recognition sequence having at least 6bp overlap, (b) annealing of the two overlapped portion of the first recombinase recognition sequence, (c) synthesis of the second strand comprising the full sequence of the first recombinase recognition 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.

Recombinase recognition sequence

[0250] The term “recombinase recognition sequence”, or equivalently as “recombinase recognition sequence” or “recombinase recognition sequence,” as used herein, refers to a nucleotide sequence target recognized by a recombinase, and which undergoes strand exchange with another DNA molecule having the recombinase recognition sequence that results in excision, integration, inversion, or exchange of DNA fragments between the recombinase recognition sequences. In various embodiments, the prime editors can install one or more recombinase recognition sequences in a target sequence, or in more than one target sequence. When more than one recombinase recognition sequence is installed by a prime editor, the recombinase recognition sequences can be installed at adjacent target sites or non-adjacent target sites (e.g., separate nucleic acid molecules or separate chromosomes). In various embodiments, installed recombinase recognition sequences can be used as “landing sites” or "landing pads" for a recombinase- mediated reaction between the genomic recombinase recognition sequence and a second recombinase recognition sequence within an exogenously supplied nucleic acid molecule, e.g., a template RNA or a sequence reverse-transcribed by the template RNA described herein. This enables the targeted integration of a desired nucleic acid molecule. In other embodiments, where two recombinase recognition sequences are inserted in adjacent regions of DNA (e.g., separated by 25-50 bp, 50-100 bp, 100-200 bp, 200-300 bp, 300-400 bp, 400-500 bp, 500-600 bp, 600-700 bp, 700-800 bp, 800-900 bp, 900-1000 bp, 1000-2000 bp, 2000-3000 bp, 3000-4000 bp, 4000-5000 bp, or more), the recombinase recognition sequences can be used for recombinase-mediated excision or inversion of the intervening sequence. In some cases, a recombinase has one corresponding recombinase recognition sequence, for example, loxP site and Cre recombinase.

[0251] In some cases, a recombinase can mediate DNA inversion, deletion, and translocation between two recombinase recognition sequences, for example, Cre recombinase and two loxP sites. In some cases, the two recombinase recognition sequences are inserted by the prime editor on the same nucleic acid molecule, which can cause a deletion or inversion of the sequence between the two recombinase recognition sequences. For example, when the two recombinase recognition sequences are inserted in the same direction flanking a sequence, the sequence can be deleted or excised. In another example, when the two recombinase recognition sequences are inserted in an opposite direction flanking a sequence, the sequence can be inverted. In some cases, the two recombinase recognition sequences are located on different nucleic acid molecules, which can cause a translocation of two sequences following the 3 ’ end of two recombinase recognition sequence. When the two or more recombinase recognition sequences are installed by prime editors on two different chromosomes, translocation of the intervening sequence can occur from a first chromosomal location to a second chromosomal locations.

[0252] In some cases, a recombinase recognizes two corresponding recombinase recognition sequences that work in pairs, for example, attP site and attB site recognized by Bxbl recombinase. In some cases, one recombinase recognition sequence (e.g., an attP or an attB site in an attB/P pair, or an attL or an attR in an attL/R pair) within a pair of the recombinase recognition sequences is inserted in DNA of a host cell, into which a lipid delivery particle described in the present disclosure is delivered. In some cases, the other recombinase recognition sequence (e.g, an attB or an attP site in an attB/P pair, or an attL or an attR in an attL/R pair) within a pair of the recombinase recognition sequences is on a template nucleic acid molecule. In some embodiments, a first recombinase recognition sequence can be inserted in DNA of a host cell and used for recombinase-mediated cassette exchange with exogenous nucleic acid molecule (e.g., a template RNA or a sequence reverse-transcribed by a template RNA) having a second recombinase recognition sequence that works in pairs with the first recombinase recognition sequence. In some cases, a first set of two recombinase recognition sequences are inserted in DNA of a host cell where a lipid delivery particle described in the present disclosure is delivered. In some cases, the two recombinase recognition sequences are the same recombinase recognition sequence within a pair of recombinase recognition sequences, (e.g., two attB sequences or two attP sequences). In some cases, the two recombinase recognition sequences are adjacent to each other, flanking a sequence to be replaced by an exogenous nucleic acid molecule (e.g., a template nucleic acid molecule. In some cases, the template nucleic acid molecule comprises a second set of two recombinase recognition sequences that work in pairs with the first set of two recombinase recognition sequences (e.g., two attP sequences or two attB sequences). In some cases, the second set of two recombinase recognition sequences are the same recombinase recognition sequence within a pair of recombinase recognition sequences, (e.g., two attP sequences or two attB sequences). In some cases, the second set of two recombinase recognition sequences are adjacent to each other on the template nucleic acid molecule, flanking a sequence to be inserted between the first two recombinase recognition sequence on the DNA of the host cell. In some cases, a recombinase (e.g., a Bxbl) mediate a DNA translocation between the DNA of the host cell that comprises the first two recombinase recognition sequences (e.g. , two attP sites) and the template nucleic acid molecule that comprises the second two recombinase recognition sequences (e.g., two attB sites). In some cases, two corresponding recombinase recognition sequences that work in pairs can comprise a left recombinase recognition sequence and a right recombinase recognition sequence after recombination has been completed, for example attL site and attR site flanking a sequence inserted by the Bxb 1 recombinase.

[0253] In other cases, a prime editor can insert two corresponding recombinase recognition sequences that work in pairs to complete a deletion of a sequence at the target site. The two corresponding recombinase recognition sequences can be a left recombinase recognition sequence and a right recombinase recognition sequence (e.g., attL site and attR site). The two corresponding recombinase recognition sequences can be a pair of recombinase recognition sequences, (e.g., attB and attP sequences). A recombinase (e.g., a Bxbl) and optionally corresponding recombination directionality factors can mediate deletion of a sequence at the target site as guided by the two corresponding recombinase recognition sequences present in the target sequence (e.g., the recombinase recognition sequences inserted via prime editing), thereby deleting a sequence in the target sequence flanked by the recombinase recognition sequences. The two corresponding recombinase recognition sequence inserted to the target sequence can be positioned in the same direction, flanking a sequence to be deleted at the target site. In another case, a prime editor can insert two corresponding recombinase recognition sequences that work in pairs to complete an inversion of a sequence at the target site. The two corresponding recombinase recognition sequences can be a left recombinase recognition sequence and a right recombinase recognition sequence (e.g., attL site and attR site). A recombinase (e.g., a Bxbl) and corresponding recombination directionality factors can mediate inversion of a sequence at the target site as guided by the two recombinase recognition sequences present in the target sequence (e.g., the recombinase recognition sequences inserted via prime editing), thereby inverting a sequence in the target sequence flanked by the recombinase recognition sequences. The two corresponding recombinase recognition sequence inserted to the target sequence can be positioned in an opposite direction, flanking a sequence to be inverted at the target site.

[0254] In some embodiments, a recombinase recognition sequence can include a Cre recombinase loxP site, a Bxbl recombinase attB (about 38 bp) and/or attP (about 50 bp) site, or a recombinase recognition sequence recognized by Hin recombinase, Gin recombinase, Tn3 recombinase, I3-six recombinase, CinH recombinase, ParA recombinase, 76 recombinase, OC31 recombinase, TP901 recombinase, TGI recombinase, pBTl recombinase, R4 recombinase, pRVl recombinase, pFCl recombinase, MR11 recombinase, Al 18 recombinase, U153 recombinase, and gp29 recombinase, FLP recombinase, R recombinase, Lambda recombinase, HK101 recombinase, HK022 recombinase, and pSAM2 recombinase. Examples of tyrosine recombinases and corresponding recombinase recognition sequences are listed in Table 5-A. Examples of serine recombinases and corresponding recombinase recognition sequences are listed in Table 5-B. Examples of serine resolvases and corresponding recombinase recognition sequences are listed in Table 5-C. Examples of tyrosine integrases and corresponding recombinase recognition sequences are listed in Table 5-D. Examples of yeast recombinases and corresponding recombinase recognition sequences are listed in Table 5-E. Examples of bacterial recombinases and corresponding recombinase recognition sequences are listed in Table 5-F. Additional exemplary recombinase recognition sequences are listed in Table 5-G. In some embodiments, a recombinase recognition sequence can include any sequences set forth in Tables 5-A to 5-G. In some embodiments, a recombinase recognition sequence includes any one of SEQ ID NOs: 105-317 or 515- 538. In some embodiments, a recombinase recognition sequence includes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to any one of SEQ ID NOs: 105-317 or 515-538. In some cases, the recombinase recognition sequence comprises a nucleic acid sequence that has at least about 80% sequence identity to a sequence set forth in any one of SEQ ID NOs: 515-538. In some cases, the recombinase recognition sequence comprises a nucleic acid sequence that has at least about 85% sequence identity to a sequence set forth in any one of SEQ ID NOs: 515-538. In some cases, the recombinase recognition sequence comprises a nucleic acid sequence that has at least about 90% sequence identity to a sequence set forth in any one of SEQ ID NOs: 515-538. In some cases, the recombinase recognition sequence comprises a nucleic acid sequence that has at least about 95% sequence identity to a sequence set forth in any one of SEQ ID NOs: 515-538. In some cases, the recombinase recognition sequence comprises a nucleic acid sequence that has at least about 96% sequence identity to a sequence set forth in any one of SEQ ID NOs: 515-538. In some cases, the recombinase recognition sequence comprises a nucleic acid sequence that has at least about 97% sequence identity to a sequence set forth in any one of SEQ ID NOs: 515-538. In some cases, the recombinase recognition sequence comprises a nucleic acid sequence that has at least about 98% sequence identity to a sequence set forth in any one of SEQ ID NOs: 515-538. In some cases, the recombinase recognition sequence comprises a nucleic acid sequence that has at least about 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 515-538. In some cases, the recombinase recognition sequence comprises a mutant AttP that have improved recombination efficiency relative to wild type AttP. In some cases, the recombinase recognition sequence comprises a truncated AttB that have varied length so that the recombination efficiency is improved over full-length AttB.

Examples of the prime editing system used in combination with a recombinase, compositions, methods of use, and systems include those described in international publication no. WO2021226558, which is incorporated herein by reference in its entirety. Additional examples of recombinase recognition sequences and recombinases include those described in U.S. Patent No. 11,572,556 and International Publication No. WO2023122764, each of which is incorporated herein by reference in its entirety.

Table 5- A. Exemplary tyrosine recombinases and corresponding recombinase recognition sequences.

Table 5-B. Exemplary serine recombinases and corresponding recombinase recognition sequence pairs.

I l l

Table 5-C. Exemplary serine resolvases and corresponding recombinase recognition sequence pairs.

Table 5-D. Exemplary recombinases and corresponding recombinase recognition sequence pairs.

Table 5-E. Exemplary yeast integrases and corresponding recombinase recognition sequences

Table 5-F. Exemplary bacterial integrases and corresponding recombinase recognition sequences.

Table 5-G. Exemplary recombinase recognition sequences

Recombinase

[0255] In some aspects, the lipid delivery particles disclosed herein can be used to deliver a payload, such as a recombinase in its free form or a chimeric protein comprising a recombinase. In some embodiments, the recombinase or a chimeric protein comprising a recombinase is within the inside cavity of the protein core of the lipid delivery particles disclosed herein. “Recombinase” as used herein refers to a group of enzymes that can facilitate site-specific recombination between defined DNA sites. Such defined DNA sites are also referred to as “recombinase recognition sequences,” which are physically separated on a single DNA molecule, or each reside on a different DNA molecule. The DNA sequences of the defined recombination sites can be identical. The DNA sequences of the defined recombination sites are not necessarily identical. A recombinase can mediate the recombination of DNA between its cognate recognition sequences. A recombinase can cause DNA excision, integration, inversion, or exchange, such as translocation between two recombinase recognition sequences. Initiation of recombination depends on protein-DNA interaction, within the group there are large number of proteins that catalyze phage integration and excision (e.g., integrase, <pC31), resolution of circular plasmids (e.g., Tn3, gamma delta, Cre, Flp), DNA inversion for expression of alternate genes (e.g, Hin, Gin, Pin), assembly of genes during development (e.g., Anabaena nitrogen fixation genes), and transposition (e.g., IS607 transposon).

[0256] In some cases, a recombinase or a chimeric protein comprising a recombinase is enclosed or packaged within an inside cavity of the protein core of the lipid delivery particle. In some cases, a recombinase or a chimeric protein comprising a recombinase can be delivered to a host cells by the lipid delivery particle.

[0257] Recombinases can include serine recombinases, for example, resolvases and invertases, or a variant thereof that is functionally equivalent or functions in a substantially similar way. Recombinases can also include tyrosine recombinase, such as integrases, or a variant thereof that is functionally equivalent or functions in a substantially similar way. Examples of serine recombinases include, Hin, Gin, Tn3, -six, CinH, ParA, y5, Bxbl, C31, TP901, TGI, cpBTl, R4, cpRVl, cpFCl, MR11, Al 18, U153, and gp29. Examples of tyrosine recombinases include Cre, FLP, R, Lambda, HK101, HK022, and pSAM2. The serine and tyrosine recombinase names stem from the conserved nucleophilic amino acid residue that the recombinase uses to attack the DNA, and which becomes covalently linked to the DNA during strand exchange. Recombinases have numerous applications, including the creation of gene knockouts/knock-ins and gene therapy applications. See, e.g. , Brown et al., “Serine recombinases as tools for genome engineering.” Methods.2011;53(4):372-9; Hirano et al., “Site-specific recombinases as tools for heterologous gene integration.” Appl. Microbiol. Biotechnol.2011; 92(2):227-39; Chavez and Calos, “Therapeutic applications of the <DC31 integrase system.” Curr. Gene Ther. 2011;11(5):375-81; Turan and Bode, “Site-specific recombinases: from tag-and-target- to tag-and- exchange-based genomic modifications.” FASEB J.2011; 25(12):4088-107; Venken and Bellen, “Genome-wide manipulations of Drosophila melanogaster with transposons, Flp recombinase, and OC31 integrase.” Methods Mol. Biol.2012; 859:203-28; Murphy, “Phage recombinases and their applications.” Adv. Virus Res.2012; 83:367-414; Zhang et al., “Conditional gene manipulation: Creating a new biological era.” J. Zhejiang Univ. Sci. B.2012; 13(7):511-24; Karpenshif and Bernstein, “From yeast to mammals: recent advances in genetic control of homologous recombination.” DNA Repair (Amst).2O12; 1;11 (10) :781 -8; the entire contents of each are hereby incorporated by reference in their entirety. The recombinases provided herein are not meant to be exclusive examples of recombinases that can be used in embodiments of the disclosure. The methods and compositions of the disclosure can be expanded by mining databases for new orthogonal recombinases or designing synthetic recombinases with defined DNA specificities (See, e.g., Groth et al., “Phage integrases: biology and applications.” J. Mol. Biol.2004; 335, 667-678; Gordley et al., “Synthesis of programmable integrases.” Proc. Natl. Acad. Sci. U S A.2009; 106, 5053-5058; the entire contents of each are hereby incorporated by reference in their entirety). Other examples of recombinases that are useful in the methods and compositions described herein are known to those of skill in the art, and any new recombinase that is discovered or generated is expected to be able to be used in the different embodiments of the disclosure.

[0258] In some embodiments, the catalytic domains of a recombinase are fused to a nuclease-inactivated RNA-programmable nuclease (e.g. , dCas9, or a fragment thereof), such that the recombinase domain does not comprise a nucleic acid binding domain or is unable to bind to a target nucleic acid (e.g. , the recombinase domain is engineered such that it does not have specific DNA binding activity). Recombinases lacking DNA binding activity and methods for engineering such are known, and include those described by Klippel et al., “Isolation and characterization of unusual gin mutants.” EMBO J.1988; 7: 3983-3989: Burke et al., “Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation. Mol Microbiol.2004; 51: 937-948; Olorunniji et al., “Synapsis and catalysis by activated Tn3 resolvase mutants.” Nucleic Acids Res.2008; 36: 7181-7191; Rowland et al., “Regulatory mutations in Sin recombinase support a structure-based model of the synaptosome.” Mol Microbiol.2009; 74: 282-298; Akopian et al., “Chimeric recombinases with designed DNA sequence recognition.” Proc Natl Acad Sci USA.2003;100: 8688-8691; Gordley et al., “Evolution of programmable zinc finger- recombinases with activity in human cells. J Mol Biol.2007; 367: 802-813; Gordley et al., “Synthesis of programmable integrases.” Proc Natl Acad Sci USA.2009;106: 5053-5058; Arnold et al., “Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity.” EMBO J. 1999; 18: 1407-1414; Gaj et al., “Structure-guided reprogramming of serine recombinase DNA sequence specificity.” Proc Natl Acad Sci USA.2011;108(2):498-503; and Proudfoot et al., “Zinc finger recombinases with adaptable DNA sequence specificity.” PLoS

One.201 l;6(4):el9537; the entire contents of each are hereby incorporated by reference. For example, serine recombinases of the resolvase-invertase group, e.g. , Tn3 and y8 resolvases and the Hin and Gin invertases, have modular structures with autonomous catalytic and DNA-binding domains (See, e.g., Grindley et al., “Mechanism of site-specific recombination.” Ann Rev Biochem.2006; 75: 567-605, the entire contents of which are incorporated by reference). The catalytic domains of these recombinases are thus amenable to being recombined with nuclease-inactivated RNA-programmable nucleases (e.g., dCas9, or a fragment thereof) as described herein, e.g. , following the isolation of ‘activated’ recombinase mutants which do not require any accessory factors (e.g., DNA binding activities) (See, e.g., Klippel et al., “Isolation and characterization of unusual gin mutants.” EMBO J.1988; 7: 3983- 3989: Burke et al., “Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation. Mol Microbiol.2004; 51: 937-948; Olorunniji et al., “Synapsis and catalysis by activated Tn3 resolvase mutants.” Nucleic Acids Res.2008; 36: 7181- 7191; Rowland et al., “Regulatory mutations in Sin recombinase support a structure-based model of the synaptosome.” Mol Microbiol.2009; 74: 282- 298; Akopian et al., “Chimeric recombinases with designed DNA sequence recognition.” Proc Natl Acad Sci USA.2003;100: 8688-8691). Additionally, many other natural serine recombinases having an N- terminal catalytic domain and a C- terminal DNA binding domain are known (e.g., phiC31 integrase, TnpX transposase, IS607 transposase), and their catalytic domains can be co-opted to engineer programmable site-specific recombinases as described herein (See, e.g., Smith et al., “Diversity in the serine recombinases.” Mol Microbiol.2002;44: 299-307, the entire contents of which are incorporated by reference). Similarly, the core catalytic domains of tyrosine recombinases (e.g., Cre, X integrase) are known, and can be similarly co-opted to engineer programmable site-specific recombinases as described herein (See, e.g., Guo et al., “Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse.” Nature.1997; 389:40-46; Hartung et al., “Cre mutants with altered DNA binding properties.” J Biol Chem 1998; 273:22884-22891; Shaikh et al., “Chimeras of the Flp and Cre recombinases: Tests of the mode of cleavage by Flp and Cre. J Mol Biol.2000; 302:27-48; Rongrong et al., “Effect of deletion mutation on the recombination activity of Cre recombinase.” Acta Biochim Pol.2005; 52:541-544; Kilbride et al., “Determinants of product topology in a hybrid Cre-Tn3 resolvase site-specific recombination system.” J Mol Biol.2006; 355: 185-195; Warren et al., “A chimeric cre recombinase with regulated directionality.” Proc Natl Acad Sci SA.2008105: 18278- 18283; Van Duyne, “Teaching Cre to follow directions.” Proc Natl Acad Sci USA.2009 Jan 6; 106(l):4-5; Numrych et al., “A comparison of the effects of single-base and triple-base changes in the integrase arm-type binding sites on the site-specific recombination of bacteriophage X.” Nucleic Acids Res.1990; 18:3953-3959; Tirumalai et al., “The recognition of core-type DNA sites by X integrase.” J Mol Biol.1998; 279:513-527; Aihara et al., “A conformational switch controls the DNA cleavage activity of X integrase.” Mol Cell.2003; 12:187- 198; Biswas et al., “A structural basis for allosteric control of DNA recombination by X integrase.” Nature.2005; 435: 1059-1066; and Warren et al., “Mutations in the amino-terminal domain of X-integrase have differential effects on integrative and excisive recombination.” Mol Microbiol.2005; 55: 1104-1112; the entire contents of each are incorporated by reference).

[0259] In some cases, the lipid delivery particle comprises one or more recombinases. In some cases, each of the one or more recombinase is independently selected from the group consisting of: Cre, Bxb 1 , FLP, Al 18, Abrogate, Airmid, Anglerfish, B2, B3, Benedict, BL3, Bob3, Bred, BxZ2, Cin, Conceptll, CreALSHG, Cre-R3M3, Doom, Dre, Fre, Gin, Hin, Hinder, HK022, ICleared, IntlO, Inti 1, Intl2, Intl3, Int3, Int4, Int8, Int9, Inti, K38, Kd, KSSJEB, LI, L5, LI, Lockley, Mariner (Himarl), Mariner (mosl), Min, Minos, MJ1 (phiFCl), MR11, Mundrea, Museum, Nigri, P22, Panto, PattyP, Peaches, phi370.1, phiBTl, phiC31, phiJoe, phiK38, phiRVl, R, Rl, R2, R3, R4, R5, RDF, Rebeuca, retrotransposases encoded by R2, Sarfire, Scowl, SCre, Severus, Sheen, Sin, SkiPole, SPBc, SprA, SV1, Switzer, Tc3, TD1-40, TGI, Theia, Tol2Tcl, TP901-1, Tre, Troube, U153, VCre, Veracruz, Vika, WB, W0, <p370.1, cpBTl, cpCl, cpC31, cpFCl, and (pRV. In some cases, the lipid delivery particle comprises two different recombinases. In some cases, the two different recombinases can mediate recombination at a different rate. In some cases, the two different recombinases are FLP and Cre. In some cases, the two different recombinases are FLP and Bxbl.

[0260] In some embodiments, a recombinase delivered by the lipid delivery particle described in the present disclosure is Bxb 1 recombinase or a mutant thereof. In some embodiments, a chimeric protein comprising a recombinase delivered by the lipid delivery particle described in the present disclosure is a Bxb 1 coupled to a plasma membrane recruitment element. In some embodiments, a chimeric protein comprising a recombinase delivered by the lipid delivery particle described in the present disclosure is a Bxbl-gag. In some embodiments, a chimeric protein comprising a recombinase delivered by the lipid delivery particle described in the present disclosure is a Bxb 1 coupled to a pleckstrin homology (PH) domain. In some cases, the Bxbl recombinase delivered by the lipid delivery particle described in the present disclosure can recognize attB recombinase recognition sequence. In some cases, the Bxbl recombinase delivered by the lipid delivery particle described in the present disclosure can recognize attP recombinase recognition sequence. In some cases, the attB and the attP are located separately on one DNA molecule. In some cases, the attB and the attP are located on two separate DNA molecules, such as a DNA molecule of a host cell receiving a lipid delivery particle described herein and a DNA molecule reverse -transcribed by a template RNA described herein (e.g., a circular template DNA encoding a therapeutic molecule described herein). In some cases, the Bxbl recombinase delivered by the lipid delivery particle described in the present disclosure can mediate a DNA recombination, such as an inversion, a deletion, or a translocation between the attP and the attB recombinase recognition sequence. [0261] In some embodiments, a recombinase delivered by the lipid delivery particle described in the present disclosure is Cre recombinase or a mutant thereof. In some embodiments, a chimeric protein comprising a recombinase delivered by the lipid delivery particle described in the present disclosure is a Cre recombinase coupled to a plasma membrane recruitment element. In some embodiments, a chimeric protein comprising a recombinase delivered by the lipid delivery particle described in the present disclosure is a Cre-gag. In some embodiments, a chimeric protein comprising a recombinase delivered by the lipid delivery particle described in the present disclosure is a Cre recombinase coupled to a pleckstrin homology (PH) domain. In some cases, the Cre recombinase delivered by the lipid delivery particle described in the present disclosure can recognize loxP recombinase recognition sequence. In some cases, the Cre recombinase delivered by the lipid delivery particle described in the present disclosure can recognize loxP recombinase recognition sequence. In some cases, two loxP sites are located separately on one DNA molecule. In some cases, two loxP sites are located on two separate DNA molecules, such as a DNA molecule of a host cell receiving a lipid delivery particle described herein and a DNA molecule reverse -transcribed by a template RNA described herein (e.g., a circular template DNA encoding a therapeutic molecule described herein). In some cases, the Cre recombinase delivered by the lipid delivery particle described in the present disclosure can mediate a DNA recombination, such as an inversion, a deletion, or a translocation between the two loxP recombinase recognition sequence sequences.

Template nucleic acid molecule

[0262] In some aspects, the lipid delivery particles disclosed herein can be used to deliver a payload, such as a template nucleic acid molecule. In some embodiments, the template nucleic acid molecule is encapsulated by the lipid containing membrane. In some embodiments, the template nucleic acid molecule is within the inside cavity of the protein core of the lipid delivery particles disclosed herein. “Template nucleic acid molecule” as used herein can refer to a nucleic acid molecule (e.g. , DNA or RNA sequences) that comprises a donor nucleic acid molecule or a donor sequence nucleic acid sequence or encodes a donor nucleic acid molecule or a donor sequence. The template nucleic acid molecule can be a DNA sequence. The template nucleic acid molecule can be an RNA sequence, or a template RNA. The template nucleic acid molecule can get packaged and/or incorporated into lipid delivery particles (e.g., VLPs, e.g., heVLPs). In some cases, the lipid delivery particles disclosed herein are capable of packaging and delivering a wide variety of template nucleic acid molecule comprising a donor nucleic acid molecule or a donor sequence or encodes a donor nucleic acid molecule or a donor sequence. In some cases, the template nucleic acid molecule is enclosed inside cavity of the protein core of the lipid delivery particles described by the present disclosure. In some embodiments, the template nucleic acid molecule packaged in and delivered by the lipid delivery particles is used as an intermediate to deliver a donor sequence to a cell.

[0263] In some embodiments, the template nucleic acid molecule is a template RNA. The template RNA disclosed herein can be derived from a retroviral RNA genome, for example, from oncoretroviruses, lentiviruses, or spumaviruses, or any other suitable source. In some embodiments, the template RNA can be derived from a lentivirus, such as human immunodeficiency virus (HIV). In some cases, the template RNA is derived from a human endogenous retrovirus. The template RNA can be a linear single strand RNA sequence. The template RNA can be a circular single strand RNA sequence.

Long terminal repeat (LTR)

[0264] In one aspect, a template nucleic acid molecule described herein comprises a long terminal repeat (LTR) sequence. In some cases, the template nucleic acid molecule comprises at least two LTR sequences. In some cases, the template nucleic acid molecule comprises at least two LTR sequences flanking a donor nucleic acid molecule or a donor sequence. In some embodiments, the template nucleic acid molecule is a DNA sequence. In some embodiments, the template nucleic acid molecule is an RNA sequence, or a template RNA.

[0265] The LTR sequence can comprise a U3 region. The LTR sequence can comprise a R region. The LTR sequence can comprise a U5 region. The LTR sequence can be a truncated version that lacks U3 region. The U3 region can comprise viral promoters. The U3 region can comprise viral enhancer elements. The U3 region can bind to transcription factors when the template RNA is delivered to a host cell by the lipid delivery particles described in the present disclosure. In some cases, the U3 region regulates the expression of the donor sequence that is flanked by the LTR sequences. The R region can include a mRNA initiation site. The R region can be used as a primer during reverse transcription. The U5 region can contain a polyadenylation signal. In some cases, the U3 region, R region, and U5 region are directly linked from a 5’ to 3’ direction. In some cases, the U3 region, R region, and U5 region are operably linked from a 5’ to 3’ direction.

[0266] In some cases, the template RNA can be reverse transcribed into a DNA sequence comprising two LTR sequences flanking a donor sequence. In some cases, the template RNA can be reverse transcribed into a sequence comprising one LTR sequence at 5’ end of a donor sequence. In some cases, the template RNA can be reverse transcribed into a sequence comprising one LTR sequence operably linked to 5’ end of a donor sequence. In some cases, the template RNA can be reverse transcribed into a sequence comprising one LTR sequence directly linked to 5’ end of a donor sequence. In some cases, the LTR sequence operably linked or directly linked at 5’ end of a donor sequence is a 5’ LTR. In some cases, the U5 region of the 5’ LTR is operably linked at the 5’ end of a donor sequence. In some cases, the U5 region of the 5’ LTR is directly linked at the 5’ end of a donor sequence.

[0267] In some cases, the template RNA encodes a sequence comprising one LTR sequence at 3 ’ end of a donor sequence. In some cases, the template RNA encodes a sequence comprising one LTR sequence operably linked at 3’ end of a donor sequence. In some cases, the template RNA encodes a sequence comprising one LTR sequence directly linked at 3’ end of a donor sequence. In some cases, the LTR sequence operably linked or directly linked at 3’ end of a donor sequence is a 3’ LTR. In some cases, the U3 region of the 3’ LTR is operably linked at the 3’ end of a donor sequence. In some cases, the U3 region of the 3’ LTR is directly linked at the 3’ end of a donor sequence.

[0268] In some cases, the template RNA encodes a sequence comprising a first LTR sequence at 5’ end of a donor sequence and a second LTR sequence at 3’ end of the donor sequence. In some cases, the template RNA encodes a sequence comprising a first LTR sequence at 3 ’ end of a donor sequence and a second LTR sequence at 5’ end of the donor sequence. In some embodiments, a first LTR sequence at 3’ end of a donor sequence is a 3 ’ LTR and a second LTR sequence at 5 ’ end of the donor sequence is a 5 ’ LTR. In some cases, the 3’ LTR and 5’ LTR have identical sequences. In some cases, the U5 region of the 5’ LTR is operably linked at the 5’ end of a donor sequence and the U3 region of the 3’ LTR is operably linked at the 3’ end of a donor sequence. In some cases, the U5 region of the 5’ LTR is directly linked at the 5’ end of a donor sequence and the U3 region of the 3’ LTR is directly linked at the 3’ end of a donor sequence.

[0269] In some embodiments, the LTR sequence comprises a double-stranded polynucleotide, such as a DNA, that has a length of at least 50 base pairs (bp), at least 100 base pairs (bp), at least 150 bp, at least 200 bp, at least 250 bp, at least 300 bp, at least 350 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1000 bp. In some embodiments, the LTR sequence comprises a double-stranded polynucleotide that has a length of about 200 bp. In some embodiments, the LTR sequence comprises a double -stranded polynucleotide that has a length of about 250 bp. In some embodiments, the LTR sequence comprises a double-stranded polynucleotide that has a length of about 300 bp. In some embodiments, the LTR sequence comprises a double -stranded polynucleotide that has a length of about 350 bp. In some embodiments, the LTR sequence comprises a double -stranded polynucleotide that has a length of about 400 bp. In some embodiments, the LTR sequence comprises a double -stranded polynucleotide that has a length of about 450 bp. In some embodiments, the LTR sequence comprises a double-stranded polynucleotide that has a length of about 500 bp. In some embodiments, the LTR sequence comprises a double-stranded polynucleotide that has a length of about 550 bp. In some embodiments, the LTR sequence comprises a double -stranded polynucleotide that has a length of about 600 bp. In some embodiments, the LTR sequence comprises a double -stranded polynucleotide that has a length of about 650 bp.

[0270] The template RNA can be reverse transcribed into a template DNA comprising the LTR. The template nucleic acid molecule can be a template DNA comprising the LTR. In some cases, the LTRs can facilitate self-circularization of a DNA molecule comprising such LTRs. In some cases, the linear DNA is circularized into a circular DNA. In some cases, the linear DNA is circularized into a circular DNA by various mechanisms available within the recipient cell receiving the lipid delivery particle. In some cases, the linear DNA comprises a 5’ LTR (long terminal repeat). In some cases, the linear DNA comprises a 3’ LTR. In some cases, the linear DNA comprises a 5’ LTR and a 3’ LTR. In some cases, the linear DNA comprises a 5’ LTR and a 3’ LTR, flanking a sequence encoding a payload. In some cases, the linear DNA further comprises a non-coding sequence comprising a promoter. In some cases, the circularization of linear DNA occurs by nonhomologous end-joining. In some cases, the nonhomologous end-joining mechanism brings together and ligates the 3’ and 5’ ends of the linear DNA. In some cases, the nonhomologous end-joining mechanism turns a linear DNA into a circular DNA with two LTRs. In some cases, the circularization of linear DNA occurs by homologous recombination via strand-invasion. In some cases, the circularization of linear DNA occurs by homologous recombination via single strand annealing. In some cases, the homologous recombination mechanism turns a linear DNA into a circular DNA with one LTR. In some cases, the circularization of linear DNA occurs by closure of intermediate products of reverse transcription. In some cases, the circularization of linear DNA occurs by autointegration. In some cases, the circular DNA comprises a LTR sequence. In some cases, the circular DNA comprises one LTR sequence. In some cases, the circular DNA comprises two LTR sequences. In some cases, the circular DNA comprises a nucleic acid sequence encoding a payload. In some cases, the nonhomologous end-joining mechanism is active in different cell cycle stages, such as, G1 and early S phase. In some cases, the homologous recombination mechanism is active in different cell cycle stages, such as, late S phase and G2. In some cases, the circular DNA comprises a non-coding sequence comprising a promoter. In some cases, the circular DNA is not integrated into genome of the cell. In some cases, the circular DNA has the advantage of posing lower immunological risk than an integrating nucleic acid molecule. In some cases, the lower immunological risk is due to low copy-number persistence. In some cases, the circular DNA is not subject to epigenetic silencing. In some cases, the circular DNA does not degrade.

[0271] In some cases, the LTR derived is from human endogenous retrovirus (ERV) family, such as ERV1, ERV3, ERV9, ERVK, ERVK3, ERVK14, MaLR, ERVL, or any other suitable source. In some embodiments, a LTR from ERV1 comprises LTR7, MER39, MER41, LTR12C. In some embodiments, a LTR from ERV3 comprises MER19C. In some embodiments, a LTR from ERVL comprises MLT2A1, MLT2B3, LTR16A. Examples of LTR sequences are listed in Table 6-A. In some embodiments, a LTR comprises a nucleic acid sequence set forth in any one of SEQ ID NOs: 345-352. In some embodiments, a LTR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in any one of SEQ ID NOs: 345-352. Exemplary configurations, sequences, functions, classifications for LTRs that can be encoded by a template RNA delivered by a lipid delivery particle disclosed herein also include those described in Thompson PJ et al. Molecular Cell 62, June 2, 2016; Havecker ER et al. Genome Biology 2004, 225.3; Krebs FC et al. Lentiviral LTR-Directed Expression, Sequence Variation, and Disease Pathogenesis. Los Alamos, NM: National Laboratory HIV Sequence Compendium; 2001; De Baar MP et al. April 2000 AIDS Research and Human Retroviruses 16(5):499-504; Gifford RJ et al. Retrovirology 15, 59 (2018); Van Beveren et al. Proc. Natl. Acad. Sci. USA 77 (1980). each of which is incorporated herein by reference in its entirety.

Table 6- A. Exemplary LTR sequences.

A second recombinase recognition sequence

[0272] In one aspect, a template RNA described herein comprises a sequence encoding a second recombinase recognition sequence. The second recombinase recognition sequence can be the same as the first recombinase recognition described in the present disclosure (e.g., a loxP site in Cre-lox system). The second recombinase recognition sequence can be different from the first recombinase recognition described in the present disclosure but work as a pair of recognition sites (e.g., attB/attP site recognized by Bxbl). The second recombinase recognition sequence can locate on a different nucleic acid molecule than the first recombinase recognition sequence. The second recombinase recognition sequence and the first recombinase recognition sequence together with a recombinase recognizing both recombinase recognition sequences can mediate a DNA recombination, e.g, inserting a portion of DNA following the second recombinase recognition sequence into the location of the first recombinase recognition sequence. In some cases, the portion of DNA inserted by the recombination comprises an LTR-flanked donor sequence.

[0273] The sequence encoding a second recombinase recognition sequence can be operably linked to a sequence encoding a donor sequence via a sequence encoding the 5’ LTR described herein. The sequence encoding a second recombinase recognition sequence can be operably linked to a sequence encoding a donor sequence via a sequence encoding the 3’ LTR described herein. The sequence encoding a second recombinase recognition sequence can be any part of the template RNA that is upstream of a 5 ’ LTR. The sequence encoding a second recombinase recognition sequence can be any part of the template RNA that is downstream of a 3 ’ LTR.

Donor nucleic acid molecule

[0274] Template nucleic acid molecules described herein, such as template RNA, can encode a donor nucleic acid molecule. Donor nucleic acid molecule can comprise donor sequence for genomic integration that encodes a therapeutic molecule. “Donor sequence” or “donor nucleic acid sequence” as used herein can refer to nucleic acid sequence, e.g., DNA or RNA, which encodes a therapeutic molecule, e.g., a therapeutic protein for therapeutic or diagnostic use. In some cases, the donor sequence can be a portion of a template RNA described herein that is packaged and/or incorporated into lipid delivery particles (e.g., VLPs, e.g., heVLPs). In addition, the donor sequence can be encoded by a portion of a template RNA described herein that is packaged and/or incorporated into lipid delivery particles (e.g., VLPs, e.g., heVLPs).

[0275] In some embodiments, the donor sequence disclosed herein comprises a polynucleotide, e.g., a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA) molecule. Examples of the nucleic acid molecules include DNA, nDNA (nuclear DNA), mtDNA (mitochondrial DNA), protein coding DNA, gene, operon, chromosome, genome, transposon, retrotransposon, viral genome, intron, exon, modified DNA, mRNA (messenger RNA), tRNA (transfer RNA), modified RNA, microRNA, siRNA (small interfering RNA), tmRNA (transfer messenger RNA), rRNA (ribosomal RNA), mtRNA (mitochondrial RNA), snRNA (small nuclear RNA), small nucleolar RNA (snoRNA), SmY RNA (mRNA trans-splicing RNA), gRNA (guide RNA), TERC (telomerase RNA component), aRNA (antisense RNA), cis-NAT (Cis-natural antisense transcript), CRISPR RNA (crRNA), IncRNA (long noncoding RNA), piRNA (pi wi -interacting RNA), shRNA (short hairpin RNA), tasiRNA (trans-acting siRNA), eRNA (enhancer RNA), satellite RNA, pcRNA (protein coding RNA), dsRNA (double stranded RNA), RNAi (interfering RNA), circRNA (circular RNA), reprogramming RNAs, aptamers, and any combination thereof. In some embodiments, the nucleic acid is a wild-type nucleic acid. In some embodiments, the nucleic acid is a mutant nucleic acid. In some embodiments, the nucleic acid is a fusion or chimera of multiple nucleic acid sequences.

[0276] In some cases, the donor sequence encoded by a portion of a template RNA described herein comprises a nucleic acid molecule. The nucleic acid molecule can have a coding sequence that encodes a protein or polypeptide described herein. For instance, the nucleic acid molecule can be delivered by the lipid delivery particle disclosed herein for the purpose of delivering a protein encoded by the nucleic acid molecule. In some cases, the nucleic acid molecule is a functional nucleic acid molecule, for instance, that nucleic acid molecule can have a non-coding sequence or a coding sequence that has biological functions other than being used as a template for protein synthesis. For instance, the nucleic acid molecule can regulate RNA splicing, regulate translation of mRNA, target genomic DNA for transcriptional regulation, or bind to a protein or organelle.

[0277] In some embodiments, the donor sequence disclosed herein encodes a polypeptide, e.g. , a nuclear transport polypeptide, a nucleic acid binding polypeptide, a reprogramming polypeptide, a DNA editing polypeptide, a DNA repair polypeptide, a DNA recombination polypeptide, atransposase polypeptide, a DNA integration polypeptide, a targeted endonuclease (e.g., a Zinc -finger nuclease (ZFN), a transcription-activator-like nuclease (TALENs), Cas9 or a homolog thereof), a recombinase, an enzyme, a structural polypeptide, a signaling polypeptide, a regulatory polypeptide, a transport polypeptide, a sensory polypeptide, a motor polypeptide, a defense polypeptide, a storage polypeptide, a transcription factor, an antibody, a cytokine, a hormone, a catabolic polypeptide, an anabolic polypeptide, a proteolytic polypeptide, a metabolic polypeptide, a kinase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, an enzyme modulator polypeptide, a protein binding polypeptide, a lipid binding polypeptide, a membrane fusion polypeptide, a cell differentiation polypeptide, an epigenetic polypeptide, a cell death polypeptide, or any combination thereof. In some embodiments, the donor sequence contained in the lipid delivery particles disclosed herein encodes a protein that targets a protein in the cell for degradation. In some cases, the donor sequence contained in the lipid delivery particles disclosed herein encodes a chimeric antigen receptor (CAR), an antibody, a T cell receptor, or a functional fragment thereof, or any combination thereof. In some cases, the donor sequence comprises a guide nucleic acid molecule described herein.

[0278] In some embodiments, the donor sequence described herein, e.g. , a DNA sequence, is edited to correct a genetic mutation. For instance, the edited nucleic acid has been edited using a gene editing technology, e.g., a guide RNA and CRISPR-Cas9/Cpfl, or using a different targeted endonuclease (e.g., Zinc-finger nucleases, transcription-activator-like nucleases (TALENs)). In some cases, the nucleic acid is synthesized in vitro. In some embodiments, the genetic mutation is linked to a disease in a subject. Examples of edits to DNA include small insertions/deletions, large deletions, gene corrections with template DNA, or large insertions of DNA. In some embodiments, gene editing is accomplished with non-homologous end joining (NHEJ) or homology directed repair (HDR). In some embodiments, the edit is a knockout. In some embodiments, the edit is a knock-in. In some embodiments, both alleles of DNA are edited. In some embodiments, a single allele is edited. In some embodiments, multiple edits are made. [0279] In some embodiments, the donor sequence includes a nucleic acid. For example, the donor sequence can comprise RNA to enhance expression of an endogenous protein, or a siRNA or miRNA that inhibits protein expression of an endogenous protein. For example, the endogenous protein can modulate structure or function in the target cells. In some embodiments, the donor sequence can include a nucleic acid encoding an engineered protein that modulates structure or function in the target cells. In some embodiments, the donor sequence is a nucleic acid that targets a transcriptional activator that modulate structure or function in the target cells.

[0280] In some embodiments, the lipid delivery particle provided herein can deliver a template RNA encoding a donor sequence that encodes dominant-negative forms of proteins in order to elicit a therapeutic effect.

[0281] In some cases, a donor sequence protein loaded in the lipid delivery particle functions to bind to another donor sequence molecule to be delivered by the lipid delivery particle. In some cases, a donor sequence protein loaded in the lipid delivery particle encodes a polypeptide that functions to bind to another donor sequence molecule to be delivered by the lipid delivery particle.

[0282] In some embodiments, the donor sequence disclosed herein encodes a mixture of proteins e.g., multiple polypeptides, such as a polyprotein component of ribonucleoprotein complexes (e.g., Cas9- gRNA complex); multiple transcription factors, multiple epigenetic factors, reprogramming factors (e.g., Oct4, Sox2, cMyc, and Klf4); and any combination thereof.

[0283] In some cases, the donor sequence disclosed herein comprises a double-stranded nucleic acid molecule, such as a DNA, that has a length of at least 50 base pairs (bp), at least 100 base pairs (bp), at least 150 bp, at least 200 bp, at least 250 bp, at least 300 bp, at least 350 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1000 bp, at least 1200 bp, at least 1400 bp, at least 1500 bp, at least 1800 bp, at least 2000 bp, at least 2500 bp, at least 3000 bp, at least 4000 bp, at least 5000 bp, at least 6000 bp, at least 8000 bp, at least 10000 bp, at least 12000 bp, at least 14000 bp, or at least 15000 bp. In some cases, the donor sequence disclosed herein comprises a double -stranded nucleic acid molecule, such as a DNA, that has a length of about 20 bp, about 30 bp, about 50 bp, about 70 bp, about 80 bp, about 100 bp, about 120 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1200 bp, about 1400 bp, about 1500 bp, about 1800 bp, about 2000 bp, about 2500 bp, about 3000 bp, about 4000 bp, about 5000 bp, about 6000 bp, about 8000 bp, about 10000 bp, about 12000 bp, about 14000 bp, or about 15000 bp.

[0284] In some cases, the donor sequence disclosed herein comprises a double-stranded polynucleotide that has a length of at least 50 nucleotides, at least 80 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, at least 900 nucleotides, at least 1000 nucleotides, at least 1200 nucleotides, at least 1400 nucleotides, at least 1500 nucleotides, at least 1800 nucleotides, at least 2000 nucleotides, at least 2500 nucleotides, at least 3000 nucleotides, at least 4000 nucleotides, at least 5000 nucleotides, at least 6000 nucleotides, at least 8000 nucleotides, at least 10000 nucleotides, at least 12000 nucleotides, at least 14000 nucleotides, or at least 15000 nucleotides. In some cases, the donor sequence disclosed herein comprises a single-stranded polynucleotide encoding a polypeptide that has a length of about 20 nucleotides, about 30 nucleotides, about 50 nucleotides, about 70 nucleotides, about 80 nucleotides, about 100 nucleotides, about 120 nucleotides, about 150 nucleotides, about 200 nucleotides, about 250 nucleotides, about 300 nucleotides, about 350 nucleotides, about 400 nucleotides, about 500 nucleotides, about 600 nucleotides, about 700 nucleotides, about 800 nucleotides, about 900 nucleotides, about 1000 nucleotides, about 1200 nucleotides, about 1400 nucleotides, about 1500 nucleotides, about 1800 nucleotides, about 2000 nucleotides, about 2500 nucleotides, about 3000 nucleotides, about 4000 nucleotides, about 5000 nucleotides, about 6000 nucleotides, about 8000 nucleotides, about 10000 nucleotides, about 12000 nucleotides, about 14000 nucleotides, or about 15000 nucleotides. [0285] In some cases, the donor sequence contained in and to be delivered by the lipid delivery particles of the present disclosure encodes a polypeptide that has a length of at least 10 amino acids (aa), at least 20 aa, at least 30 aa, at least 50 aa, at least 80 aa, at least 100 aa, at least 150 aa, at least 200 aa, at least 250 aa, at least 300 aa, at least 350 aa, at least 400 aa, at least 500 aa, at least 600 aa, at least 700 aa, at least 800 aa, at least 900 aa, at least 1000 aa, at least 1200 aa, at least 1400 aa, at least 1500 aa, at least 1800 aa, at least 2000 aa, at least 2500 aa, at least 3000 aa, at least 4000 aa, or at least 5000 aa. In some cases, the donor sequence disclosed herein comprises a polypeptide that has a length of about 20 aa, about 30 aa, about 50 aa, about 80 aa, about 100 aa, about 150 aa, about 200 aa, about 250 aa, about 300 aa, about 350 aa, about 400 aa, about 500 aa, about 600 aa, about 700 aa, about 800 aa, about 900 aa, about 1000 aa, about 1200 aa, about 1400 aa, about 1500 aa, about 1800 aa, about 2000 aa, about 2500 aa, about 3000 aa, about 4000 aa, or about 5000 aa.

[0286] In some cases, the donor sequence disclosed herein encodes a polynucleotide encoding a polypeptide that has a length of at least 20 aa, at least 30 aa, at least 50 aa, at least 80 aa, at least 100 aa, at least 150 aa, at least 200 aa, at least 250 aa, at least 300 aa, at least 350 aa, at least 400 aa, at least 500 aa, at least 600 aa, at least 700 aa, at least 800 aa, at least 900 aa, at least 1000 aa, at least 1200 aa, at least 1400 aa, at least 1500 aa, at least 1800 aa, at least 2000 aa, at least 2500 aa, at least 3000 aa, at least 4000 aa, or at least 5000 aa. In some cases, the donor sequence disclosed herein comprises a polynucleotide encoding a polypeptide that has a length of about 20 aa, about 30 aa, about 50 aa, about 80 aa, about 100 aa, about 150 aa, about 200 aa, about 250 aa, about 300 aa, about 350 aa, about 400 aa, about 500 aa, about 600 aa, about 700 aa, about 800 aa, about 900 aa, about 1000 aa, about 1200 aa, about 1400 aa, about 1500 aa, about 1800 aa, about 2000 aa, about 2500 aa, about 3000 aa, about 4000 aa, or about 5000 aa.

Additional pair of recombinase recognition sequences

[0287] The donor nucleic acid molecule can further comprise an additional pair of recombinase recognition sequences. In some cases, the lipid delivery particle comprises a recombinase and an additional recombinase that is different from the recombinase. In some cases, the recombinase mediates the recombination between the first recognition recombinase sequence inserted to the genome of a cell receiving lipid delivery particle. In some cases, the additional pair of recombinase recognition sequences comprises a third recombinase recognition sequence located at a 3 ’ end of the donor nucleic acid molecule and a fourth recombinase recognition sequence located at a 5 ’ end of the donor nucleic acid molecule. In some cases, the additional recombinase mediates the recombination between the additional pair of recombinase recognition sequences thereby allows the additional pair of recombinase recognition to self-circularize when contacted with the additional recombinase. In some cases, the additional pair of recombinase recognition sequences has a faster integration rate than the first recombinase recognition sequence and the second recombinase recognition sequence, thereby the additional pair of recombinase recognition sequences recombines prior to recombination of the first recombinase recognition sequence and the second recombinase recognition sequence in the presence of the recombinase and the additional recombinase. In some cases, the recombinase is Bxbl, and the additional recombinase is FLP. In some cases, the circularized donor nucleic acid molecule comprises the donor sequence encoding a therapeutic molecule and the second recombinase recognition sequence. In some cases, the lipid delivery particle comprises a nucleic acid molecule that comprises a first nucleic acid sequence encoding the prime editor described herein, the second nucleic acid sequence encoding a guide RNA described herein comprising a sequence encoding at least a portion of the first recombinase recognition sequence, the third nucleic acid sequence encoding the recombinase, the fourth nucleic acid sequence encoding the additional recombinase, and the donor nucleic acid sequence. In some cases, the lipid delivery particle further comprises an envelope protein (e.g., HERV envelopes described herein). In some cases, the lipid delivery particle further comprises a plasma membrane recruitment element (e.g., HERV gag or pH domains described herein). Examples of the additional pair of recombinase recognition sequence, additional recombinase, and the mechanism for the two recombinases to work together with a prime editor to complete genomic insertion of a payload include those described in International Publication No. WO2023077148, which is hereby incorporated herein by reference in its entirety.

PAYLOAD

[0288] In some aspects, the lipid delivery particles described herein (e.g. , heVLPs) can be used to deliver a payload. In some cases, the payload is a component of a prime editor described herein. In some cases, the payload is a recombinase. In some cases, the payload is a template nucleic acid molecule. In some cases, the payload is not integrated into the genome of a cell receiving the lipid delivery particle. The payload can be one or more of chemicals, e.g., combination of DNA, RNA, and protein, a combination of RNA and protein, a combination of DNA and protein, or a protein, to be delivered by the lipid delivery particle disclosed herein.

[0289] 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.

[0290] 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 acid-binding 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.

[0291] 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.

[0292] The payload can include any therapeutically or diagnostically useful protein, DNA, RNP, or combination of DNA, protein and/or RNP, or any binding partners thereof. See, e.g., US20180298359A1; US10137206; US20180339166; US5892020A; EP2134841B1; W02007020965A1. For example, payload encoding or composed of nuclease or base editor proteins or RNPs or derivatives thereof can be delivered to retinal cells for the purposes of correcting a splice site defect responsible for Leber Congenital Amaurosis type 10. In the mammalian inner ear, the delivery vehicle provided herein can deliver base editing reagents or HDR promoting payload to sensory cells such as cochlear supporting cells and hair cells for the purposes of editing b-catenin (b- catenin Ser 33 edited to Tyr, Pro, or Cys) in order to better stabilize b-catenin could help reverse hearing loss. In some cases, the payload comprises a component of a prime editor, an epigenetic editor, or an RNA editor. In some cases, the RNA editor comprises an RNA editase. In some cases, the RNA editase comprises at least a functional portion of AD ARI, ADRA2, ADRA3, or APOBEC1. Addition exemplary RNA editase is described in Kung et al, Front. Endocrinol., 18 December 2018, which is which is incorporated herein by reference in its entirety. [0293] In some cases, a lipid delivery particle can deliver more than one payload (e.g, various components of a prime editor and recombinase). Each of the payloads can independently comprise a nuclease, a ribonucleoprotein complex (optionally a base editor or a prime editor), an epigenetic editor, a restriction endonuclease (optionally a Type IIS restriction enzyme), a recombinase, a transcription factor, an antibody, a chimeric antigen receptor, a T cell receptor, an organelle, a nucleic acid molecule, a DNA, a RNA, a retrotransposon, a reverse transcriptase, an oligonucleotide, an aptazyme, an aptamer, or a ribozyme, or any combinations thereof.

Therapeutic molecule

[0294] In some aspects, the lipid delivery particles described herein (e.g., heVLPs) can be used to deliver a template nucleic acid molecule comprising a nucleic acid sequence encoding a therapeutic molecule. In some cases, the template nucleic acid molecule encodes a sequence (i.e., encoding the therapeutic molecule) that is integrated into the genome of a cell receiving the lipid delivery particle. In some cases, the lipid delivery particles de-liver a template nucleic acid molecule that comprises a nucleic acid sequence encoding a payload in a target cell. In some embodiments, the template nucleic acid molecule comprising a sequence encoding the therapeutic molecule is encapsulated in the lipid containing membrane of the lipid delivery particle. In some embodiments, the template nucleic acid molecule comprising a sequence encoding the therapeutic molecule is within the inside cavity of the protein core of the lipid delivery particle. In some cases, the therapeutic molecule encoded by the template nucleic acid molecule described herein comprises at least a functional portion of a hormone, a cytokine, a ligand, a receptor, an antibody, an enzyme, a transcription factor, a chimeric antigen receptor, a T cell receptor, an antigen, a secreted protein, an RNA or any combination thereof.

[0295] The therapeutic molecule can be encoded by the donor sequence described herein or the template DNA encoded by the template RNA described herein. In some cases, the donor sequence can be inserted into the genome of a cell receiving the lipid delivery particle described herein comprising the template RNA encoding the donor sequence, thereby allows for stable expression of the therapeutic molecule in the cell. In some cases, the therapeutic molecule comprises any therapeutically or diagnostically useful protein, DNA, RNP, or combination of DNA, protein and/or RNP.

[0296] In some embodiments, the therapeutic molecule comprises a polypeptide, e.g., enzymes, structural polypeptides, signaling polypeptides, regulatory polypeptides, transport polypeptides, sensory polypeptides, motor polypeptides, defense polypeptides, storage polypeptides, transcription factors, antibodies, cytokines, hormones, catabolic polypeptides, anabolic polypeptides, proteolytic polypeptides, metabolic polypeptides, kinases, transferases, hydrolases, lyases, isomerases, ligases, enzyme modulator polypeptides, protein binding polypeptides, lipid binding polypeptides, membrane fusion polypeptides, cell differentiation polypeptides, epigenetic polypeptides, cell death polypeptides, nuclear transport polypeptides, nucleic acid binding polypeptides, reprogramming polypeptides, DNA editing polypeptides, DNA repair polypeptides, DNA recombination polypeptides, transposase polypeptides, DNA integration polypeptides, targeted endonucleases (e.g., Zinc -finger nucleases, transcription-activator-like nucleases (TALENs), cas9 and homologs thereof), recombinases, and any combination thereof. In some embodiments the protein targets a protein in the cell for degradation. In some embodiments the protein targets a protein in the cell for degradation by localizing the protein to the proteasome. In some embodiments, the protein is a wild-type protein. In some embodiments, the protein is a mutant protein. In some embodiments the protein is a fusion or chimeric protein.

[0297] In some cases, the therapeutic molecule comprises decoy proteins for binding to dis-ease-causing target proteins; peptides or proteins for inducing endosomal escape, such as HA2; peptides or proteins for targeting the exosome to a tissue or organ or cell type of interest; antibodies, intrabodies, single chain variable fragments (scFv), affibodies, bispecific or multispecific antibodies or binders, receptors, etc; enzymes such as alpha-glucosidase and/or glucocerebrosidase for enzyme re-placement therapy; transport proteins such as NPC1 or cystinosin; peptides or proteins for optimizing the in vivo behavior of exosomes (e.g., their circulation time or immune system recognition), e.g., CD47 and/or CD55 or parts of these proteins; cytokines or chemokines; a targeting peptide or protein, such as an RVG peptide, a VSV-G peptide, a p-selectin binding peptide, or an e-selectin binding peptide; a cell-penetrating peptide (CPP) (e.g., Tat, penetratin, TP10, CADY); or tumor suppressors.

[0298] In some cases, the therapeutic molecule is a protein that is at least 1 kDa, at least 2 kDa, at least 5 kDa, at least 10 kDa, at least 15 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 35 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 100 kDa, at least 120 kDa, at least 150 kDa, at least 180 kDa, at least 200 kDa, at least 220 kDa, at least 250 kDa, at least 280 kDa, at least 300 kDa, at least 320 kDa, at least 350 kDa, at least 400 kDa, at least 500 kDa, at least 600 kDa, at least 700 kDa, at least 800 kDa, at least 900 kDa, or at least 1000 kDa. In some cases, the therapeutic molecule is a protein that is about 1 kDa, about 2 kDa, about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80 kDa, about 100 kDa, about 120 kDa, about 150 kDa, about 180 kDa, about 200 kDa, about 220 kDa, about 250 kDa, about 280 kDa, about 300 kDa, about 320 kDa, about 350 kDa, about 400 kDa, about 500 kDa, about 600 kDa, about 700 kDa, about 800 kDa, about 900 kDa, or about 1000 kDa.

[0299] In some cases, the therapeutic molecule delivered by the lipid delivery particle of the present disclosure comprises a recombinant protein. The therapeutic molecule can be a diagnostic imaging agent, such as a contrast agent. In some cases, the therapeutic molecule comprises 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, a transposon, 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, or any combination thereof. The therapeutic molecule can be a prophylactic agent. In some cases, the therapeutic molecule comprises a biomarker. The therapeutic molecule can also comprise an exogenous antigen or an enzyme. In some cases, the therapeutic molecule comprises a metabolite molecule. In some cases, the therapeutic molecule comprises a lipid molecule. In some cases, the therapeutic molecule comprises a structural protein. In some cases, the therapeutic molecule comprises a hormone or a hormonal protein.

Transcription factors

[0300] In some embodiments, the therapeutic molecule comprises a transcription factor. The transcription factor can be fused to a DNA binding domain described herein.

[0301] Examples of transcription factor can include a transcription activator or a transcription repressor domain (e.g., the Kruppel associated box (KRAB or SKD); the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), etc.); zine-finger- based artificial transcription factors (see, e.g., Sera (2009) Adv. Drug Deliv. 61:513); TALE- based artificial transcription factors (see, e.g., Liu et al. (2013) Nat. Rev. Genetics 14:781); CRISPR/Cas-based artificial transcription factors (see, e.g.. Pandelakis M, et al. Cell Syst. 2020 Jan 22; 10(1): 1-14; Martinez-Escobar, et al. Frontiers in oncology vol. 10 604948. 3 Feb. 2021), and the like.

[0302] In some cases, the transcription factor comprises a VP64 polypeptide (transcriptional activation). In some cases, the transcription factor comprises a Kriippel-associated box (KRAB) polypeptide (transcriptional repression). In some cases, the transcription factor comprises a Mad mSIN3 interaction domain (SID) polypeptide (transcriptional repression). In some cases, the transcription factor comprises an ERF repressor domain (ERD) polypeptide (transcriptional repression). For example, in some cases, the transcription factor is a transcriptional activator, where the transcriptional activator is GAL4-VP16.

Antibodies

[0303] In some embodiments, the therapeutic molecule comprises an antibody or a functional fragment thereof, or a chimeric protein that comprises an antigen-binding domain. [0304] In some embodiments, the antibody or a functional fragment thereof disclosed herein, or antigenbinding domain disclosed herein binds to an antigen associated with a disease such as a viral, bacterial, and/or parasitic infection; inflammatory and/or autoimmune disease; or neoplasm such as a cancer and/or tumor. In some embodiments, the antibody or a functional fragment thereof disclosed herein, or antigenbinding domain disclosed herein binds a tumor associated antigen (e.g. , protein or polypeptide). In some embodiments, the antibody or a functional fragment thereof disclosed herein, or antigen-binding domain disclosed herein is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or a functional derivative, variant or fragment thereof, including a Fab, a Fab', a F(ab')2, an Fc, an Fv, a scFv, minibody, a diabody, and a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived Nanobody.

[0305] In some embodiments, the antibody or a functional fragment thereof disclosed herein, or antigenbinding domain disclosed herein comprises, or is derived from, or is functional equivalent to an antibody selected from the group consisting of: 20-(74)-(74) (milatuzumab; veltuzumab), 20-2b-2b, 3F8, 74-(20)- (20) (milatuzumab; veltuzumab), 8H9, A33, AB-16B5, abagovomab, abciximab, abituzumab, ABP 494 (cetuximab biosimilar), abrilumab, ABT-700, ABT-806, Actimab-A (actinium Ac-225 lintuzumab), actoxumab, adalimumab, ADC-1013, ADCT-301, ADCT-402, adecatumumab, aducanumab, afelimomab, AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, AMG 228, AMG 820, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, APN301, APN311, apolizumab, APX003/ SIM-BD0801 (sevacizumab), APX005M, arcitumomab, ARX788, ascrinvacumab, aselizumab, ASG- 15ME, atezolizumab, atinumab, ATL101, atlizumab (also referred to as tocilizumab), atorolimumab, Avelumab, B-701, bapineuzumab, basiliximab, bavituximab, BAY1129980, BAY1187982, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, Betalutin (177Lu-tetraxetan- tetulomab), bevacizumab, BEVZ92 (bevacizumab biosimilar), bezlotoxumab, BGB -A317, BHQ880, BI 836880, BI-505, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, BIW-8962, blinatumomab, blosozumab, BMS-936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, bococizumab, brentuximab vedotin, BrevaRex, briakinumab, brodalumab, brolucizumab, brontictuzumab, C2-2b-2b, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, CBR96-doxorubicin immunoconjugate, CBT124 (bevacizumab), CC-90002, CDX-014, CDX-1401, cedelizumab, certolizumab pegol, cetuximab, CGEN- 15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch.14.18, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, CM-24, codrituzumab, coltuximab ravtansine, conatumumab, concizumab, Cotara (iodine 1-131 derlotuximab biotin), cR6261, crenezumab, DA-3111 (trastuzumab biosimilar), dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, Daratumumab Enhanze (daratumumab), Darleukin, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, Depatuxizumab, Depatuxizumab mafodotin, derlotuximab biotin, detumomab, DI-B4, dinutuximab, diridavumab, DKN-01, DMOT4039A, dorlimomab aritox, drozitumab, DS-1123, DS-8895, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, FF-21101, FGFR2 Antibody-Drug Conjugate, Fibromun, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, FPA144, fresolimumab, FS102, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, Gerilimzumab, gevokizumab, girentuximab, glembatumumab vedotin, GNR-006, GNR-011, golimumab, gomiliximab, GSK2849330, GSK2857916, GSK3174998, GSK3359609, guselkumab, Hul4.18K322A MAb, hu3S193, Hu8F4, HuL2G7, HuMab-5Bl, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, IGN002, IGN523, igovomab, IMAB362, IMAB362 (claudiximab), imalumab, IMC-CS4, IMC-D11, imciromab, imgatuzumab, IMGN529, IMMU-102 (yttrium Y-90 epratuzumab tetraxetan), IMMU-114, ImmuTune IMP701 Antagonist Antibody, INCAGN1876, inclacumab, INCSHR1210, indatuximab ravtansine, indusatumab vedotin, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, Ipafricept, IPH4102, ipilimumab, iratumumab, isatuximab, Istiratumab, itolizumab, ixekizumab, JNJ-56022473, JNJ-61610588, keliximab, KTN3379, L19IL2/L19TNF, Labetuzumab, Labetuzumab Govitecan, LAG525, lambrolizumab, lampalizumab, L-DOS47, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, Leukotuximab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, LKZ145, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, LY3164530, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, MB311, MCS-110, MEDI0562, MEDI-0639, MEDI0680, MEDI-3617, MEDI-551 (inebilizumab), MEDI-565, MEDI6469, mepolizumab, metelimumab, MGB453, MGD006/ S80880, MGD007, MGD009, MGD011, milatuzumab, Milatuzumab-SN-38, minretumomab, mirvetuximab soravtansine, mitumomab, MK-4166, MM-111, MM-151, MM-302, mogamulizumab, MOR202, MOR208, MORAb-066, morolimumab, motavizumab, moxetumomab pasudotox, muromonab- CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, namatumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, NOV-10, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, OMP-131R10, OMP-305B83, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otlertuzumab, 0X002/ MEN 1309, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pankomab, PankoMab-GEX, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PAT-SC1, PAT-SM6, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, PF-05082566 (utomilumab), PF-06647263, PF-06671008, PF-06801591, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, PRO 140, Proxinium, PSMA ADC, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, REGN1400, REGN2810/ SAR439684, reslizumab, RFM-203, RG7356, RG7386, RG7802, RG7813, RG7841, RG7876, RG7888, RG7986, rilotumumab, rinucumab, rituximab, RM-1929, R07009789, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, SAR408701, SAR566658, sarilumab, SAT 012, satumomab pendetide, SCT200, SCT400, SEA-CD40, secukinumab, seribantumab, setoxaximab, sevirumab, SGN-CD19A, SGN-CD19B, SGN-CD33A, SGN-CD70A, SGN-LIV1A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, SYD985, SYM004 (futuximab and modotuximab), Sym015, TAB08, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, Tanibirumab, taplitumomab paptox, tarextumab, TB-403, tefibazumab, Teleukin, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, TG-1303, TGN1412, Thorium-227- Epratuzumab Conjugate, ticilimumab, tigatuzumab, tildrakizumab, Tisotumab vedotin, TNX-650, tocilizumab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, TRC105, tregalizumab, tremelimumab, trevogrumab, TRPH Oi l, TRX518, TSR-042, TTI-200.7, tucotuzumab celmoleukin, tuvirumab, U3-1565, U3-1784, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, Vadastuximab Talirine, vandortuzumab vedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, VB6-845, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, YYB-101, zalutumumab, zanolimumab, zatuximab, ziralimumab, and zolimomab aritox. In certain embodiments, the ligand interacting domain binds an Fc domain of an aforementioned antibody.

[0306] In some embodiments, the antibody or a functional fragment thereof disclosed herein, or antigenbinding domain disclosed herein binds an antibody selected from the group consisting of: 20-(74)-(74) (milatuzumab; veltuzumab), 20-2b-2b, 3F8, 74-(20)-(20) (milatuzumab; veltuzumab), 8H9, A33, AB- 16B5, abagovomab, abciximab, abituzumab, ABP 494 (cetuximab biosimilar), abrilumab, ABT-700, ABT-806, Actimab-A (actinium Ac -225 lintuzumab), actoxumab, adalimumab, ADC-1013, ADCT-301, ADCT-402, adecatumumab, aducanumab, afelimomab, AFM13, afutuzumab, AGEN1884, AGS15E, AGS-16C3F, AGS67E, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, AMG 228, AMG 820, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, APN301, APN311, apolizumab, APX003/ SIM-BD0801 (sevacizumab), APX005M, arcitumomab, ARX788, ascrinvacumab, aselizumab, ASG-15ME, atezolizumab, atinumab, ATL101, atlizumab (also referred to as tocilizumab), atorolimumab, Avelumab, B-701, bapineuzumab, basiliximab, bavituximab, BAY1129980, BAY1187982, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, Betalutin (177Lu-tetraxetan-tetulomab), bevacizumab, BEVZ92 (bevacizumab biosimilar), bezlotoxumab, BGB -A317, BHQ880, BI 836880, BI-505, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, BIW-8962, blinatumomab, blosozumab, BMS- 936559, BMS-986012, BMS-986016, BMS-986148, BMS-986178, BNC101, bococizumab, brentuximab vedotin, BrevaRex, briakinumab, brodalumab, brolucizumab, brontictuzumab, C2-2b-2b, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, CBR96-doxorubicin immunoconjugate, CBT124 (bevacizumab), CC-90002, CDX-014, CDX-1401, cedelizumab, certolizumab pegol, cetuximab, CGEN-15001T, CGEN-15022, CGEN-15029, CGEN-15049, CGEN-15052, CGEN-15092, Ch.14. 18, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, CM-24, codrituzumab, coltuximab ravtansine, conatumumab, concizumab, Cotara (iodine 1-131 derlotuximab biotin), cR6261, crenezumab, DA-3111 (trastuzumab biosimilar), dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, Daratumumab Enhanze (daratumumab), Darleukin, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, Depatuxizumab, Depatuxizumab mafodotin, derlotuximab biotin, detumomab, DI-B4, dinutuximab, diridavumab, DKN-01, DMOT4039A, dorlimomab aritox, drozitumab, DS-1123, DS-8895, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, FF-21101, FGFR2 Antibody-Drug Conjugate, Fibromun, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, FPA144, fresolimumab, FS102, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, Gerilimzumab, gevokizumab, girentuximab, glembatumumab vedotin, GNR-006, GNR-011, golimumab, gomiliximab, GSK2849330, GSK2857916, GSK3174998, GSK3359609, guselkumab, Hul4.18K322A MAb, hu3S193, Hu8F4, HuL2G7, HuMab-5Bl, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, IGN002, IGN523, igovomab, IMAB362, IMAB362 (claudiximab), imalumab, IMC-CS4, IMC-D11, imciromab, imgatuzumab, IMGN529, IMMU-102 (yttrium Y-90 epratuzumab tetraxetan), IMMU-114, ImmuTune IMP701 Antagonist Antibody, INCAGN1876, inclacumab, INCSHR1210, indatuximab ravtansine, indusatumab vedotin, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, Ipafricept, IPH4102, ipilimumab, iratumumab, isatuximab, Istiratumab, itolizumab, ixekizumab, JNJ-56022473, JNJ-61610588, keliximab, KTN3379, L19IL2/L19TNF, Labetuzumab, Labetuzumab Govitecan, LAG525, lambrolizumab, lampalizumab, L- DOS47, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, Leukotuximab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, LKZ145, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, LY3164530, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, MB311, MCS-110, MEDI0562, MEDI-0639, MEDI0680, MEDI-3617, MEDI-551 (inebilizumab), MEDI-565, MEDI6469, mepolizumab, metelimumab, MGB453, MGD006/ S80880, MGD007, MGD009, MGD011, milatuzumab, Milatuzumab-SN-38, minretumomab, mirvetuximab soravtansine, mitumomab, MK-4166, MM-111, MM-151, MM-302, mogamulizumab, MOR202, MOR208, MORAb-066, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, namatumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, NOV-10, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, OMP-131R10, OMP-305B83, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otlertuzumab, 0X002/ MEN 1309, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pankomab, PankoMab- GEX, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PAT-SC1, PAT-SM6, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, PF-05082566 (utomilumab), PF-06647263, PF-06671008, PF-06801591, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, PRO 140, Proxinium, PSMA ADC, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, REGN1400, REGN2810/ SAR439684, reslizumab, RFM-203, RG7356, RG7386, RG7802, RG7813, RG7841, RG7876, RG7888, RG7986, rilotumumab, rinucumab, rituximab, RM-1929, R07009789, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, SAR408701, SAR566658, sarilumab, SAT 012, satumomab pendetide, SCT200, SCT400, SEA-CD40, secukinumab, seribantumab, setoxaximab, sevirumab, SGN-CD19A, SGN-CD19B, SGN-CD33A, SGN- CD70A, SGN-LIV1A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, SYD985, SYM004 (futuximab and modotuximab), Sym015, TAB08, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, Tanibirumab, taplitumomab paptox, tarextumab, TB-403, tefibazumab, Teleukin, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, TG-1303, TGN1412, Thorium -227-Epratuzumab Conjugate, ticilimumab, tigatuzumab, tildrakizumab, Tisotumab vedotin, TNX-650, tocilizumab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab emtansine, TRBS07, TRC105, tregalizumab, tremelimumab, trevogrumab, TRPH Oi l, TRX518, TSR-042, TTI-200.7, tucotuzumab celmoleukin, tuvirumab, U3-1565, U3-1784, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, Vadastuximab Talirine, vandortuzumab vedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, VB6-845, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, YYB-101, zalutumumab, zanolimumab, zatuximab, ziralimumab, and zolimomab aritox. In certain embodiments, the ligand interacting domain binds an Fc domain of an aforementioned antibody.

[0307] In some embodiments, the antibody or a functional fragment thereof disclosed herein, or antigenbinding domain disclosed herein binds an antigen selected from the group consisting of: 1-40- -amyloid, 4-1BB, 5AC, 5T4, activin receptor-like kinase 1, ACVR2B, adenocarcinoma antigen, AGS-22M6, alphafetoprotein, angiopoietin 2, angiopoietin 3, anthrax toxin, AOC3 (VAP-1), B7-H3, Bacillus anthracis anthrax, BAFF, beta-amyloid, B-lymphoma cell, C242 antigen, C5, CA-125, Canis lupus familiaris IL31, carbonic anhydrase 9 (CA-IX), cardiac myosin, CCL11 (eotaxin-1), CCR4, CCR5, CD11, CD18, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD25 (a chain of IL-2receptor), CD27, CD274, CD28, CD3, CD3 epsilon, CD30, CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD74, CD79B, CD80, CEA, CEA-related antigen, CFD, ch4D5, CLDN18.2, Clostridium difficile, clumping factor A, CSF1R, CSF2, CTLA-4, C-X-C chemokine receptor type 4, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, E. coli shiga toxin type-1, E. coli shiga toxin type-2, EGFL7, EGFR, endotoxin, EpCAM, episialin, ERBB3, Escherichia coli, F protein of respiratory syncytial virus, FAP, fibrin II beta chain, fibronectin extra domain-B, folate hydrolase, folate receptor 1, folate receptor alpha, Frizzled receptor, ganglioside GD2, GD2, GD3 ganglioside, glypican 3, GMCSF receptor a-chain, GPNMB, growth differentiation factor 8, GUCY2C, hemagglutinin, hepatitis B surface antigen, hepatitis B virus, HER1, HER2/neu, HER3, HGF, HHGFR, histone complex, HIV-1, HLA-DR, HNGF, Hsp90, human scatter factor receptor kinase, human TNF, human beta-amyloid, ICAM-1 (CD54), IFN-a, IFN-y, IgE, IgE Fc region, IGF-1 receptor, IGF-1, IGHE, IL 17A, IL 17F, IL 20, IL-12, IL-13, IL-17, IL-ip, IL-22, IL-23, IL-3 IRA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, ILGF2, influenza A hemagglutinin, influenza A virus hemagglutinin, insulin-like growth factor I receptor, integrin a407, integrin a4, integrin a5|31, integrin a7 07, integrin allb03, integrin av03, interferon a/0 receptor, interferon gamma-induced protein, ITGA2, ITGB2 (CD 18), KIR2D, Lewis-Y antigen, LFA-1 (CDl la), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), LTA, MCP-1, mesothelin, MIF, MS4A1, MSLN, MUC1, mucin CanAg, myelin-associated glycoprotein, myostatin, NCA-90 (granulocyte antigen), neural apoptosis-regulated proteinase 1, NGF, N-glycolylneuraminic acid, NOGO-A, Notch receptor, NRP1, Oryctolagus cuniculus, OX-40, oxLDL, PCSK9, PD-1, PDCD1, PDGF-R a, phosphate-sodium co-transporter, phosphatidylserine, platelet-derived growth factor receptor beta, prostatic carcinoma cells, Pseudomonas aeruginosa, rabies virus glycoprotein, RANKL, respiratory syncytial virus, RHD, Rhesus factor, RON, RTN4, sclerostin, SDC1, selectin P, SLAMF7, SOST, sphingosine- 1 -phosphate, Staphylococcus aureus, STEAP1, TAG-72, T-cell receptor, TEM1, tenascin C, TFPI, TGF-0 1, TGF-0 2, TGF-0, TNF-a, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, tumor specific glycosylation of MUC1, tumor-associated calcium signal transducer 2, TWEAK receptor, TYRP1 (glycoprotein 75), VEGFA, VEGFR1, VEGFR2, vimentin, and VWF.

IMMUNOMODULATOR

[0308] In some cases, the lipid delivery particle provided herein comprises one or more immunomodulators, e.g., immunosuppressive molecules, in the lipid containing membrane. For instance, in the case of viral-like particle, lipid nanoparticle, or proteo-lipid vehicle, it can have one or more immunomodulators in the lipid-based external layer (e.g., envelope of some heVLPs). In the case of exosomes, the one or more immunomodulators can be present in the lipid bilayer membrane that forms the enclosure.

[0309] The lipid containing membrane of a lipid delivery particle disclosed herein can comprise one or more immunomodulators (e.g., immunosuppressive molecules or immuno stimulatory molecules). In some embodiments, the lipid containing membrane comprises immunosuppressive molecules. The immunosuppressive molecules can be associated with the lipid containing membrane in any manner. In some embodiments, the immunosuppressive molecule is embedded within or on the lipid containing membrane. For instance, the immunosuppressive molecule can comprise, either naturally or synthetically, a transmembrane domain, which integrates into the lipid containing membrane. In some embodiments, the transmembrane domain is embedded in the lipid containing membrane and at least a portion (e.g., a functional portion) of the immunosuppressive molecule is displayed on the exterior of the lipid delivery particle. In some embodiments, the transmembrane domain spans the lipid containing membrane and at least a portion (e.g. , a functional portion) of the immunosuppressive molecule is displayed on the exterior of the lipid delivery particle. Transmembrane domains are known in the art including the PDGFR transmembrane domain, the EGFR transmembrane domain, or the murine CTLA4 transmembrane domain. In some embodiments, the transmembrane domain is any domain that efficiently traffics the immunosuppressive molecule and/or a targeting molecule to the plasma membrane of the producer cell. Methods of incorporating transmembrane domains (e.g., by generating fusion proteins) can include those known in the art.

[0310] The immunosuppressive molecule can be any molecule that reduces the host immune response (immune response from a host body when the lipid delivery particle is administered to the host) to a therapeutic agent as compared to the same agent without co-administering of the lipid delivery particle or with a lipid delivery particle that is not engineered to contain immunosuppressive molecules. The immunosuppressive molecules include molecules (e.g., proteins) that down-regulate immune function of a host by any mechanism, such as by stimulating or up-regulating immune inhibitors or by inhibiting or down-regulating immune stimulating molecules and/or activators. Immunosuppressive molecules include immune checkpoint receptors and ligands. Examples of immunosuppressive molecules include, for instance, CTLA-4 and its ligands (e.g., B7-1 and B7-2), PD-1 and its ligands (e.g., PDL-1 and PDL-2), VISTA, TIM-3 and its ligand (e.g., GAL9), TIGIT and its ligand (e.g., CD155), LAG3, VISTA, and BTLA and its ligand (e.g., HVEM). Also included are active fragments and derivatives of any of the foregoing checkpoint molecules; agonists of any of the foregoing checkpoint molecules, such as agonistic antibodies to any of the foregoing checkpoint molecules; antibodies that block immune stimulatory receptors (co-stimulatory receptors) or their ligands, such as anti-CD28 antibodies; or peptides that mimic the immune functions of immune checkpoint molecules. To the extent a desired immunosuppressive molecule does not natively include a transmembrane domain, the immunosuppressive molecules can be engineered to embed in an lipid containing membrane by creating chimeric molecules comprising an extracellular domain, a transmembrane domain, and, optionally, either full length intracellular domains, or any minimal intercellular domain that can play a role in maintaining chimeric molecule expression and binding to its ligand or receptor. The transmembrane domains and intercellular domains of effector molecules can comprise immunoglobulin Fc receptor domains (or transmembrane region thereof) or any other functional domain that can play a role in maintaining expression and ligand binding activities. In some embodiments, the immunosuppressive molecule inhibits the function of B cells. In some embodiments, the immunosuppressive molecule is an antagonist of CD40 or its ligand, CD40L (also known as CD 154). In some embodiments, the immunosuppressive molecule is an antibody that specifically binds CD40 or its ligand, CD40L (also known as CD154).

[0311] The lipid containing membrane can comprise any one or more different types of immunosuppressive molecules. In some embodiments, the lipid containing membrane comprises a combination of two or more different immunosuppressive molecules (e.g, three or more different immunosuppressive molecules, four or more different immunosuppressive molecules, or even five or more different immunosuppressive molecules). For example, in some embodiments, the lipid containing membrane comprises a combination of two or more different immune checkpoint molecules (e.g, three or more different immune checkpoint molecules, four or more different immune checkpoint molecules, or even five or more different immune checkpoint molecules), optionally two or more (e.g. , three or more, four or more, or even five or more) molecules selected from CTLA-4 and its ligands (e.g., B7-1 and B7- 2), PD-1 and its ligands (e.g, PDL-1 and PDL-2), VISTA, TIM-3 and its ligand (e.g., GAL9), TIGIT and its ligand (e.g., CD155), LAG3, VISTA, and BTLA and its ligand (e.g., HVEM); active fragments and derivatives of any of the foregoing checkpoint molecules; agonists of any of the foregoing checkpoint molecules, such as agonistic antibodies to any of the foregoing checkpoint molecules; antibodies that block immune stimulatory receptors (co stimulatory receptors) or their ligands, such as anti-CD28 antibodies; or peptides that mimic the immune functions of immune checkpoint molecules. In some embodiments, the lipid containing membrane comprises CTLA-4 and PD-L1 and PD-L2 and VISTA, or any combination of these, or other immune suppressing molecules, singly or in combinations of up to four different molecules. In some embodiments, the lipid containing membrane comprises CTLA-4 and PD- Ll, CTLA-4 and PD-L2, CTLA-4 and PD-1, CTLA-4 and VISTA, CTLA-4 and anti-CD28, PD-1 and VISTA, B7-1 and PD-L1, B7-1 and PD-L2, B7-land PD-1, B7-1 and VISTA, B7-1 and anti- CD28, B7-2 and PD-L1, B7-2 and PD-L2, B7-2and PD-1, B7-2 and VISTA, B7-2 and anti- CD28, PD-1 and VISTA, PD-1 and anti-CD-28, VISTA and anti-CD28, PD-L1 and VISTA, PD-L1 and anti-CD-28, PD-L2 and VISTA, PD-L2 and anti-CD-28, or VISTA and anti- CD28. In some embodiments, the lipid containing membrane comprises CTLA4 and PD-L1, CTLA and PD-L2 CTLA-4 and VISTA, PD-L1 and PD-L2, PD-L1 and VISTA, PD-L2 and VISTA, CTLA4 and PD-L1 and PD-L2, CTLA4 and PD-L1 and VISTA, CTLA4 and PD-L2 and VISTA, PD-L1 and PD-L2 and VISTA, or CTLA4 and PD-L1 and PD-L1 and VISTA.

[0312] In some embodiments, the immunosuppressive molecules are engineered to include a transmembrane domain. The immunosuppressive molecule used in the lipid delivery particle can be that of the species of mammal to which the lipid delivery particle is to be administered. Thus, for use in humans, the human ortholog of the immunosuppressive molecule can be used. In one embodiment, the immunosuppressive molecules included in the lipid containing membrane comprise, consist essentially of, or consist of, CTLA-4 and PD-L1. Human CTLA-4 is provided, for instance, by the protein identified by NCBI Reference Sequence: NP_005205.2, and PD-L1 is provided, for instance, by the protein identified by NCBI Reference Sequence: NP_054862.1. [0313] The lipid containing membrane of a lipid delivery particle disclosed herein can comprise the immunosuppressive molecules in any suitable amount or concentration that is functionally greater than produced by the producer cell in the absence of introduction of exogenous nucleic acids encoding the immunosuppressive molecules. In some embodiments, the lipid containing membrane comprises the immunosuppressive molecules in an amount sufficient to improve delivery and expression of the transgene encoded by a lipid delivery particle as compared to the same lipid delivery particle that is not administered in conjunction with a lipid delivery particle engineered to contain the immunosuppressive molecules. As explained in greater detail in connection with the method of producing the lipid delivery particles, the lipid delivery particles comprising sufficient concentration of immunosuppressive molecules in the lipid containing membrane can be provided by engineering the host (producer) cell to overexpress the immunosuppressive molecules as compared to the native producer cell. Thus, in some embodiments, the lipid containing membrane of the lipid delivery particles provided herein comprises one or more (or all) of the immunosuppressive molecules in an amount greater than the same lipid delivery particle produced from the same producer cell that has not been engineered to overexpress the immunosuppressive molecules. For instance, the lipid containing membrane provided herein comprises one or more (or all) of the immunosuppressive molecules in an amount greater than the same lipid delivery particle produced from the same producer cell that has not been engineered to overexpress the immunosuppressive molecules by about 2x or more, by about 3x or more, by about 5x or more, by about lOx or more, by about 20x or more, by about 50x or more, or even about lOOx or more (e.g., about lOOOx or more). In some embodiments, the producer cell is engineered to overexpress one or more (or all) of the immunosuppressive molecules by about 2x or more, about 3x or more, about 5x or more, about lOx or more, about 20x or more, about 50x or more, or even about lOOx or more (e.g. , about lOOOx or more) than the same producer cell that is not engineered to overexpress the immunosuppressive molecules. In some embodiments, the producer cell is a non-tumor producer cell engineered to overexpress the immunosuppressive molecules, and the lipid containing membrane is a non-tumor lipid delivery particle lipid containing membrane. In some embodiments, the lipid containing membrane is an external lipid bilayer (e.g., an exosomal external lipid bilayer or an lipid containing membrane of a VLP disclosed herein) from a 293 cell (e.g, HEK293 or any variation thereof, such as HEK293E, HEK293F, HEK293T, etc.) engineered to overexpress the immunosuppressive molecules.

[0314] The lipid delivery particles provided herein can further include additional moieties in the lipid containing membrane as desired to provide different functions. For instance, as discussed above, the lipid containing membrane can be engineered to contain membrane surface proteins that target the vehicle to a desired cell or tissue type, for instance, a molecule that specifically binds to a ligand or receptor on a desired cell type. By engineering the lipid delivery particles provided herein to contain lipid containing membrane -associated targeting moieties (e.g. targeting proteins) that bind to ligands or receptors on a desired cell type, the lipid delivery particles can enable more precise targeting to tolerogenic environments; for example, the liver, spleen or thymus. In some embodiments, the lipid containing membrane of the lipid delivery particle can be engineered to include a moiety that specifically or preferentially binds a surface protein expressed specifically or preferentially on liver cells (e.g., a protein, such as a membrane -bound antigen binding domain (e.g., domain of clone 8D7, BD Biosciences), that specifically binds asialoglycoprotein receptor l(ASGRl)). In some embodiments, the targeting molecules is an antibody or antigen binding fragment thereof, such as scFvs (single-chain variable fragments, composed of a fusion of the variable regions of the heavy and light chains of an immunoglobulin) or Fabs (antigen-binding fragments, composed of one constant and one variable domain from each heavy and light chain of the antibody). In some embodiments, the targeting molecule is a nanobodies: an antibody fragment consisting of a single monomeric variable antibody domain that targets specific proteins or cell types. In some embodiments, the targeting molecule is a protein, a polypeptide or a polysaccharide that specifically bind to desired targets or target cells. In some embodiments, the targeting molecule targets MHC class I or MHC class II mismatches between donor tissue and a recipient. Such targeting can be used in treating or preventing tissue rejection or graft versus host disease. Such an lipid containing membrane can be provided by engineering producer cells to express high levels of a membrane bound targeting moiety.

[0315] The lipid delivery particle can further comprise additional elements that improve effectiveness or efficiency of the lipid delivery particle, or improve production. For example, the lipid delivery particles can include CD9 in the lipid containing membrane. Exogenous expression of Tetraspanin CD9 in producer cells can improve production of lipid delivery particles (e g., VLP or exosome) without degrading their delivery performance (Shifter et al., Mol Ther Methods Clin Dev, (2018) 9:278-287). [0316] The lipid delivery particles containing immunomodulators in the lipid containing membrane that are provided herein can be produced by any suitable method. Example are provided by US 9829483B2 and US 2013/0202559, incorporated herein by reference. One particularly advantageous method involves producing the lipid delivery particles from a producer cell line that has been engineered to overexpress the immunosuppressive molecules desired to be included in the lipid containing membrane of the lipid delivery particles. In some aspects, provided herein is a method of preparing a lipid delivery particle (e.g., an exosome or a VLP disclosed herein) with an lipid containing membrane comprising immunosuppressive molecules, as described herein, by (a) culturing producer cells under conditions to generate the lipid delivery particles, wherein the producer cells comprise a nucleic acid encoding one or more one or more membrane-bound immunosuppressive molecules, and (b) collecting the lipid delivery particles.

[0317] Expression of the immunosuppressive molecules in the producer cells can be driven by a promoter, such as a constitutive promoter (e.g., a CMV promoter). In some embodiments, the gene encoding the effector molecule is followed by polyadenylation signal (e.g. , a hemoglobin polyadenylation signal) downstream of the effector molecule coding region. In some embodiments, an intron is inserted downstream of the promoter. For example, a hemoglobin derived artificial intron downstream of the promoter can be employed to increase effector molecule production. The method for transient transfections includes calcium phosphate transfection. The method to produce stable cell lines expressing single or combined immune modulators includes retroviral gene transfer or concatemer transfection followed by selection (Throm et al. (2009) Blood, 113(21): 5104- 5110). The producer cells are engineered in this way to express individual immunosuppressive molecules, or to express different combinations of immunosuppressive molecules, as can be desired in the lipid delivery particle. The producer cells also can be engineered in other ways known in the art to increase productivity. For example, the producer cells can be engineered to overexpress Tetraspanin CD9 to improve vector production (Shiller et al., (2018) Mol Ther Methods Clin Dev, 9:278-287).

PRODUCTION OF LIPID DELIVERY PARTICLES

[0318] In some aspects, provided herein are composition, methods of production, methods of purification related to the lipid delivery particles provided herein. In some cases, the lipid delivery particles can be produced from producer cell lines that are either transiently transfected with at least one plasmid or stably expressing constructs that have been integrated into the producer cell line genomic DNA. In some cases, the lipid delivery particles can be produced from contacting producer cell with a template nucleic acid molecule or a nucleic acid molecule encoding a payload (e.g., one or more components of a prime editor or a recombinase) or a chimeric protein.

[0319] Producer cell lines can be generated by stably integrating genetic material with a gene of interest into a host cell line. In some cases, the genetic material is transiently expressed in a producer cell line. In some cases, the genetic material is expressed via viral methods. In some cases, the genetic material is expressed via non-viral methods. In some cases, a producer cell line grows in a serum-free medium or in suspension. A producer cell line can be grown in serum-free medium and suspension simultaneously. In some cases, producer cell lines can be generated with adherent cells (e.g., cells cultured in media and attached to a substrate).

[0320] Producer cells can be used to produce the lipid delivery particles described herein. In some cases, generating a producer cell line comprises transfecting cells (e.g., cells of a mammalian cell type) with genetic material of the present disclosure, culturing the cells to produce the lipid delivery particles, obtaining a media from the mammalian cell producing the lipid delivery particles, collecting and filtering the harvested media, and, optionally, purifying the lipid delivery particles to retain structural integrity. In some cases, the method of producing the lipid delivery particle further comprises providing new media to promote transient production of the lipid delivery particles. In some cases, the mammalian cell type includes a HT1080 cell, a COS cell, a HeLa cell, a Chinese Hamster Ovary (CHO) cell, or a HEK 293 cell. HEK293 cells are cells derived from human embryonic kidney cells grown in tissue culture. In some cases, the HEK293 cell is a HEK293, 293E, 293T, 293F, 293FT, or 293T Gesicle cell. The producer cell line can be transformed with a viral vector or non-viral method in any number of means including calcium phosphate and the like.

[0321] Following transfection, the cells can be cultured under conditions for production of lipid delivery particles. Exemplary culturing conditions can include refeeding cells in appropriate media, addition of CO2, and humidity. In some cases, culturing conditions includes addition of antibiotics, anti-fungals, and/or growth factors. The medium can be harvested after 24, 48, 72, or 96 hours, or at any appropriate time point to allow sufficient production of the lipid delivery particles. [0322] Optionally, the lipid delivery particles in the media can be isolated and collected using any number of techniques known in the art. In some cases, the lipid delivery particles are purified, wherein the lipid delivery particles are washed or resuspended in an appropriate buffer or media or at particular concentration.

[0323] In an aspect, disclosed herein are methods of manufacturing producer cell lines that comprise the lipid delivery particles of the present disclosure. Adherent cells can be first transfected to produce lipid delivery particles. In some cases, transfection occurs by the addition or expression of exogenous nucleic acid sequences via non-viral methods (e.g., by electroporation, microinjection, or a chemical system such as DEAE-dextran or cationic polymers). In some cases, transfection occurs by the addition or expression of exogenous nucleic acid sequences via viral methods (e.g. , by infecting the cells with a viral vector, such as an adenoviral vector, adeno-associate viral vector, a lentiviral vector, a herpes viral vector, or a HSV vector). In some cases, the cells are from a HEK293 cell line (e.g., HEK293, 293E, or 293T). In some cases, to transfect DNA into the host cells, the cells are cultured in a medium. In some cases, cells can be cultured in the medium for 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 hours. In some cases, cells can be cultured in the medium for between 10-20 hours. In some cases, cells can be cultured in the medium for 18 hours.

[0324] Following incorporation into the transfection medium, cells are transferred to a new solution. In some cases, the new solution is new media. In some cases, the new media promotes the production of the lipid delivery particles. In some cases, the cells incorporate into the new media for between 10-50 hours. In some cases, the cells incorporate into the new media for 10, 20, 30, 35, 40, 45, or 50 hours. In some cases, the cells incorporate into the new media for 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 hours. The media can then be harvested. The harvested media can be filtered, and the lipid delivery particles can be collected. Filtration can comprise microfiltration and/or depth filtration. In some cases, the lipid delivery particles can undergo further purification and/or concentration methods that maintain the structural integrity of the particles.

[0325] In some aspects, provided herein is a method of loading a lipid delivery particle with components such as a payload and/or a template nucleic acid molecule. RNA and protein from a producer cell can get packaged and/or incorporated into lipid delivery vehicles of the present disclosure. In some cases, the components of the lipid delivery particles, such as a payload, is loaded via the packaging and assembly process of the lipid delivery particle. For instance, the payload can be a polypeptide or protein that is packaged into the lipid delivery particle as a part of a chimeric protein as disclosed herein.

[0326] In some cases, the payload is assembled into the lipid delivery particle as an independent entity, e.g., not as a part of a chimeric protein. In other embodiments, the lipid delivery particle provided herein is loaded with a payload by utilizing any suitable method for delivering a biological or chemical payload through a lipid membrane, such as nucleofection, electroporation, lipid-based, polymer-based, or CaC12 transfection, sonication, freeze thaw, incubation at various temperatures, or heat shock of lipid delivery particles mixed with payload. In some cases, the nucleic acid molecules, such as a template RNA described herein, are loaded into the lipid delivery particle by direct loading, such as electroporation of the lipid delivery particle in vitro. In some cases, the nucleic acid molecules are loaded into the lipid delivery particle by binding to a nucleic acid binding protein (e.g., Cas protein) that is part of the lipid delivery particle or is already loaded into the lipid delivery particle.

[0327] There can be more than one type of loading techniques utilized for loading payloads (e.g, for loading more than one type of payloads) into the lipid delivery particle. For instance, in some cases, a first payload is a polypeptide that is assembled into the lipid delivery particle as a part of a chimeric protein, and a second payload is a separate protein or nucleic acid (RNA or DNA) that interacts with (e.g., binds) the first payload, and thus is loaded into the lipid delivery particle via the interaction between the first payload and the second payload. Alternatively, the second payload can be loaded into the lipid delivery particle via a transfection-like technique or any other suitable method.

[0328] In aspects, also provided herein are methods of using a lipid delivery particle (e.g. , a heVLP) or pharmaceutical composition according to some embodiments of the present disclosure, comprising contacting a cell with the lipid delivery particle (e.g. , a heVLP) described herein. In some cases, the cell is a mammalian cell, such as a human cell. In some cases, the cell is within a subject in need of treatment for a disease or a condition. In some cases, contact comprising administering the lipid delivery particle (e.g. , a heVLP) described herein to the subject, such as via injections.

[0329] In aspects, also provided herein are methods of administering a lipid delivery particle (e.g. , a heVLP), systems, or pharmaceutical compositions according to some embodiments of the present disclosure. In some cases, the method comprises administering the lipid delivery particle (e.g., a heVLP), system, or pharmaceutical composition described herein to a subject in need thereof, such as via injections.

[0330] In aspects, also provided herein are methods of producing a lipid delivery particle (e.g., a heVLP) or pharmaceutical composition according to some embodiments of the present disclosure. In some cases, the method comprises contacting a producer cell with compositions described herein.

[0331] In aspects, also provided herein are methods of using a lipid delivery particle (e.g., a heVLP) or pharmaceutical composition according to some embodiments of the present disclosure, comprising contacting a cell with a lipid delivery particle described herein, generating a template DNA in the cell using at least a portion of the template nucleic acid molecule described herein as a template. In some cases, the template DNA encodes a therapeutic molecule. In some cases, the template DNA is circularized. In some cases, the circularization is achieved via LTR sequences. In some cases, the circularization is achieved via additional pair of recombinase recognition sequences located at each end of the template DNA, wherein the additional pair of recombinase recognition is capable of self-circularizing when contacted with the additional recombinase and has a faster integration rate than the first recombinase recognition sequence introduced to the target site and the second recombinase recognition sequence located between the additional pair of recombinase recognition sequences on the template DNA. The mechanism for the circularization mediated by two pairs of recombinase recognition sequences and two recombinases are described in International Publication No. WO2023077148, which is hereby incorporated herein by reference in its entirety. [0332] Examples of lipid delivery particle and payload configurations include the following:

[0333] (1) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle either by producer cells expressing payload or particles being loaded by various particle loading methods described herein, such as electroporation. [0334] (2) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle either by producer cells expressing payload-gag chimera or particles being loaded by various particle loading methods described herein, such as electroporation.

[0335] (3) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle either by producer cells expressing payload-PH chimera or particles being loaded by various particle loading methods described herein, such as electroporation.

[0336] (4) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle either by producer cells expressing payload- gag/PH chimera or particles being loaded by various particle loading methods described herein, such as electroporation.

[0337] (5) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle in the presence of a dimerization molecule (A/C heterodimerizer) either by producer cells expressing payload and gag fused to DmrA or DmrC or particles being loaded by various particle loading methods described herein, such as electroporation.

[0338] (6) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle in the presence of a dimerization molecule (A/C heterodimerizer) either by producer cells expressing payload and PH fused to DmrA or DmrC or particles being loaded by various particle loading methods described herein, such as electroporation.

[0339] (7) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle in the presence of a dimerization molecule (A/C heterodimerizer) either by producer cells expressing payload and gag/PH fused to DmrA or DmrC or particles being loaded by various particle loading methods described herein, such as electroporation. [0340] (8) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle either by producer cells expressing payload and gag fused to an RNA binding protein (RBP), MS2, that binds to its MS2 RNA stem loop (MS2 SL) that is complexed with payload or particles being loaded by various particle loading methods described herein, such as electroporation.

[0341] (9) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle either by producer cells expressing payload and PH fused to an RNA binding protein (RBP), MS2, that binds to its RNA stem loop (MS2 SL) that is complexed with payload or particles being loaded by various particle loading methods described herein, such as electroporation. [0342] (10) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle either by producer cells expressing payload and gag/PH fused to an RNA binding protein (RBP), MS2, that binds to its RNA stem loop (MS2 SL) that is complexed with payload or particles being loaded by various particle loading methods described herein, such as electroporation.

[0343] (11) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle in the presence of dimerization molecule (A/C Heterodimerizer) either by producer cells expressing payload and gag and an RNA binding protein (RBP), MS2, fused to DmrA or DmrC that binds to its RNA stem loop (MS2 SL) that is complexed with payload or particles being loaded by various particle loading methods described herein, such as electroporation.

[0344] (12) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle in the presence of dimerization molecule (A/C Heterodimerizer) either by producer cells expressing payload and PH and an RNA binding protein (RBP), MS2, fused to DmrA or DmrC that binds to its RNA stem loop (MS2 SL) that is complexed with payload or particles being loaded by various particle loading methods described herein, such as electroporation. [0345] (13) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle in the presence of dimerization molecule (A/C Heterodimerizer) either by producer cells expressing payload and gag/PH and an RNA binding protein (RBP), MS2, fused to DmrA or DmrC that binds to its RNA stem loop (MS2 SL) that is complexed with payload or particles being loaded by various particle loading methods described herein, such as electroporation.

[0346] (14) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle either by producer cells expressing payload and gag fused to a repetitive GCN4 domain that is bound by an scFv that is fused with payload or particles being loaded by various particle loading methods described herein, such as electroporation.

[0347] (15) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle either by producer cells expressing payload and PH fused to a repetitive GCN4 domain that is bound by an scFv that is fused with payload or particles being loaded by various particle loading methods described herein, such as electroporation.

[0348] (16) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle either by producer cells expressing payload and gag/PH fused to a repetitive GCN4 domain that is bound by an scFv that is fused with payload or particles being loaded by various particle loading methods described herein, such as electroporation.

[0349] (17) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle in the presence of a dimerization molecule (A/C Heterodimerizer) by producer cells expressing gag and a repetitive GCN4 domain that are fused to DmrA or DmrC. GCN4 is bound by an scFv that is fused with payload that is also being expressed in producer cells. Particles can also be loaded by various particle loading methods described herein, such as electroporation.

[0350] (18) A lipid delivery particle (e.g, VLP, e.g., heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle in the presence of a dimerization molecule (A/C Heterodimerizer) by producer cells expressing PH and a repetitive GCN4 domain that are fused to DmrA or DmrC. GCN4 is bound by an scFv that is fused with payload that is also being expressed in producer cells. Particles can also be loaded by various particle loading methods described herein, such as electroporation.

[0351] (19) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload is packaged inside the particle in the presence of a dimerization molecule (A/C Heterodimerizer) by producer cells expressing gag/PH and a repetitive GCN4 domain that are fused to DmrA or DmrC. GCN4 is bound by an scFv that is fused with payload that is also being expressed in producer cells. Particles can also be loaded by various particle loading methods described herein, such as electroporation.

[0352] (20) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (AAV particles) is packaged inside the particle either by producer cells expressing payload or particles being loaded by various particle loading methods described herein, such as electroporation.

[0353] (21) A lipid delivery particle (e.g, VLP, e.g., heVLP) is created by producer cells expressing an envelope protein. Payload (AAV particles) is packaged inside the particle either by producer cells expressing payload and gag or particles being loaded by various particle loading methods described herein, such as electroporation.

[0354] (22) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (AAV particles) is packaged inside the particle either by producer cells expressing payload and PH or particles being loaded by various particle loading methods described herein, such as electroporation.

[0355] (23) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (AAV particles) is packaged inside the particle either by producer cells expressing payload and gag/PH or particles being loaded by various particle loading methods described herein, such as electroporation.

[0356] (24) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (AAV particles with DmrB inserted in the Capsid protein, VP2) is packaged inside the particle in the presence of DmrB dimerizer molecule either by producer cells expressing payload and gag fused to DmrB or particles being loaded by various particle loading methods described herein, such as electroporation.

[0357] (25) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (AAV particles with DmrB inserted in the Capsid protein, VP2) is packaged inside the particle in the presence of DmrB dimerizer molecule either by producer cells expressing payload and PH fused to DmrB or particles being loaded by various particle loading methods described herein, such as electroporation.

[0358] (26) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (AAV particles with DmrB inserted in the Capsid protein, VP2) is packaged inside the particle in the presence of DmrB dimerizer molecule either by producer cells expressing payload and gag/PH fused to DmrB or particles being loaded by various particle loading methods described herein, such as electroporation.

[0359] (27) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (AAV particles with DmrB inserted in the Capsid protein, VP2) is packaged inside the particle in the presence of DmrB dimerizer and A/C Heterodimerizer molecules either by producer cells expressing payload and gag fused to DmrA, DmrB, or DmrC, or particles being loaded by various particle loading methods described herein, such as electroporation.

[0360] (28) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (AAV particles with DmrB inserted in the Capsid protein, VP2) is packaged inside the particle in the presence of DmrB dimerizer and A/C Heterodimerizer molecules either by producer cells expressing payload and PH fused to DmrA, DmrB, or DmrC, or particles being loaded by various particle loading methods described herein, such as electroporation.

[0361] (29) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (AAV particles with DmrB inserted in the Capsid protein, VP2) is packaged inside the particle in the presence of DmrB dimerizer and A/C Heterodimerizer molecules either by producer cells expressing payload and gag/PH fused to DmrA, DmrB, or DmrC, or particles being loaded by various particle loading methods described herein, such as electroporation.

[0362] (30) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (single-stranded RNA, such as a template RNA) can be packaged inside the particle by various particle loading methods described herein, such as electroporation.

[0363] (31) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein and gag. Payload (single -stranded DNA, such as a template nucleic acid molecule) can be packaged inside the particle by various particle loading methods described herein, such as electroporation.

[0364] (32) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein and PH. Payload (single-stranded RNA, such as a template RNA) can be packaged inside the particle by various particle loading methods described herein, such as electroporation.

[0365] (33) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein and gag/PH. Payload (single -stranded RNA, such as a template RNA) can be packaged inside the particle by various particle loading methods described herein, such as electroporation.

[0366] (34) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (double -stranded DNA, such as a template nucleic acid molecule) can be packaged inside the particle by various particle loading methods described herein, such as electroporation. [0367] (35) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein and gag. Payload (double -stranded DNA, such as a template nucleic acid molecule) can be packaged inside the particle by various particle loading methods described herein, such as electroporation.

[0368] (36) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein and PH. Payload (double-stranded DNA, such as a template nucleic acid molecule) can be packaged inside the particle by various particle loading methods described herein, such as electroporation.

[0369] (37) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein and gag/PH. Payload (double -stranded DNA, such as a template nucleic acid molecule) can be packaged inside the particle by various particle loading methods described herein, such as electroporation.

[0370] (38) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA, such as a template RNA and PEgRNA) is packaged inside the particle either by producer cells expressing payload or particles being loaded by various particle loading methods described herein, such as electroporation.

[0371] (39) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA, such as a template RNA and PEgRNA) is packaged inside the particle either by producer cells expressing payload and gag or particles being loaded by various particle loading methods described herein, such as electroporation.

[0372] (40) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA, such as a template RNA and PEgRNA) is packaged inside the particle either by producer cells expressing payload and PH or particles being loaded by various particle loading methods described herein, such as electroporation.

[0373] (41) A lipid delivery particle (e.g, VLP, e.g., heVLP) is created by producer cells expressing an envelope protein. Payload (RNA, such as a template RNA and PEgRNA) is packaged inside the particle either by producer cells expressing payload and gag/PH or particles being loaded by various particle loading methods described herein, such as electroporation.

[0374] (42) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA, with MS2 stem loop(s)) is packaged inside the particle either by producer cells expressing payload and gag fused to MS2 or particles being loaded by various particle loading methods described herein, such as electroporation.

[0375] (43) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA with MS2 stem loop(s)) is packaged inside the particle either by producer cells expressing payload and PH fused to MS2 or particles being loaded by various particle loading methods described herein, such as electroporation.

[0376] (44) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA with MS2 stem loop(s)) is packaged inside the particle either by producer cells expressing payload and gag/PH fused to MS2 or particles being loaded by various particle loading methods described herein, such as electroporation.

[0377] (45) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA with MS2 stem loop(s)) is packaged inside the particle either by producer cells expressing payload and gag and MS2 fused to DmrA or DmrC in the presence of A/C heterodimerizer, or particles being loaded by various particle loading methods described herein, such as electroporation.

[0378] (46) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA with MS2 stem loop(s)) is packaged inside the particle either by producer cells expressing payload and PH and MS2 fused to DmrA or DmrC in the presence of A/C heterodimerizer, or particles being loaded by various particle loading methods described herein, such as electroporation.

[0379] (47) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA with MS2 stem loop(s)) is packaged inside the particle either by producer cells expressing payload and gag/PH and MS2 fused to DmrA or DmrC in the presence of A/C heterodimerizer, or particles being loaded by various particle loading methods described herein, such as electroporation.

[0380] (48) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA with RBP stem loop(s)) is packaged inside the particle either by producer cells expressing payload fused to an RBP and gag fused to another RBP or particles being loaded by various particle loading methods described herein, such as electroporation.

[0381] (49) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA with RBP stem loop(s)) is packaged inside the particle either by producer cells expressing payload fused to an RBP and PH fused to another RBP or particles being loaded by various particle loading methods described herein, such as electroporation.

[0382] (50) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA with RBP stem loop(s)) is packaged inside the particle either by producer cells expressing payload fused to an RBP and gag/PH fused to another RBP or particles being loaded by various particle loading methods described herein, such as electroporation.

[0383] (51) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA with RBP stem loop(s)) is packaged inside the particle either by producer cells expressing payload fused to an RBP and gag and another RBP fused to DmrA or DmrC in the presence of A/C Heterodimerizer molecule, or particles being loaded by various particle loading methods described herein, such as electroporation.

[0384] (52) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA with RBP stem loop(s)) is packaged inside the particle either by producer cells expressing payload fused to an RBP and PH and another RBP fused to DmrA or DmrC in the presence of A/C Heterodimerizer molecule, or particles being loaded by various particle loading methods described herein, such as electroporation.

[0385] (53) A lipid delivery particle (e.g. , VLP, e.g. , heVLP) is created by producer cells expressing an envelope protein. Payload (RNA with RBP stem loop(s)) is packaged inside the particle either by producer cells expressing payload fused to an RBP and gag/PH and another RBP fused to DmrA or DmrC in the presence of A/C Heterodimerizer molecule, or particles being loaded by various particle loading methods described herein, such as electroporation.

Methods of purification

[0386] In an aspect, described herein are methods of purifying lipid delivery particles . In some cases, the lipid delivery particles are produced from producer cell lines that are either transiently transfected with at least one plasmid or stably expressing constructs that have been integrated into the producer cell line genomic DNA. In some cases, the producer cell culture medium is harvested 24-, 48-, 72-, or 96- hours post-transfection. In some cases, the producer cell culture medium is harvested between 40- and 48- hours post-transfection. The harvested medium can undergo centrifugation steps to remove producer cell debris while maintaining the structural integrity of the lipid delivery particle. In some cases, during harvesting, the producer cell medium is centrifuged, e.g., at 500g for 5 minutes. The clarified lipid delivery particle containing supernatant can then be collected and filtered. In some cases, the lipid delivery particles are further concentrated. In some cases, the lipid delivery particles are further concentrated by ultracentrifugation. In some cases, the lipid delivery particles are concentrated 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 3000-fold, or 5000-fold. In some cases, the concentrated lipid delivery particles are resuspended, e.g., in cold PBS. In some cases, the concentrated lipid delivery particles are frozen, e.g., frozen at a rate of -l°C/min and stored at -80°C.

[0387] In some cases, the purification methods can comprise chromatographic methods (e.g., anion exchange chromatography), ultrafiltration methods (e.g, tangential flow filtration), clarifying normal flow filtration, and/or sterilizing membrane filtration. Anion exchange chromatography can separate substances based on net-surface charge, using an ion-exchange resin. Tangential flow filtration can separate molecules using ultrafiltration membranes. In some cases, the membrane pore size used for tangential flow filtration can retain a biological product of a size less than 1000 kDa, less than 750 kDa, less than 500 kDa, less than 250 kDa, less than 200 kDa, less than 150 kDa, less than 100 kDa, or less than 50 kDa. Normal flow filtration assists in the clarification of biofluid by convecting the substance directly toward a membrane under an applied pressure. In some cases, normal flow filtration can comprise a membrane pore size of greater than 0.1 pm, greater than 0.2 pm, greater than 0.3 pm, greater than 0.4 pm, greater than 0.5 pm, greater than 0.6 pm, greater than 0.7 pm, greater than 0.8 pm, greater than 0.9 pm, greater than 1.0 pm, greater than 1.5 pm, or greater than 2.0 pm. In some cases, normal flow filtration can comprise a membrane pore size of 0.2 pm, 0.45 pm, 0.8 pm, 1.2 pm, or 2.0 pm. Sterilizing membrane filtration can be used to sterilize heat-sensitive liquid without exposure to denaturing hear. In some cases, sterilizing membrane filtration can comprise a membrane pore size of about 0.1 pm, about 0.2 pm, about 0.3 pm, about 0.4 pm, or about 0.5 pm. In some cases, sterilizing membrane filtration can comprise a membrane pore size of about 0.2 pm or 0.22 pm.

COMPOSITIONS AND SYSTEMS

[0388] In aspects, also provided herein are nucleic acid molecules that encode one or more of the components of the lipid delivery particles of the present disclosure. For instance, a nucleic acid molecule encoding the chimeric protein is provided. A nucleic acid molecule encoding the envelope protein is also provided. In aspects, provided herein are compositions or systems that include nucleic acid molecules that encode one or more of the components of the lipid delivery particles of the present disclosure.

[0389] A composition can comprise a first nucleic acid sequence encoding a chimeric protein. The chimeric protein can comprise a plasma membrane recruitment element coupled to a prime editor. The prime editor can comprise a nucleic acid-guided polypeptide (e.g., derived from a Cas nuclease) coupled to a nucleic acid polymerase (e.g, a reverse transcriptase). The composition can comprise a guide nucleic acid molecule (e.g., PEgRNA) or a second nucleic acid sequence encoding the guide nucleic acid molecule. The guide nucleic acid molecule can comprise an edit template, which can comprise a sequence corresponding to a recombinase recognition sequence (e.g., attB/P or loxP site). In some cases, the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence (e.g. , attB/P or loxP site) into a target nucleic acid molecule. The target nucleic acid molecule can be a DNA sequence of a cell receiving the lipid delivery particles. The composition can comprise a third nucleic acid sequence encoding a chimeric protein comprising a plasma membrane recruitment element coupled to a recombinase (e.g., Bxbl or Cre recombinase). The recombinase can mediate recombination between the first recombinase recognition sequence and a second recombinase recognition sequence. In some cases, the second recombinase recognition sequence is also introduced to the target nucleic acid molecule. In some cases, the second recombinase recognition sequence is adjacent to the first recombinase recognition sequence. In some cases, the second recombinase recognition sequence is located on an exogenous nucleic acid molecule. In some cases, the recombinase is Cre, and the first and the second recombinase recognition sequence are both loxP. In some cases, the recombinase is Bxbl, the first and the second recombinase recognition sequence are different from each other and are either attB or attP. The recombination can comprise an inversion of a sequence between the first and the second recognition sequence that are adjacent to each other. The recombination can comprise a deletion of a sequence between the first and the second recognition sequence that are adjacent to each other. The recombination can comprise a translocation between a sequence following the 3 ’ end of the first recognition sequence and a sequence following the 3 ’ end of the second recognition sequence located on an exogenous nucleic acid molecule. The exogenous nucleic acid molecule can be a template nucleic acid molecule.

[0390] A composition can comprise a first nucleic acid sequence encoding a prime editor. The prime editor can comprise a nucleic acid-guided polypeptide (e.g. , derived from a Cas nuclease) coupled to a nucleic acid polymerase (e.g, a reverse transcriptase). The composition can comprise a guide nucleic acid molecule (e.g., PEgRNA) or a second nucleic acid sequence encoding the guide nucleic acid molecule. The guide nucleic acid molecule can comprise an edit template, which can comprise a sequence corresponding to a recombinase recognition sequence (e.g., attB/P or loxP site). In some cases, the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence (e.g. , attB/P or loxP site) into a target nucleic acid molecule. The target nucleic acid molecule can be a DNA sequence of a cell receiving the lipid delivery particles. The composition can comprise a third nucleic acid sequence encoding a chimeric protein comprising a plasma membrane recruitment element coupled to a recombinase (e.g., Bxbl or Cre recombinase). In some embodiments, the composition further comprises a template nucleic acid molecule, such as a template RNA. The template RNA can comprise an RNA sequence that codes a second recombinase recognition sequence (e.g., attP/B or loxP site). The template RNA can comprise a sequence that codes a donor nucleic acid molecule. The recombinase can mediate recombination between the first recombinase recognition sequence and a second recombinase recognition sequence. In some cases, the recombinase is Cre, and the first and the second recombinase recognition sequence are both loxP. In some cases, the recombinase is Bxbl, the first and the second recombinase recognition sequence are different from each other and are either attB or attP. In some cases, the recombinase and the first and the second recombinase recognition sequence causes the donor nucleic acid molecule to be inserted into the target nucleic molecule.

[0391] A system can comprise a lipid delivery particle described herein. The lipid delivery particle can comprise a lipid containing membrane. In some cases, the lipid containing membrane encapsulates a protein core. The lipid delivery particle can comprise a ribonucleoprotein complex. The ribonucleoprotein complex can comprise a prime editor, which can comprise a nucleic acid-guided polypeptide (e.g., a Cas nuclease) coupled to a nucleic acid polymerase (e.g., a reverse transcriptase); and a guide nucleic acid molecule (e.g., PEgRNA). The ribonucleoprotein complex can be within an inside cavity encapsulated by the lipid containing membrane (e.g., inside the protein core). The system can also comprise a recombinase (e.g., Bxbl or Cre) or a nucleic acid sequence encoding the recombinase. The recombinase or a nucleic acid sequence encoding the recombinase can be outside of the cavity encapsulated by the lipid containing membrane. The recombinase or a nucleic acid sequence encoding the recombinase can be outside of the lipid delivery particle. The recombinase or a nucleic acid sequence encoding the recombinase can be within an inside cavity encapsulated by the lipid containing membrane (e.g., inside the protein core) of a second lipid delivery particle. In some cases, the second lipid delivery particle does not comprise a ribonucleoprotein complex within the inside cavity encapsulated by the lipid containing membrane (e.g., inside the protein core). In some cases, the first lipid delivery particle and the second lipid delivery particle are delivered together to a cell. In some cases, the first lipid delivery particle and the second lipid delivery particle are delivered separately to a cell.

[0392] A system can comprise a lipid delivery particle that can comprise a lipid containing membrane encapsulating a protein core. The lipid delivery particle can comprise a recombinase (e.g., Bxbl or Cre). The recombinase can be within an inside cavity encapsulated by the lipid containing membrane. The system can further comprise a ribonucleoprotein complex. The ribonucleoprotein can comprise a prime editor, which can comprise a nucleic acid-guided polypeptide (e.g., a Cas nuclease) coupled to a nucleic acid polymerase (e.g., a reverse transcriptase); and a guide nucleic acid molecule (e.g., PEgRNA). The ribonucleoprotein complex can be outside of the cavity encapsulated by the lipid containing membrane. The ribonucleoprotein complex or a nucleic acid sequence encoding the prime editor can be outside of the cavity encapsulated by the lipid containing membrane. The ribonucleoprotein complex or a nucleic acid sequence encoding the prime editor can be outside of the lipid delivery particle. The guide nucleic acid molecule or a sequence coding the guide nucleic acid molecule can be outside of the cavity encapsulated by the lipid containing membrane. The guide nucleic acid molecule or a sequence coding the guide nucleic acid molecule can be outside of the lipid delivery particle. The ribonucleoprotein complex or a nucleic acid sequence encoding the prime editor can be within an inside cavity of a protein core of a second lipid delivery particle. The guide nucleic acid molecule or a sequence coding the guide nucleic acid molecule can be within the inside cavity encapsulated by the lipid containing membrane of the second lipid delivery particle. In some cases, the second lipid delivery particle does not comprise a recombinase within an inside cavity encapsulated by the lipid containing membrane. In some cases, the first lipid delivery particle and the second lipid delivery particle are delivered together to a cell. In some cases, the first lipid delivery particle and the second lipid delivery particle are delivered separately to a cell.

[0393] The compositions or systems can be used for producing a lipid delivery particle of the present disclosure, for instance, by transfecting or otherwise delivering the nucleic acid molecules in the compositions or systems into a producer cell. The nucleic acid molecules can be expressed in the producer cell, the result of which assemble, package, and subsequently cause the producer cell to release the lipid delivery particle.

[0394] In some cases, provided herein are producer cell lines that have been genetically modified to produce the lipid delivery particles of the present disclosure. For instance, the producer cell can have one or more nucleic acid molecules that encode one or more of the components of the lipid delivery particles of the present disclosure. The producer cells can be a stable cell line, or temporarily genetically modified. In some cases, provided herein are systems comprising the producer cells from which the lipid delivery particles of the present disclosure are produced. In some cases, the systems further comprise the produced lipid delivery particles. In some cases, the producer cell is a suitable cell line, e.g., a human cell line, such as VERO, WI38, MRC5, A549, HEK293, HEK293T, B-50 or any other HeLa cells, HepG2, Saos-2, HuH7, Chinese Hamster Ovary (CHO) cells, and HT1080 cell lines.

[0395] In some cases, a lipid delivery particle of the present disclosure facilitates gene editing efficiency greater than 70%. In some cases, a lipid delivery particle of the present disclosure facilitates gene editing efficiency comprising 8-fold increase of base editing efficiency when compared to conventional VLP (e.g., the VLPs described in Mangeot, P. E. et al. Genome editing in primary cells and in vivo using viral- derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins. Nat. Commun. 10, 45 (2019).). In some cases, a lipid delivery particle of the present disclosure exhibits reduced immunogenicity in transduced target cells. In some cases, a lipid delivery particle of the present disclosure produces reduced off-target genome editing in target cells when delivering genome editing system into the target cells. In some cases, a lipid delivery particle of the present disclosure leads to more than 100-fold reduction in Cas- independent off-target editing. In some cases, a lipid delivery particle of the present disclosure leads to at least 10-fold, such as 12- to 900-fold, lower Cas-dependent off-target editing.

PHARMACEUTICAL COMPOSITION

[0396] A lipid delivery particle provided herein can find use in a variety of fields and methods. In some cases, the lipid delivery particle of the present disclosure can be used to deliver one or more payloads, such as a ribonucleoprotein complex, a recombinase, or a template nucleic acid molecule, to a cell. In some cases, the target cells to which the lipid delivery particles are delivered are in vitro cells, ex vivo cells, or in vivo cells. The lipid delivery particles of the present disclosure can be applicable for delivery of freights into a variety of cell types, such as, animal cells, plant cells, bacteria cells, algal cells, or fungal cells.

[0397] In an aspect, disclosed herein is a pharmaceutical formulation comprising the lipid delivery particle disclosed herein and optionally further comprising a pharmaceutically acceptable carrier, excipient, or additive. The term “pharmaceutical formulation”, as used herein, refers to a composition formulated for pharmaceutical use. The terms such as “excipient”, “carrier,” “pharmaceutically acceptable carrier” or the like are used interchangeably herein. Pharmaceutical formulations comprise an immunologically effective amount of one or more cells, vectors, lipid delivery particles, or compositions disclosed herein, and optionally one or more other components which are pharmaceutically acceptable. In some cases, the pharmaceutical formulation comprises additional agents, e.g., for specific delivery, increasing half-life, or other therapeutic benefit. In some cases, the pharmaceutical formulation may comprise one or more of dimethylsulfoxide (DMSO), dextrose, water, succinate, poly I: poly C, poly-L- lysine, carboxymethylcellulose, and/or chloride.

[0398] As used herein, a “pharmaceutically acceptable carrier” is an agent that is compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.) In some cases, a pharmaceutically acceptable carrier comprises any vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g. , the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body).

[0399] Some exemplary materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanthin; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.

[0400] Pharmaceutical formulation disclosed herein can comprise one or more pH buffering compounds to maintain the pH of the formulation at a predetermined level that reflects physiological pH, such as in the range of about 5.0 to about 8.0. The pH of the pharmaceutical formulation can be about 4, about 5, about 6, about 7, about 8 or about 9. The pH buffering compound used in the aqueous liquid formulation can be an amino acid or mixture of amino acids, such as histidine or a mixture of amino acids such as histidine and glycine. The pH buffering compound can be an agent which does not chelate calcium ions. Exemplary pH buffering compounds include imidazole and acetate ions. The pH buffering compound can be present in any amount suitable to maintain the pH of the formulation at a predetermined level.

[0401] The pharmaceutical formulations described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and/or one or more other accessory ingredients, and then, optionally, shaping and/or packaging the product into a desired single- or multidose unit. Pharmaceutical formulations can additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as suited to the particular dosage form desired.

METHOD OF TREATMENT, PREVENTION, OR DIAGNOSIS

[0402] In aspects, also provided herein are methods of treating a subject by administering a lipid delivery particle (e.g., a heVLP) described herein, a system described herein, a composition described herein, or pharmaceutical composition according to some embodiments of the present disclosure.

[0403] In some cases, the present disclosure provides methods of treating, preventing, or diagnosing a condition, disease, or disorder. In some cases, a composition, kit, or method described herein can be used to treat, prevent, or diagnose a condition, disease, or disorder. The condition, disease, or disorder can comprise a cancer, an immune disorder, an autoimmune disorder, a metabolic disorder, a hormonal disorder, an inflammatory disorder, a developmental disorder, a reproductive disorder, an imprinting disorder, a genetic disorder, a neurological disorder, or a neurodegenerative disorder. In some cases, the condition, disease, or disorder comprises a liver disorder, an eye disorder, a heart disorder, a kidney disorder, a skin disorder, a blood disorder, a fibrotic disorder, a skeletal disorder, or a muscle order. In some cases, the condition, disease, or disorder is caused by a genetic mutation (e.g, an insertion, deletion, or point mutation). In some cases, the condition, disease, or disorder is hereditary. In some cases, the condition, disease, or disorder is caused by a virus or bacteria or fungus. In some cases, the condition, disease, or disorder is caused by aberrant gene expression. In some cases, the condition, disease, or disorder is a result of age. In some embodiments, the condition, disease, or disorder is chronic.

[0404] The subject in the method of present disclosure can be an animal. In some embodiments, the subject is an animal cell. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is an aquaculture animal (fish, crabs, shrimp, oysters etc.), a mammal, e.g., from a pet or zoo animal (cats, dogs, lizards, birds (e.g., parrots), lions, tigers and bears etc.), from a farm or working animal (horses, cows (e.g, dairy and beef cattle) pigs, chickens, turkeys, hens or roosters, goats, sheep, etc.), or a human. In some embodiments, the target cell as disclosed herein is in a subject to whom the method of the present disclosure is applicable.

[0405] The methods described herein can be therapeutic or veterinary methods for treating a subject. In some embodiments, the methods described herein are used to treat a disease resulting from a nonfunctional, poorly functional, or poorly expressed protein or gene product, for instance, resulting from a genetic mutation in one or more cells of the subject. In some embodiments, the methods described herein are used to treat a genetic disease (e.g. , a mutation, a substitution, a deletion, an expansion, or a recombination), a monogenic disease, an inherited metabolic disease, a cancer, a neurodegenerative disease, a cardiovascular disease, a pulmonary disease, a renal disease, a liver disease, a genetic disease, a vascular disease, ophthalmic disease, musculoskeletal disease, lymphatic disease, auditory and inner ear disease, a metabolic disease, an inflammatory disease, an autoimmune disease, or an infectious disease. In some cases, provided herein are pharmaceutical compositions and methods for treating a retinal disease, e.g., Leber congenital amaurosis, by administering a pharmaceutical composition formulated for subretinal injection.

KIT

[0406] In aspects, also provided herein are kits comprising the unit doses containing the lipid delivery particles (e.g., a VLP or heVLP), systems, compositions or pharmaceutical compositions of the present disclosure. In some embodiments, the kit comprises the lipid delivery particles, compositions, or pharmaceutical formulations of the present disclosure; and an informational medium containing instructions for administering the lipid delivery particle, composition, or pharmaceutical formulation to a subject. The kit can include a label indicating the intended use of lipid delivery particle, composition, or pharmaceutical formulation in the kit. Label can include any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0407] A kit of the present disclosure can include, alternatively or additionally, diagnostic agents and/or other therapeutic agents. In some cases, the kit includes cells or pharmaceutical formulations of the present disclosure and a diagnostic agent that can be used in a diagnostic method for diagnosing a condition, disease, or disorder in a subject.

EXAMPLES

[0408] The following examples are provided to further illustrate some embodiments of the present disclosure but are not intended to limit the scope of the disclosure; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art can alternatively be used.

Example 1: Materials and methods for inserting a donor sequence (e.g., photoreceptor-specific orphan nuclear receptor gene NR2E3) via PE-VLP in vitro

[0409] This example provides an experimental illustration of the preparation and delivery of lipid delivery particles in vitro according to some embodiments of the present disclosure.

[0410] This example describes materials and methods that will use PE-VLP as a delivery platform to insert a donor sequence, for example NR2E3, in vitro. NR2E3 gene encodes a protein that is a photoreceptor-specific orphan nuclear receptor with about 322 amino acids. Mutations in NR2E3 gene can play a role in inherited human retinal degenerations, including retinitis pigmentosa and visual loss. Using the materials and materials described below, a donor sequence, such as NR2E3 gene, can be inserted into the genome of a cell via PE-VLP delivery platform. First, PE-VLP containing VSV-G, MLVgag-pro-pol, chimeric proteins including MLVgag-PE (e.g., MLVgag-Cas9-RT) and MLVgag- recombinase (e.g., MLVgag-human Cre), a PEgRNA that contains an edit template comprising a sequence corresponding to a recombinase recognition sequence (e.g., loxP), and a template RNA (e.g., loxP-LTR-human NR2E3-LTR) will be produced and purified. The target cells will be transduced with PE-VLP. PE-VLP will be fused to the cell membrane and will release components involved in a two-step insertion of the donor sequence. Prime editor and recombinase will be in their free form after cleavable linkers connecting gag -PE and gag -recombinase are cleaved by MLV protease. The DNA of the target cell will first be prime-edited by the Cas9-RT and PEgRNA, leaving a recombinase recognition sequence (e.g., loxP site) at a desirable location for insertion of the donor sequence. The template RNA will be reverse transcribed into a DNA template sequence by the MLV pol, which can then be self-circularized via LTR sequences to form a circular DNA comprising the donor sequence and the recombinase recognition sequence. The recombinase will recognize the recombinase recognition sequence edited in the DNA of the target cell and the recombinase recognition sequence located on the circular DNA template and mediate a translocation between the DNA of the target cell and the circular DNA template containing the donor sequence (e.g. , LTR-flanked human NR2E3). The donor sequence will be inserted to the target cell DNA following the recombinase recognition sequence inserted by the prime editor. The target cells will be lysed for genomic sequencing and protein expression analysis to confirm the successful insertion of the donor sequence (e.g. , LTR-flanked human NR2E3 gene), using the method and procedures described below.

[0411] Cells

[0412] HEK293T cells, Gesicle Producer 293T cells, 3T3 cells, and Neuro-2a cells will be maintained in suitable cell media. Primary human and mouse fibroblasts will be maintained in suitable cell media. Cells will be cultured at 37 °C with 5% carbon dioxide and will be confirmed to be negative for mycoplasma by testing with MycoAlert (Lonza Biologies).

[0413] Primary human T cells and peripheral blood mononuclear cells will be isolated.

[0414] Cloning [0415] All plasmids to be used in this study will be cloned using either USER cloning or KLD cloning. DNA will be PCR-amplified and Maehl (Thermo Fisher Scientific) chemically competent E. coli will be used for plasmid propagation.

[0416] PE-VLP production and purification

[0417] PE-VLPs will be produced by transient transfection of Gesicle Producer 293T cells. Gesicle cells will be seeded in T-75 flasks (Coming) at a density of 5xl0⁶ cells per flask. After 20-24 h, cells will be transfected. For producing PE-VLPs, a mixture of plasmids expressing an membrane-fusion or envelope protein (e.g., VSV-G), MLVgag-pro-pol, chimeric proteins including MLVgag-PE (e.g., MLVgag-Cas9- RT) and MLVgag-recombinase (e.g., MLVgag-human Cre), a PEgRNA that contains an edit template comprising a sequence corresponding to a recombinase recognition sequence (e.g., loxP), and a template RNA (e.g., loxP-LTR-human NR2E3-LTR) will be co-transfected per T-75 flask. The sequences for various components of the PE-VLP are provided in Tables 1-A, 1-B, 2-A, 2-B, 2-C, 3, 4-A, 4-B, 5-A, 5- B, 5-C, 5-D, 5-E, 5-F, 5-G, and 6-A.

[0418] 40-48 h post-transfection, producer cell supernatant will be harvested and centrifuged to remove cell debris. The clarified VLP-containing supernatant will be filtered through a 0.45 -pm PVDF filter. For PE-VLPs that will be used in cell culture, the filtered supernatant will be concentrated 100-fold using PEG-it Vims Precipitation Solution (System Biosciences; LV825A-1) according to the manufacturer’s protocols. For PE-VLPs that will be injected into mice, the filtered supernatant will be concentrated 1000-3000-fold by ultracentrifugation using a cushion of 20% (w/v) sucrose in PBS. Following ultracentrifugation, PE-VLP pellets will be resuspended and centrifuged to remove debris. PE-VLPs will be frozen and stored at -80°C. PE-VLPs will be thawed on ice immediately prior to use.

[0419] PE-VLP transduction in cell culture and genomic DNA isolation

[0420] Cells will be plated for transduction in 48-well plates at a density of 30,000-40,000 cells per well. After 20-24 h, PE-VLPs will be added directly to the culture media in each well. 48-72 h posttransduction, cellular genomic DNA will be isolated. Cells will be washed and lysed at 37 °C for 1 h followed by heat inactivation at 80°C for 30 min.

[0421] High-throughput sequencing of genomic DNA of a cell receiving PE-VLP

[0422] Genomic DNA will be isolated. Following genomic DNA isolation, 1 pL of the isolated DNA (1-

10 ng) will be used as input for the first of two PCR reactions.

[0423] Genomic loci will be amplified in PCR1 using PhusionU polymerase (Thermo Fisher Scientific). PCR1 products will be confirmed on a 1% agarose gel. 1 pL of PCR1 will be used as an input for PCR2 to install Illumina barcodes. PCR2 will be conducted for nine cycles of amplification using a Phusion HS

11 kit (Life Technologies). Following PCR2, samples will be pooled and gel purified in a 1% agarose gel using a Qiaquick Gel Extraction Kit (Qiagen). Library concentration will be quantified using the Qubit High-Sensitivity Assay Kit (Thermo Fisher Scientific). Samples will be sequenced on an Illumina MiSeq instrument using an Illumina MiSeq 300 v2 Kit (Illumina).

[0424] High-throughput sequencing data analysis [0425] Sequencing reads will be demultiplexed using the MiSeq Reporter software (Illumina) and will be analyzed using CRISPResso2. Reads will be filtered by minimum average quality score (Q > 30) prior to analysis. Prime editing efficiencies are reported as the percentage of sequencing reads containing a given sequence insertion (e.g., a recombinase recognition sequence) at a specific position. Prism 9 (GraphPad) will be used to generate dot plots and bar plots.

[0426] Immunoblot analysis of PE-VLP protein content

[0427] PE-VLPs will be lysed and spotted onto a dry nitrocellulose membrane and dry for 30 min. The membrane will be blocked for 1 h at room temperature with rocking in blocking buffer. After blocking, the membrane will be incubated overnight at 4°C with rocking with one of the following primary antibodies diluted in blocking buffer: mouse anti-Cas9, mouse anti-MLV p30, mouse anti-VSV-G, or mouse anti -recombinase (e.g., anti-Cre or anti-Bxbl). The membrane will be washed three times with IxTBST (Tris-buffered saline + 0.5% Tween-20) for 10 min each time at room temperature, then will be incubated with goat anti -mouse antibody for 1 h at room temperature with rocking. The membrane will be washed as before and imaged using an Odyssey Imaging System (LI-COR).

[0428] Western blot analysis of PE-VLP protein content

[0429] PE-VLPs will be lysed and protein extracts will be separated by electrophoresis at 150 V for 45 min on a NuPAGE 3-8% Tris-Acetate gel (Thermo Fisher Scientific) in NuPAGE Tris-Acetate SDS running buffer (Thermo Fisher Scientific). Transfer to a PVDF membrane will be performed using an iBlot 2 Gel Transfer Device (Thermo Fisher Scientific) at 20 V for 7 min. The membrane will be blocked for 1 h at room temperature with rocking in blocking buffer: 1% bovine serum albumin (BSA) in TBST (150 mM NaCl, 0.5% Tween-20, and 50 mM Tris-HCl). After blocking, the membrane will be incubated overnight at 4°C with rocking with mouse anti-Cas9. The membrane will be washed three times with IxTBST for 10 min each time at room temperature, then incubated with goat anti-mouse antibody for 1 h at room temperature with rocking. The membrane will be washed as before and imaged using an Odyssey Imaging System (LI-COR). The relative amounts of cleaved PE and full-length gag-PE will be quantified by densitometry using ImageJ, and the fraction of cleaved PE relative to total (cleaved + full-length) PE will be calculated. The relative amounts of cleaved recombinase and full-length gag-recombinase will be quantified by densitometry using ImageJ, and the fraction of cleaved recombinase relative to total (cleaved + full-length) PE will be calculated.

[0430] Immunofluorescence microscopy of producer cells

[0431] Gesicle Producer 293T cells will be seeded and co-transfected with Ing of PE-VLP plasmids, 40 ng of mouse NR2E3 -targeting PEgRNA plasmid, and 40 ng of pUC19 plasmid using the jetPRIME transfection reagent (Polyplus) according to the manufacturer’s protocols. After 40 h, 32% aqueous paraformaldehyde (Electron Microscopy Sciences) will be added dropwise directly into the cellular media to a final concentration of 4% paraformaldehyde. Cells will be subsequently fixed for 20 min at room temperature. After fixation, cells will be washed three times with PBS and then permeabilized with IxPBST (PBS + 0.1% Triton X-100) for 30 min at room temperature. Cells will then be blocked in blocking buffer (3% w/v BSA in IxPBST) for 30 min at room temperature. After blocking, cells will be incubated overnight at 4°C with mouse anti-Cas9, mouse anti-recombinase, and rabbit anti-tubulin diluted in blocking buffer. Cells will be washed four times with IxPBST, then incubated for 1 h at room temperature with goat anti-mouse AlexaFluor® 647-conjugated antibody (abeam; 150115, 1:500 dilution), goat anti-rabbit AlexaFluor® 488-conjugated antibody (abeam; 150077, 1:500 dilution), and 1 pM DAPI diluted in blocking buffer. Cells will be washed three times with IxPBST and two times with PBS before imaging using an Opera Phenix High-Content Screening System (PerkinElmer). Images will be acquired using a 20x water immersion objective in a confocal mode. Automated image analysis will be performed using the Harmony software (PerkinElmer). The normalized cytoplasmic intensity will be determined by calculating the ratio of the mean cytoplasmic intensity of Cas9 signal and recombinase signal per cell to the mean cytoplasmic intensity of tubulin signal per cell, respectively.

[0432] Negative-stain transmission electron microscopy

[0433] Negative-stain TEM will be performed at the Koch Nanotechnology Materials Core Facility of MIT. PE-VLPs will be centrifuged for 5 min at 15,000 g to remove debris. From the clarified supernatant, 10 pL of sample and buffer containing solution will be added to 200 mesh copper grid coated with a continuous carbon film. The sample will be allowed to adsorb for 60 seconds after which excess solution will be removed with kimwipes. 10 pL of negative staining solution containing 1% aqueous phosphotungstic acid will be added to the TEM grid and the stain will be immediately blotted off with kimwipes. The grid will then be air-dried at room temperature in the chemical hood. The grid will be then mounted on a JEOL single tilt holder equipped within the TEM column. The specimen will be cooled down by liquid-nitrogen and then observed using JEOL 2100 FEG microscope at 200kV with a magnification of 10,000-60,000. Images will be taken using Gatan 2kx2k UltraScan CCD camera.

[0434] PE-VLP protein content quantification

[0435] For protein quantification, PE-VLPs will be lysed. The concentration of PE protein in purified PE-VLPs will be quantified using the FastScanTM Cas9 (.S', pyogenes) ELISA kit (Cell Signaling Technology; 29666C) according to the manufacturer’s protocols. Recombinant Cas9 (.S', pyogenes) nuclease protein (New England Biolabs; M0386) and recombinase protein will each be used to generate the standard curve for quantification. The concentration of MLV p30 protein in purified PE-VLPs will be quantified using the MuLV Core Antigen ELISA kit (Cell Biolabs; VPK-156) according to the manufacturer’s protocols. The concentration of VLP-associated p30 protein will be calculated with the assumption that 20% of the observed p30 in solution is associated with VLPs, as will be previously reported for MLV particles (Renner et al., 2020). The number of PE protein molecules and recombinase protein molecules per VLP will be calculated by assuming a copy number of 1800 molecules of p30 per VLP, as will be previously reported for MLV particles (Renner et al., 2020). The same analysis will be used to determine VLP titers for all therapeutic application experiments.

[0436] PE-VLP PEgRNA and template RNA extraction and quantification

[0437] RNA will be extracted from PE-VLPs using the QIAmp Viral RNA Mini Kit (Qiagen; 52904) according to the manufacturer’s protocols. Extracted RNA will be reverse transcribed using SuperScriptTM III First-Strand Synthesis SuperMix (Thermo Fisher Scientific; 18080400) and an sgRNA-specific DNA primer according to the manufacturer’s protocols. qPCR will be performed using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with SYBR green dye (Lonza; 50512). The amount of cDNA input will be normalized to MLV p30 content, and the PEgRNA abundance and template RNA abundance per VLP will be calculated as log2[fold change] (DCq) relative to PE-VLPs.

[0438] Plasmid transfections

[0439] HEK293T cells will be plated for transfection in 48-well plates (Coming) at a density of 40,000 cells per well. After 20-24 h, cells will be transfected with 1 pg total DNA using 1.5 pL of Lipofectamine 2000 (Thermo Fisher Scientific) per well according to the manufacturer’s protocols.

[0440] Quantification of PE-encoding DNA and recombinase-encoding DNA

[0441] For quantifying the amount of PE-encoding DNA and recombinase-encoding DNA in PE-VLP preparations, PE-VLPs will be lysed, and the lysate will be used as input into a qPCR reaction with PE- specific primers and recombinase-specific primers. For quantifying the amount of PE-encoding DNA and recombinase-encoding DNA in VLP-transduced vs. plasmid-transfected HEK293T cells, DNA will be isolated from cell lysate as described above and used as input into a qPCR reaction with PE-specific primers and recombinase-specific primers. In both cases, a standard curve will be generated with PE- encoding plasmid and recombinase-encoding standards of known concentration and will be used to infer the amount of PE-encoding DNA and recombinase-encoding DNA present in the original samples.

Example 2: Methods for inserting a donor sequence (e.g., photoreceptor-specific orphan nuclear receptor gene NR2E3) in vivo via PE-VLP delivery platform

[0442] This example provides an experimental illustration of the delivery and use of lipid delivery particles according to some embodiments of the present disclosure.

[0443] This example describes one way of using PE-VLP as a delivery platform to insert a donor sequence, for example NR2E3, using mice as a model. Using the materials and materials described below, NR2E3 gene can be an exemplary donor sequence delivered by PE-VLP in vivo to cure genetic disease such as retinitis pigmentosa.

[0444] PE-VLP that can deliver mouse NR2E3 gene will be produced using the same methods and materials described in Example 1. Engineered mice with mutations m NR2E3 gene that exhibit retinitis pigmentosa can be used as subjects. The PE-VLP will be given to one eye of a mouse, and blank VLP without template RNA will be given to the other eye as a negative control. Mice will be analyzed for progressive improvement in regaining visual function using techniques such as Electroretinography (ERG). The eyes of the mouse will be removed and processed for genomic sequencing and protein expression analysis to confirm the successful insertion of the donor sequence (e.g., LTR-flanked mouse NR2E3 gene), using the same method and procedures described in Example 1 and the method described below.

[0445] Mouse subretinal injection

[0446] Mice will be anesthetized by intraperitoneal injection of a cocktail consisting of 20 mg/mL ketamine and 1.75 mg/mL xylazine in phosphate-buffered saline at a dose of 0.1 mL per 20 g body weight, and their pupils will be dilated with topical administration of 1% tropicamide ophthalmic solution. Subretinal injections will be performed under an ophthalmic surgical microscope (Zeiss). An incision will be made through the cornea adjacent to the limbus at the nasal side using a 25-gauge needle. A 34-gauge blunt-end needle will be inserted through the corneal incision while avoiding the lens and advanced through the retina. Each mouse will be injected with 1 pL of experimental reagent (PE-VLPs) or control (blank VLP without template RNA), respectively, for each eye. PE-VLPs will be normalized to a titer of 4xlO¹⁰ PE-VLPs/pL, corresponding to an encapsulated PE protein content of 3 pmol/pL. After injections, pupils will be hydrated with the application of GenTeal Severe Lubricant Eye Gel (0.3% Hypromellose, Alcon) and kept for recovery.

[0447] RPE dissociation and genomic DNA and RNA preparation

[0448] Under a light microscope, mouse eyes will be dissected and immediately immersed in 350 pl of RLT Plus tissue lysis buffer provided with AllPrep DNA/RNA Mini Kit (Qiagen; 80284). After 1 min incubation, cells will be detached in the lysis buffer by gentle pipetting. The lysis buffer containing cells will be further processed for DNA and RNA extraction using the AllPrep DNA/RNA Mini Kit protocol. The final DNA and RNA will be eluted in 30 pL and 15 pL water, respectively. cDNA synthesis will be performed using the SuperScriptTM III First-Strand Synthesis SuperMix (Thermo Fisher; 18080400).

[0449] Western blot analysis of mouse eye tissue extracts

[0450] To prepare the protein lysate from the mouse tissue, the dissected mouse eyes, will be transferred to a microcentrifuge tube containing 30 pL of RIPA buffer with protease inhibitors and homogenized with a motor tissue grinder and centrifuged for 30 min at 20,000 g at 4°C. The resulting supernatant will be subject to Western blotting analysis for prime editor, recombinase, protein encoded by the donor sequence (e.g. , mouse NR2E3 gene encoding a photoreceptor-specific orphan nuclear receptor NR2E3), and p-actin expression using mouse anti-Cas9 monoclonal antibody, anti-recombinase monoclonal antibody, anti -mouse NR2E3 monoclonal antibody, and rabbit anti- -actin polyclonal antibody (1:1,000; Cell Signaling Technology; 4970S), following the same protocol. Corresponding secondary antibodies will be goat anti-mouse IgG-HRP antibody (1:5,000; Cell Signaling Technology; 7076S) and goat antirabbit IgG-HRP antibody (1:5,000; Cell Signaling Technology; 7074S).

[0451] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the present disclosure may be employed in practicing the present disclosure. It is intended that the following claims define the scope of the present disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Numbered Embodiments.

Embodiment 1. A lipid delivery particle, comprising:

(a) a lipid containing membrane;

(b) a recombinase; and (c) a ribonucleoprotein complex that comprises:

(i) a prime editor comprising a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; and

(ii) a guide nucleic acid molecule, wherein the recombinase and the ribonucleoprotein complex are within an inside cavity encapsulated by the lipid containing membrane.

Embodiment 2. The lipid delivery particle of embodiment 1, wherein the lipid delivery particle has a diameter that is less than 5 pm, less than 4 pm, less than 3 pm, less than 2 pm, or less than 1 pm.

Embodiment 3. The lipid delivery particle of embodiment 1 or 2, wherein the lipid containing membrane encapsulates a protein core.

Embodiment 4. The lipid delivery particle of any one of embodiments 1-3, wherein the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule.

Embodiment 5. The lipid delivery particle of embodiment 4, wherein the recombinase mediates recombination between the first recombinase recognition sequence and a second recombinase recognition sequence.

Embodiment 6. The lipid delivery particle of embodiment 5, wherein the lipid delivery particle further comprises either (1) a donor nucleic acid molecule that comprises the second recombinase recognition sequence; or (2) a template RNA that encodes the donor nucleic acid molecule.

Embodiment 7. The lipid delivery particle of embodiment 6, wherein the donor nucleic acid molecule or the template RNA is within the inside cavity encapsulated by the lipid containing membrane, optionally wherein the recombinase, the ribonucleoprotein complex, the donor nucleic acid molecule, and/or the template RNA is within the inside cavity of the protein core.

Embodiment 8. A lipid delivery particle, comprising:

(a) a recombinase or a nucleic acid sequence encoding the recombinase;

(b) (i) a ribonucleoprotein complex comprising: (1) a prime editor comprising a nucleic acid- guided polypeptide coupled to a nucleic acid polymerase; and (2) a guide nucleic acid molecule, or

(ii) (1) a nucleic acid sequence encoding the prime editor; and (2) the guide nucleic acid molecule or a nucleic acid sequence encoding the guide nucleic acid molecule; and

(c) a template RNA that encodes a donor nucleic acid molecule, wherein the donor nucleic acid molecule comprises a donor nucleic acid sequence and a second recombinase recognition sequence, and wherein the recombinase mediates recombination between a first recombinase recognition sequence and the second recombinase recognition sequence.

Embodiment 9. A lipid delivery particle comprising: a first nucleic acid sequence encoding a prime editor, wherein the prime editor comprises a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; a guide nucleic acid molecule or a second nucleic acid sequence encoding the guide nucleic acid molecule; a third nucleic acid sequence encoding a recombinase; and a donor nucleic acid sequence that comprises a second recombinase recognition sequence, or a template RNA encoding the donor nucleic acid sequence, and wherein the recombinase mediates recombination between a first recombinase recognition sequence and the second recombinase recognition sequence.

Embodiment 10. The lipid delivery particle of embodiment 8 or 9, wherein the lipid delivery particle has a diameter that is less than 5 pm, less than 4 pm, less than 3 pm, less than 2 pm, or less than 1 pm.

Embodiment 11. The lipid delivery particle of any one of embodiments 8-10, wherein the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule.

Embodiment 12. The lipid delivery particle of any one of embodiments 8-11, wherein the lipid delivery particle comprises a lipid containing membrane encapsulating a protein core.

Embodiment 13. The lipid delivery particle of any one of embodiments 1-12, wherein the lipid containing membrane comprises a phospholipid bilayer.

Embodiment 14. The lipid delivery particle of any one of embodiments 6-13, wherein the template RNA comprises a long terminal repeat (LTR) sequence.

Embodiment 15. The lipid delivery particle of embodiment 14, wherein the template RNA comprises at least two LTR sequences flanking a nucleic acid sequence encoding the donor nucleic acid molecule, optionally wherein the at least two LTR sequences is capable of self-circularizing.

Embodiment 16. The lipid delivery particle of embodiment 14 or 15, wherein the LTR sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 345-352.

Embodiment 17. The lipid delivery particle of any one of embodiments 1-16, wherein the lipid delivery particle further comprises an envelope protein attached to the lipid containing membrane.

Embodiment 18. The lipid delivery particle of embodiment 17, wherein the envelope protein is a viral envelope protein.

Embodiment 19. The lipid delivery particle of embodiment 18, wherein the viral envelope protein is selected from the group consisting of: a VSV-G protein, a FuG-B2 envelope protein, a FuG-E envelope protein, an HIV-1 envelope, a baboon retroviral envelope protein, and an ecotropic murine leukemia virus (MLV) envelope protein, and functional mutants thereof.

Embodiment 20. The lipid delivery particle of embodiment 18, wherein the viral envelope protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 83-104.

Embodiment 21. The lipid delivery particle of embodiment 17, wherein the envelope protein is a human endogenous retroviral envelope protein. Embodiment 22. The lipid delivery particle of embodiment 21, wherein the human endogenous retroviral envelope protein is selected from the group consisting of hENVHl, hENVH2, hENVH3, hENVKl, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)l, and hENVK_con, and functional mutants thereof.

Embodiment 23. The lipid delivery particle of embodiment 21, wherein the human endogenous retroviral envelope protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 49-82.

Embodiment 24. The lipid delivery particle of any one of embodiment 1-23, comprising a plasma membrane recruitment element.

Embodiment 25. The lipid delivery particle of embodiment 24, wherein the plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof, optionally wherein the plasma membrane recruitment element is part of a structural protein that forms the protein core.

Embodiment 26. The lipid delivery particle of embodiment 25, wherein the structural protein further comprises a retroviral protease (pro) protein.

Embodiment 27. The lipid delivery particle of embodiment 24, wherein the plasma membrane recruitment element is a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof.

Embodiment 28. The lipid delivery particle of embodiment 24, wherein the plasma membrane recruitment element is a pleckstrin homology (PH) domain of a protein selected from the group consisting of: phospholipase C31 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3- phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin- Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, and MAPK associated protein 1 (MAPKAP1), and functional mutants thereof.

Embodiment 29. The lipid delivery particle of embodiment 24, wherein the plasma membrane recruitment element is selected from the group consisting of: a pleckstrin homology (PH) domain of human phospholipase C81, a pleckstrin homology (PH) domain of human Aktl, a pleckstrin homology (PH) domain of human Arc, human endogenous retroviral gag protein, a pleckstrin homology (PH) domain of human 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), a transmembrane domain of human CD9, a transmembrane domain of human CD47, a transmembrane domain of human CD63, a transmembrane domain of human CD81, a transmembrane domain of human Daapl, a transmembrane domain of mouse Grpl, a transmembrane domain of human Grpl, a transmembrane domain of human OSBP, a transmembrane domain of human Btkl, a transmembrane domain of human FAPP1, a transmembrane domain of human CERT, a transmembrane domain of human PKD, a transmembrane domain of human PHLPP1, a transmembrane domain of human SWAP70, and a transmembrane domain of human MAPKAP1, and functional mutants thereof.

Embodiment 30. The lipid delivery particle of embodiment 24, wherein the plasma membrane recruitment element comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-48.

Embodiment 31. The lipid delivery particle of any one of embodiments 1-30, wherein the lipid delivery particle further comprises a first chimeric protein that comprises a second prime editor and a second plasma membrane recruitment element.

Embodiment 32. The lipid delivery particle of embodiment 31, wherein the second prime editor has the same sequence as the prime editor.

Embodiment 33. The lipid delivery particle of any one of embodiments 1-31, wherein the lipid delivery particle further comprises a second chimeric protein that comprises a second recombinase and a third plasma membrane recruitment element.

Embodiment 34. The lipid delivery particle of embodiment 33, wherein the second recombinase has the same sequence as the recombinase.

Embodiment 35. The lipid delivery particle of any one of embodiments 31-34, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof, and optionally wherein the first chimeric protein or the second chimeric protein forms part of the protein core.

Embodiment 36. The lipid delivery particle of any one of embodiments 31-35, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element is a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof.

Embodiment 37. The lipid delivery particle of any one of embodiments 31-35, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element is a pleckstrin homology (PH) domain of a protein selected from the group consisting of: phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3 -phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, and MAPK associated protein 1 (MAPKAP1), and functional mutants thereof.

Embodiment 38. The lipid delivery particle of any one of embodiments 31-35, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element is selected from the group consisting of: a pleckstrin homology (PH) domain of human phospholipase C81, a pleckstrin homology (PH) domain of human Aktl, a pleckstrin homology (PH) domain of human Arc, human endogenous retroviral gag protein, a pleckstrin homology (PH) domain of human 3- phosphoinositide-dependent protein kinase 1 (hPDPKl), a transmembrane domain of human CD9, a transmembrane domain of human CD47, a transmembrane domain of human CD63, a transmembrane domain of human CD81, a transmembrane domain ofhuman Daapl, a transmembrane domain of mouse Grpl, a transmembrane domain ofhuman Grpl, a transmembrane domain ofhuman OSBP, a transmembrane domain ofhuman Btkl, a transmembrane domain ofhuman FAPP1, a transmembrane domain ofhuman CERT, a transmembrane domain ofhuman PKD, a transmembrane domain of human PHLPP1, a transmembrane domain ofhuman SWAP70, and a transmembrane domain of human MAPKAP1, and functional mutants thereof.

Embodiment 39. The lipid delivery particle of any one of embodiments 31-35, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-48.

Embodiment 40. The lipid delivery particle of any one of embodiments 1-39, wherein the nucleic acid-guided polypeptide is a Cas protein.

Embodiment 41. The lipid delivery particle of embodiment 40, wherein the Cas protein is a type I, type II, type III, type IV, type V, or type VI Cas protein.

Embodiment 42. The lipid delivery particle of embodiment 40, wherein the Cas protein is selected from the group consisting of: c2cl, Casl3a, Casl3b, Casl3c, Casl3d, c2c3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), Cas 10, CaslOd, Cas 14, Cas 10, CaslOd, CasF, CasG, CasH, Cas 12a, 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, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CasX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs and modified versions thereof.

Embodiment 43. The lipid delivery particle of any one of embodiments 1-42, wherein the nucleic acid-guided polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 318-338.

Embodiment 44. The lipid delivery particle of any one of embodiments 1-43, wherein the nucleic acid polymerase is a reverse transcriptase.

Embodiment 45. The lipid delivery particle of embodiment 44, wherein the reverse transcriptase comprises an RNase H domain.

Embodiment 46. The lipid delivery particle of embodiment 44, wherein the reverse transcriptase lacks an RNase H domain.

Embodiment 47. The lipid delivery particle of any one of embodiments 44-46, wherein the reverse transcriptase is selected from the group consisting of: murine leukemia virus reverse transcriptase (M- MLV RT) (optionally D200N, T306K, W313F, T330P, and L603W), friend murine leukemia virus reverse transcriptase (FMLV RT), human endogenous retrovirus Kcon reverse transcriptase (HERV Kcon RT), a AMV-RT, a MarathonRT, a transcription xenopolymerase (RTX), and a small reverse transcriptase (Tfl), and functional mutants thereof.

Embodiment 48. The lipid delivery particle of embodiment 44, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 339-344.

Embodiment 49. The lipid delivery particle of any one of embodiments 1-48, wherein the recombinase is selected from the group consisting of: Hin, Gin, Tn3, p-six, CinH, ParA, y8, Bxbl, C31, TP901, TGI, cpBTl, R4, cpRVl, cpFCl, MR11, Al 18, U153, gp29, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2 recombinase.

Embodiment 50. The lipid delivery particle of any one of embodiments 1-48, wherein the recombinase comprises one or more recombinases independently selected from the group consisting of: Cre, Bxbl, FLP, Al 18, Abrogate, Airmid, Anglerfish, B2, B3, Benedict, BL3, Bob3, Bred, BxZ2, Cin, Conceptll, CreALSHG, Cre-R3M3, Doom, Dre, Fre, Gin, Hin, Hinder, HK022, ICleared, IntlO, Inti 1, Intl2, Intl3, Int3, Int4, Int8, Int9, Inti, K38, Kd, KSSJEB, LI, L5, LI, Lockley, Mariner (Himarl), Mariner (mosl), Min, Minos, MH (phiFCl), MR11, Mundrea, Museum, Nigri, P22, Panto, PattyP, Peaches, phi370.1, phiBTl, phiC31, phiJoe, phiK38, phiRVl, R, Rl, R2, R3, R4, R5, RDF, Rebeuca, retrotransposases encoded by R2, Sarfire, Scowl, Sere, Severus, Sheen, Sin, SkiPole, SPBc, SprA, SV1, Switzer, Tc3, TD1-40, TGI, Theia, Tol2Tcl, TP901-1, Tre, Troube, U153, Vcre, Veracruz, Vika, WB, W , <p370.1, cpBTl, cpCl, cpC31, cpFCl, and cpRV.

Embodiment 51. The lipid delivery particle of any one of embodiments 1-48, wherein the recombinase recognizes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538.

Embodiment 52. The lipid delivery particle of any one of embodiments 1-51, wherein the lipid delivery particle is a retroviral particle or a lentiviral particle.

Embodiment 53. The lipid delivery particle of any one of embodiments 1-52, wherein the donor nucleic acid sequence encodes a therapeutic molecule, and optionally wherein the therapeutic molecule comprises at least a functional portion of a viral envelope protein, a hormone, a cytokine, a ligand, a receptor, an antibody, an enzyme, a transcription factor, a chimeric antigen receptor, a T cell receptor, an antigen, a secreted protein, or any combination thereof.

Embodiment 54. The lipid delivery particle of any one of embodiments 1-53, wherein the guide nucleic acid molecule comprises one or more guide RNA, optionally wherein each of the one or more guide RNA comprises (A) a primer binding site, (B) a clamp segment, (C) a sequence encoding at least a portion of a first recombinase recognition sequence, (D) an aptamer, € spacer, or (F) scaffold, or any combinations thereof. Embodiment 55. The lipid delivery particle of any one of embodiments 1-54, wherein the guide nucleic acid molecule comprises a first guide RNA encoding at least a first portion of a first recombinase recognition sequence and a second guide RNA encoding at least a second portion of the first recombinase recognition sequence, wherein the first guide RNA and the second guide RNA work in a pair and collectively encode the first recombinase recognition sequence, optionally wherein the first and the second portion of the first recombinase recognition sequence have at least 6bp overlap.

Embodiment 56. The lipid delivery particle of any one of embodiments 1-55, wherein the guide nucleic acid molecule comprises (A) a nicking guide RNA and (B) a guide RNA encoding a first recombinase recognition sequence.

Embodiment 57. The lipid delivery particle of any one of embodiments 5-55, wherein the first recombinase recognition sequence or the second recombinase recognition sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538, and wherein the first recombinase recognition sequence and the second recombinase recognition sequence are a pair of recombinase recognition sequences for a cognate recombinase.

Embodiment 58. The lipid delivery particle any one of embodiments 1-57, wherein the lipid delivery particle further comprises an additional recombinase or a fourth nucleic acid sequence encoding the additional recombinase, wherein the additional recombinase recognizes an additional pair of recombinase recognition sequences, and wherein the additional recombinase and the recombinase are different.

Embodiment 59. The lipid delivery particle of embodiment 58, wherein the donor nucleic acid molecule further comprises the additional pair of recombinase recognition sequences, wherein the additional pair of recombinase recognition sequences comprises a third recombinase recognition sequence located at a 3 ’ end of the donor nucleic acid molecule and a fourth recombinase recognition sequence located at a 5 ’ end of the donor nucleic acid molecule, wherein the additional pair of recombinase recognition is capable of self-circularizing when contacted with the additional recombinase, and wherein the additional pair of recombinase recognition sequences has a faster integration rate than the first recombinase recognition sequence and the second recombinase recognition sequence, thereby the additional pair of recombinase recognition sequences recombines prior to recombination of the first recombinase recognition sequence and the second recombinase recognition sequence in the presence of the recombinase and the additional recombinase.

Embodiment 60. The lipid delivery particle of embodiment 58 or 59, wherein the lipid delivery particle comprises a nucleic acid molecule that comprises the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, the fourth nucleic acid sequence, and the donor nucleic acid sequence.

Embodiment 61. The lipid delivery particle of any one of embodiments 1-60, further comprising a MLHldn protein, and optionally wherein the MLHldn protein is a part of a third chimeric protein comprising a fourth plasma membrane recruitment element, and optionally wherein the fourth plasma membrane recruitment element is the same as the plasma membrane recruitment element, the second plasma membrane recruitment element or the third plasma membrane recruitment element.

Embodiment 62. A composition comprising: a first nucleic acid sequence encoding a first chimeric protein comprising a first plasma membrane recruitment element coupled to a prime editor, wherein the prime editor comprises a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; a guide nucleic acid molecule or a second nucleic acid sequence encoding the guide nucleic acid molecule; and a third nucleic acid sequence encoding a second chimeric protein comprising a second plasma membrane recruitment element coupled to a recombinase.

Embodiment 63. The composition of embodiment 62, wherein the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule.

Embodiment 64. The composition of embodiment 62 or 63, wherein the recombinase mediates recombination between the first recombinase recognition sequence and a second recombinase recognition sequence.

Embodiment 65. The composition of any one of embodiments 62-64, wherein the composition further comprises either (1) a donor nucleic acid molecule that comprises the second recombinase recognition sequence; or (2) a template RNA that encodes the donor nucleic acid molecule.

Embodiment 66. The composition of any one of embodiments 62-65, wherein the composition further comprises a fourth nucleic acid sequence encoding an envelope protein.

Embodiment 67. A composition comprising: a first nucleic acid sequence encoding a prime editor, wherein the prime editor comprises a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; a guide nucleic acid molecule or a second nucleic acid sequence encoding the guide nucleic acid molecule; a third nucleic acid sequence encoding a recombinase; a donor nucleic acid sequence that comprises a second recombinase recognition sequence, or a template RNA encoding the donor nucleic acid sequence ; and a nucleic acid sequence encoding an envelope protein; wherein the recombinase mediates recombination between a first recombinase recognition sequence and the second recombinase recognition sequence.

Embodiment 68. The composition of embodiment 67, wherein the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule.

Embodiment 69. The composition of embodiment 67 or 68, wherein the first nucleic acid sequence encodes a first chimeric protein comprising a first plasma membrane recruitment element coupled to the prime editor. Embodiment 70. The composition of any one of embodiments 67-69, wherein the third nucleic acid sequence encodes a second chimeric protein comprising a second plasma membrane recruitment element coupled to the recombinase.

Embodiment 71. The composition of any one of embodiments 65-70, wherein the template RNA comprises a long terminal repeat (LTR) sequence.

Embodiment 72. The composition of embodiment 71, wherein the template RNA comprises at least two LTR sequences flanking a nucleic acid sequence encoding the donor nucleic acid molecule, optionally wherein the at least two LTR sequences is capable of self-circularizing.

Embodiment 73. The composition of embodiment 71 or 72, wherein the LTR sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 345-352.

Embodiment 74. The composition of any one of embodiments 66-73, wherein the envelope protein is a viral envelope protein.

Embodiment 75. The composition of embodiment 74, wherein the viral envelope protein is selected from the group consisting of: a VSV-G protein, a FuG-B2 envelope protein, a FuG-E envelope protein, an HIV-1 envelope, a baboon retroviral envelope protein, and an ecotropic murine leukemia virus (MLV) envelope protein, and functional mutants thereof.

Embodiment 76. The composition of embodiment 74, wherein the viral envelope protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 83-104.

Embodiment 77. The composition of any one of embodiments 66-73, wherein the envelope protein is a human endogenous retroviral envelope protein.

Embodiment 78. The composition of embodiment 77, wherein the human endogenous retroviral envelope protein is selected from the group consisting of hENVHl, hENVH2, hENVH3, hENVKl, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)l, and hENVK_COn, and functional mutants thereof.

Embodiment 79. The composition of embodiment 77, wherein the human endogenous retroviral envelope protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 49-82.

Embodiment 80. The composition of any one of embodiments 67-79, wherein the composition further comprises a fifth nucleic acid sequence encoding a structural protein comprising a third plasma membrane recruitment element.

Embodiment 81. The composition of embodiment 80, wherein the third plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof.

Embodiment 82. The composition of embodiment 80 or 81, wherein the structural protein further comprises a retroviral protease (pro) protein. Embodiment 83. The composition of any one of embodiments 70-82, wherein the first plasma membrane recruitment element or the second plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof, a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof.

Embodiment 84. The composition of any one of embodiments 70-82, wherein the first plasma membrane recruitment element or the second plasma membrane recruitment element is a pleckstrin homology (PH) domain of a protein selected from the group consisting of: phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3 -phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol -binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, and MAPK associated protein 1 (MAPKAP1), and functional mutants thereof.

Embodiment 85. The composition of any one of embodiments 70-82, wherein the first plasma membrane recruitment element or the second plasma membrane recruitment element is selected from the group consisting of: a pleckstrin homology (PH) domain of human phospholipase C81, a pleckstrin homology (PH) domain of human Aktl, a pleckstrin homology (PH) domain of human Arc, human endogenous retroviral gag protein, a pleckstrin homology (PH) domain of human 3- phosphoinositide-dependent protein kinase 1 (hPDPKl), a transmembrane domain of human CD9, a transmembrane domain of human CD47, a transmembrane domain of human CD63, a transmembrane domain of human CD81, a transmembrane domain ofhuman Daapl, a transmembrane domain of mouse Grpl, a transmembrane domain ofhuman Grpl, a transmembrane domain ofhuman OSBP, a transmembrane domain ofhuman Btkl, a transmembrane domain ofhuman FAPP1, a transmembrane domain ofhuman CERT, a transmembrane domain ofhuman PKD, a transmembrane domain of human PHLPP1, a transmembrane domain ofhuman SWAP70, and a transmembrane domain of human MAPKAP1, and functional mutants thereof.

Embodiment 86. The composition of any one of embodiments 70-82, wherein the first plasma membrane recruitment element or the second plasma membrane recruitment element comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-48.

Embodiment 87. The composition of any one of embodiments 62-86, wherein the nucleic acid- guided polypeptide is a Cas protein.

Embodiment 88. The composition of embodiment 87, wherein the Cas protein is a type I, type II, type III, type IV, type V, or type VI Cas protein.

Embodiment 89. The composition of embodiment 87, wherein the Cas protein is selected from the group consisting of: c2cl, Casl3a, Casl3b, Casl3c, Casl3d, c2c3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, Casl4, CaslO, CaslOd, CasF, CasG, CasH, Casl2a, 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, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CasX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs and modified versions thereof.

Embodiment 90. The composition of any one of embodiments 62-89, wherein the nucleic acid- guided polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 318-338.

Embodiment 91. The composition of any one of embodiments 62-90, wherein the nucleic acid polymerase is a reverse transcriptase.

Embodiment 92. The composition of embodiment 91, wherein the reverse transcriptase comprises an RNase H domain.

Embodiment 93. The composition of embodiment 91, wherein the reverse transcriptase lacks an

RNase H domain.

Embodiment 94. The composition of embodiment 91, wherein the reverse transcriptase is selected from the group consisting of: murine leukemia virus reverse transcriptase (M-MLV RT) (optionally D200N, T306K, W313F, T330P, and L603W), friend murine leukemia virus reverse transcriptase (FMLV RT), human endogenous retrovirus Kcon reverse transcriptase (HERV Kcon RT), a AMV- RT, a MarathonRT, a transcription xenopolymerase (RTX), and a small reverse transcriptase (Tfl), and functional mutants thereof.

Embodiment 95. The composition of embodiment 91, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 339-344.

Embodiment 96. The composition of any one of embodiments 62-95, wherein the recombinase is selected from the group consisting of: Hin, Gin, Tn3, P-six, CinH, ParA, y§, Bxbl, cpC31, TP901, TGI, cpBTl, R4, tpRVl, cpFCl, MR11, Al 18, U153, gp29, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2 recombinase.

Embodiment 97. The composition of any one of embodiments 62-96, wherein the recombinase comprises one or more recombinases independently selected from the group consisting of: Cre, Bxbl, FLP, Al 18, Abrogate, Airmid, Anglerfish, B2, B3, Benedict, BL3, Bob3, Bred, BxZ2, Cin, Conceptll, CreALSHG, Cre-R3M3, Doom, Dre, Fre, Gin, Hin, Hinder, HK022, ICleared, IntlO, Inti 1, Intl2, Intl3, Int3, Int4, Int8, Int9, Inti, K38, Kd, KSSJEB, LI, L5, LI, Lockley, Mariner (Himarl), Mariner (mosl), Min, Minos, MH (phiFCl), MR11, Mundrea, Museum, Nigri, P22, Panto, PattyP, Peaches, phi370.1, phiBTl, phiC31, phiJoe, phiK38, phiRVl, R, Rl, R2, R3, R4, R5, RDF, Rebeuca, retrotransposases encoded by R2, Sarfire, Scowl, SCre, Severus, Sheen, Sin, SkiPole, SPBc,

\T1 SprA, SV1, Switzer, Tc3, TD1-40, TGI, Theia, Tol2Tcl, TP901-1, Tre, Troube, U153, VCre, Veracruz, Vika, WB, Wp, cp370.1, cpBTl, cpCl, <pC31, cpFCl, and <pRV.

Embodiment 98. The composition of any one of embodiments 62-97, wherein the recombinase recognizes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538.

Embodiment 99. The composition of any one of embodiments 65-98, wherein the donor nucleic acid sequence encodes a therapeutic molecule, and optionally wherein the therapeutic molecule comprises at least a functional portion of a viral envelope protein, a hormone, a cytokine, a ligand, a receptor, an antibody, an enzyme, a transcription factor, a chimeric antigen receptor, a T cell receptor, an antigen, a secreted protein, or any combination thereof.

Embodiment 100. The composition of any one of embodiments 62-99, wherein the guide nucleic acid molecule comprises one or more guide RNA, optionally wherein each of the one or more guide RNA comprises (A) a primer binding site, (B) a clamp segment, (C) a nucleic acid sequence encoding at least a portion of a first recombinase recognition sequence, (D) an aptamer, (E) spacer, or (F) scaffold, or any combinations thereof.

Embodiment 101 . The composition of any one of embodiments 62-100, wherein the guide nucleic acid molecule comprises a first guide RNA encoding at least a first portion of a first recombinase recognition sequence and a second guide RNA encoding at least a second portion of the first recombinase recognition sequence, wherein the first guide RNA and the second guide RNA work in a pair and collectively encode the first recombinase recognition sequence, and optionally wherein the first and the second portion of the first recombinase recognition sequence have at least 6bp overlap.

Embodiment 102. The composition of any one of embodiments 62-101, wherein the guide nucleic acid molecule comprises a (A) nicking guide RNA and (B) a guide RNA sequence encoding a first recombinase recognition sequence.

Embodiment 103. The composition of any one of embodiments 64-102, wherein the first recombinase recognition sequence or the second recombinase recognition sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538, and wherein the first recombinase recognition sequence and the second recombinase recognition sequence are a pair of recombinase recognition sequences for a cognate recombinase.

Embodiment 104. The composition of any one of embodiments 67-103, wherein the composition further comprises a fourth nucleic acid sequence encoding an additional recombinase, wherein the additional recombinase recognizes an additional pair of recombinase recognition sequences, and wherein the additional recombinase and the recombinase are different.

Embodiment 105. The composition of embodiment 104, wherein the donor nucleic acid molecule further comprises the additional pair of recombinase recognition sequences, wherein the additional pair of recombinase recognition sequences comprises a third recombinase recognition sequence located at a 3 ’ end of the donor nucleic acid molecule and a fourth recombinase recognition sequence located at a 5 ’ end of the donor nucleic acid molecule, wherein the additional pair of recombinase recognition is capable of self-circularizing when contacted with the additional recombinase, and wherein the additional pair of recombinase recognition sequences has a faster integration rate than the first recombinase recognition sequence and the second recombinase recognition sequence, thereby the additional pair of recombinase recognition sequences recombines prior to recombination of the first recombinase recognition sequence and the second recombinase recognition sequence in the presence of the recombinase and the additional recombinase.

Embodiment 106. The composition of embodiment 104 or 105, wherein the composition comprises a nucleic acid molecule that comprises the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, the fourth nucleic acid sequence, and the donor nucleic acid sequence.

Embodiment 107. The composition of any one of embodiments 62-106, wherein the composition comprises a sixth nucleic acid sequence encoding a MLHldn protein, and optionally wherein the sixth nucleic acid sequence encodes a third chimeric protein comprising a fourth plasma membrane recruitment element and the MLHldn protein, and optionally wherein the fourth plasma membrane recruitment element is the same as the first plasma membrane recruitment element, the second plasma membrane recruitment element, or the third plasma membrane recruitment element.

Embodiment 108. A system, comprising:

(1) a lipid delivery particle comprising

(a) a lipid containing membrane; and

(b) a ribonucleoprotein complex comprising: (A) a prime editor comprising a nucleic acid- guided polypeptide coupled to a nucleic acid polymerase; and (B) a guide nucleic acid molecule, wherein the ribonucleoprotein complex is within an inside cavity encapsulated by the lipid containing membrane; and

(2) a recombinase or a nucleic acid sequence encoding the recombinase.

Embodiment 109. A system, comprising:

(1) a lipid delivery particle comprising

(a) a lipid containing membrane; and

(b) a recombinase, wherein the recombinase is within an inside cavity encapsulated by the lipid containing membrane; and

(2) (i) a ribonucleoprotein complex comprising: (A) a prime editor comprising a nucleic acid- guided polypeptide coupled to a nucleic acid polymerase; and (B) a guide nucleic acid molecule, or

(ii) (A) a nucleic acid sequence encoding the prime editor; and (B) the guide nucleic acid molecule or a nucleic acid sequence encoding the guide nucleic acid molecule.

Embodiment 110. The system of embodiment 108 or 109, wherein the lipid delivery particle has a diameter that is less than 5 pm, less than 4 pm, less than 3 pm, less than 2 pm, or less than 1 pm. Embodiment 111. The system of any one of embodiments 108-110, wherein the lipid containing membrane encapsulates a protein core.

Embodiment 112. The system of any one of embodiments 108-111, wherein the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule; and wherein the recombinase mediates recombination between the first recombinase recognition sequence and a second recombinase recognition sequence.

Embodiment 113. The system of any one of embodiments 108-112, wherein the system further comprises either (3) a donor nucleic acid molecule comprising a donor nucleic acid sequence and the second recombinase recognition sequence, or (4) a template RNA that encodes the donor nucleic acid molecule.

Embodiment 114. The system of any one of embodiments 108-113, wherein the lipid containing membrane comprises a phospholipid bilayer.

Embodiment 115. The system of embodiment 113 or 114, wherein the template RNA comprises a long terminal repeat (LTR) sequence.

Embodiment 116. The system of embodiment 115, wherein the template RNA comprises at least two LTR sequences flanking a nucleic acid sequence encoding the donor nucleic acid molecule, optionally wherein the at least two LTR sequences is capable of self-circularizing.

Embodiment 117. The system of embodiment 115 or 116, wherein the LTR sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 345-352.

Embodiment 118. The system of any one of embodiments 108-117, wherein the lipid delivery particle further comprises an envelope protein attached to the lipid containing membrane.

Embodiment 119. The system of embodiment 118, wherein the envelope protein is a viral envelope protein.

Embodiment 120. The system of embodiment 119, wherein the viral envelope protein is selected from the group consisting of: a VSV-G protein, a FuG-B2 envelope protein, a FuG-E envelope protein, an HIV-1 envelope, a baboon retroviral envelope protein, and an ecotropic murine leukemia virus (MLV) envelope protein, and functional mutants thereof.

Embodiment 121 . The system of embodiment 119, wherein the viral envelope protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 83-104.

Embodiment 122. The system of embodiment 118, wherein the envelope protein is a human endogenous retroviral envelope protein.

Embodiment 123. The system of embodiment 122, wherein the human endogenous retroviral envelope protein is selected from the group consisting of hENVHl, hENVH2, hENVH3, hENVKl, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)l, and hENVK_COn, and functional mutants thereof. Embodiment 124. The system of embodiment 122, wherein the human endogenous retroviral envelope protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 49-82.

Embodiment 125. The system of any one of embodiment 108-124, wherein the lipid delivery particle comprises a plasma membrane recruitment element.

Embodiment 126. The system of embodiment 125, wherein the plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof, optionally wherein the plasma membrane recruitment element is part of a structural protein that forms the protein core.

Embodiment 127. The system of embodiment 126, wherein the structural protein further comprises a retroviral protease (pro) protein.

Embodiment 128. The system of embodiment 125, wherein the plasma membrane recruitment element is a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof.

Embodiment 129. The system of embodiment 125, wherein the plasma membrane recruitment element is a pleckstrin homology (PH) domain of a protein selected from the group consisting of: phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3-phosphoinositide- dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, and MAPK associated protein 1 (MAPKAP1), and functional mutants thereof.

Embodiment 130. The system of embodiment 125, wherein the plasma membrane recruitment element is selected from the group consisting of: a pleckstrin homology (PH) domain of human phospholipase C81, a pleckstrin homology (PH) domain of human Aktl, a pleckstrin homology (PH) domain of human Arc, human endogenous retroviral gag protein, a pleckstrin homology (PH) domain of human 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), a transmembrane domain of human CD9, a transmembrane domain of human CD47, a transmembrane domain of human CD63, a transmembrane domain ofhuman CD81, a transmembrane domain of human Daapl, a transmembrane domain of mouse Grpl, a transmembrane domain ofhuman Grpl, a transmembrane domain ofhuman OSBP, a transmembrane domain ofhuman Btkl, a transmembrane domain of human FAPP1, a transmembrane domain ofhuman CERT, a transmembrane domain ofhuman PKD, a transmembrane domain ofhuman PHLPP1, a transmembrane domain ofhuman SWAP70, and a transmembrane domain ofhuman MAPKAP1, and functional mutants thereof.

Embodiment 131. The system of embodiment 125, wherein the plasma membrane recruitment element comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-48.

Embodiment 132. The system of any one of embodiments 108-131, wherein the lipid delivery particle further comprises a first chimeric protein that comprises a second prime editor and a second plasma membrane recruitment element.

Embodiment 133. The system of embodiment 132, wherein the second prime editor has the same sequence as the prime editor.

Embodiment 134. The system of any one of embodiments 108-131, wherein the lipid delivery particle further comprises a second chimeric protein that comprises a second recombinase and a third plasma membrane recruitment element.

Embodiment 135. The system of embodiment 134, wherein the second recombinase has the same sequence as the recombinase.

Embodiment 136. The system of any one of embodiments 132-135, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof, and optionally wherein the first chimeric protein or the second chimeric protein forms part of the protein core.

Embodiment 137. The system of any one of embodiments 132-136, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element is a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof.

Embodiment 138. The system of any one of embodiments 132-136, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element is a pleckstrin homology (PH) domain of a protein selected from the group consisting of: phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3 -phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol -binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, and MAPK associated protein 1 (MAPKAP1), and functional mutants thereof.

Embodiment 139. The system of any one of embodiments 132-136, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element is selected from the group consisting of: a pleckstrin homology (PH) domain of human phospholipase C81, a pleckstrin homology (PH) domain of human Aktl, a pleckstrin homology (PH) domain of human Arc, human endogenous retroviral gag protein, a pleckstrin homology (PH) domain of human 3- phosphoinositide-dependent protein kinase 1 (hPDPKl), a transmembrane domain of human CD9, a transmembrane domain of human CD47, a transmembrane domain of human CD63, a transmembrane domain of human CD81, a transmembrane domain ofhuman Daapl, a transmembrane domain of mouse Grpl, a transmembrane domain ofhuman Grpl, a transmembrane domain ofhuman OSBP, a transmembrane domain ofhuman Btkl, a transmembrane domain ofhuman FAPP1, a transmembrane domain ofhuman CERT, a transmembrane domain ofhuman PKD, a transmembrane domain of human PHLPP1, a transmembrane domain ofhuman SWAP70, and a transmembrane domain of human MAPKAP1, and functional mutants thereof.

Embodiment 140. The system of any one of embodiments 132-136, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-48.

Embodiment 141 . The system of any one of embodiments 108-140, wherein the nucleic acid-guided polypeptide is a Cas protein.

Embodiment 142. The system of embodiment 141, wherein the Cas protein is a type I, type II, type

III, type IV, type V, or type VI Cas protein.

Embodiment 143. The system of embodiment 141, wherein the Cas protein is selected from the group consisting of: c2cl, Casl3a, Casl3b, Casl3c, Casl3d, c2c3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, Casl4, CaslO, CaslOd, CasF, CasG, CasH, Casl2a, 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, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CasX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs and modified versions thereof.

Embodiment 144. The system of any one of embodiments 108-143, wherein the nucleic acid-guided polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 318-338.

Embodiment 145. The system of any one of embodiments 108-144, wherein the nucleic acid polymerase is a reverse transcriptase.

Embodiment 146. The system of embodiment 145, wherein the reverse transcriptase comprises an

RNase H domain.

Embodiment 147. The system of embodiment 145, wherein the reverse transcriptase lacks an RNase

H domain.

Embodiment 148. The system of embodiment 145, wherein the reverse transcriptase is selected from the group consisting of: murine leukemia virus reverse transcriptase (M-MLV RT) (optionally D200N, T306K, W313F, T330P, and L603W), friend murine leukemia virus reverse transcriptase (FMLV RT), human endogenous retrovirus Kcon reverse transcriptase (HERV Kcon RT), a AMV- RT, a MarathonRT, a transcription xenopolymerase (RTX), and a small reverse transcriptase (Tfl), and functional mutants thereof. Embodiment 149. The system of embodiment 145, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 339-344.

Embodiment 150. The system of any one of embodiments 108-149, wherein the recombinase is selected from the group consisting of: Elin, Gin, Tn3, p-six, CinH, ParA, y§, Bxbl, (|)C31, TP901, TGI, cpBTl, R4, cpRVl, cpFCl, MR11, Al 18, U153, gp29, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2 recombinase.

Embodiment 151. The system of any one of embodiments 108-150, wherein the recombinase comprises one or more recombinases independently selected from the group consisting of: Cre, Bxbl, FLP, Al 18, Abrogate, Airmid, Anglerfish, B2, B3, Benedict, BL3, Bob3, Bred, BxZ2, Cin, Conceptll, CreALSHG, Cre-R3M3, Doom, Dre, Fre, Gin, Hin, Hinder, HK022, ICleared, IntlO, Inti 1, Intl2, Intl3, Int3, Int4, Int8, Int9, Inti, K38, Kd, KSSJEB, LI, L5, LI, Lockley, Mariner (Himarl), Mariner (mosl), Min, Minos, MJ1 (phiFCl), MR11, Mundrea, Museum, Nigri, P22, Panto, PattyP, Peaches, phi370.1, phiBTl, phiC31, phiJoe, phiK38, phiRVl, Rl, R2, R3, R4, R5, RDF, Rebeuca, retrotransposases encoded by R2, Sarfire, Scowl, SCre, Severus, Sheen, Sin, SkiPole, SPBc, SprA, SV1, Switzer, Tc3, TD1-40, TGI, Theia, Tol2Tcl, TP901-1, Tre, Troube, U153, VCre, Veracruz, Vika, WB, W , <p370.1, cpBTl, cpCl, cpC31, cpFCl, and cpRV.

Embodiment 152. The system of any one of embodiments 108-151, wherein the recombinase recognizes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538.

Embodiment 153. The system of any one of embodiments 108-152, wherein the lipid delivery particle is a retroviral particle or a lentiviral particle.

Embodiment 154. The system of any one of embodiments 113-153, wherein the donor nucleic acid sequence encodes a therapeutic molecule, optionally wherein the therapeutic molecule comprises at least a functional portion of a viral envelope protein, a hormone, a cytokine, a ligand, a receptor, an antibody, an enzyme, a transcription factor, a chimeric antigen receptor, a T cell receptor, an antigen, a secreted protein, or any combination thereof.

Embodiment 155. The system of any one of embodiments 108-154, wherein the guide nucleic acid molecule comprises one or more guide RNA, optionally wherein each of the one or more guide RNA comprises (A) a primer binding site, (B) a clamp segment, (C) a sequence encoding at least a portion of a first recombinase recognition sequence, (D) an aptamer, (E) spacer, or (F) scaffold, or any combinations thereof.

Embodiment 156. The system of any one of embodiments 108-155, wherein the guide nucleic acid molecule comprises a first guide RNA encoding at least a first portion of a first recombinase recognition sequence and a second guide RNA encoding at least a second portion of the first recombinase recognition sequence, wherein the first guide RNA and the second guide RNA work in a pair and collectively encode the first recombinase recognition sequence, optionally wherein the first and the second portion of the first recombinase recognition sequence have at least 6bp overlap.

Embodiment 157. The system of any one of embodiments 108-156, wherein the guide nucleic acid molecule comprises (A) a nicking guide RNA and (B) a guide RNA encoding a first recombinase recognition sequence.

Embodiment 158. The system of any one of embodiments 112-157, wherein the first recombinase recognition sequence or the second recombinase recognition sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538, and wherein the first recombinase recognition sequence and the second recombinase recognition sequence are a pair of recombinase recognition sequences for a cognate recombinase.

Embodiment 159. The system of any one of embodiments 108-158, wherein the system further comprises an additional recombinase or a nucleic acid encoding the additional recombinase, wherein the additional recombinase recognizes an additional pair of recombinase recognition sequences, and wherein the additional recombinase and the recombinase are different.

Embodiment 160. The system of embodiment 159, wherein the donor nucleic acid molecule further comprises the additional pair of recombinase recognition sequences, wherein the additional pair of recombinase recognition sequences comprises a third recombinase recognition sequence located at a 3 ’ end of the donor nucleic acid molecule and a fourth recombinase recognition sequence located at a 5’ end of the donor nucleic acid molecule, wherein the additional pair of recombinase recognition is capable of self-circularizing when contacted with the additional recombinase, and wherein the additional pair of recombinase recognition sequences has a faster integration rate than the first recombinase recognition sequence and the second recombinase recognition sequence, thereby the additional pair of recombinase recognition sequences recombines prior to recombination of the first recombinase recognition sequence and the second recombinase recognition sequence in the presence of the recombinase and the additional recombinase.

Embodiment 161 . The system of any one of embodiments 108-160, wherein the system further comprises a MLHldn protein or a nucleic acid sequence encoding the MLHldn protein, and optionally wherein the MLHldn protein is a part of a third chimeric protein comprising a fourth plasma membrane recruitment element, and optionally wherein the fourth plasma membrane recruitment element is the same as the plasma membrane recruitment element, the second plasma membrane recruitment element or the third plasma membrane recruitment element.

Embodiment 162. A pharmaceutical composition comprising (a) the lipid delivery particle of any one of embodiments 1-61 or the system of any one of embodiments 108-161; and (b) a pharmaceutically acceptable excipient.

Embodiment 163. A kit comprising (a) the lipid delivery particle of any one of embodiments 1-61, the system of any one of embodiments 108-161, or the pharmaceutical composition of embodiment 162; and (b) an information material containing instructions for administering a dosage of the lipid delivery particle or the system, or a dosage form of the pharmaceutical composition to a subject.

Embodiment 164. A method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject the lipid delivery particle of any one of embodiments 1-61, the system of any one of embodiments 108-161, or the pharmaceutical composition of embodiment 162.

Embodiment 165. A method comprising contacting a cell with the lipid delivery particle of any one of embodiments 1-61.

Embodiment 166. The method of embodiment 165, the method comprising generating a template DNA in the cell using at least a portion of the template RNA as a template, wherein the template DNA encodes a therapeutic molecule, and optionally circularizing the template DNA in the cell; and expressing the therapeutic molecule from the template DNA in the cell.

Embodiment 167. A method comprising contacting a cell with the system of any one of embodiments 108-161.

Embodiment 168. A method comprising administering the lipid delivery particle of any one of embodiments 1-61 to a subject in need thereof.

Embodiment 169. A method of producing the lipid delivery particle of any one of embodiments 1- 61.

Embodiment 170. A method of producing a lipid delivery particle, the method comprising contacting a producer cell with the composition of any one of embodiments 62-107.

Claims

1. A lipid delivery particle, comprising:

(d) a lipid containing membrane;

(e) a recombinase; and

(f) a ribonucleoprotein complex that comprises:

(iii) a prime editor comprising a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; and

(iv) a guide nucleic acid molecule, wherein the recombinase and the ribonucleoprotein complex are within an inside cavity encapsulated by the lipid containing membrane.

2. The lipid delivery particle of claim 1, wherein the lipid delivery particle has a diameter that is less than 5 pm, less than 4 pm, less than 3 pm, less than 2 pm, or less than 1 pm.

3. The lipid delivery particle of claim 1, wherein the lipid containing membrane encapsulates a protein core.

4. The lipid delivery particle of claim 1, wherein the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule.

5. The lipid delivery particle of claim 4, wherein the recombinase mediates recombination between the first recombinase recognition sequence and a second recombinase recognition sequence.

6. The lipid delivery particle of claim 5, wherein the lipid delivery particle further comprises either (1) a donor nucleic acid molecule that comprises the second recombinase recognition sequence; or (2) a template RNA that encodes the donor nucleic acid molecule.

7. The lipid delivery particle of claim 6, wherein the donor nucleic acid molecule or the template RNA is within the inside cavity encapsulated by the lipid containing membrane, optionally wherein the recombinase, the ribonucleoprotein complex, the donor nucleic acid molecule, and/or the template RNA is within the inside cavity of the protein core.

8. A lipid delivery particle, comprising:

(d) a recombinase or a nucleic acid sequence encoding the recombinase;

(e) (i) a ribonucleoprotein complex comprising: (1) a prime editor comprising a nucleic acid- guided polypeptide coupled to a nucleic acid polymerase; and (2) a guide nucleic acid molecule, or

(f) a template RNA that encodes a donor nucleic acid molecule, wherein the donor nucleic acid molecule comprises a donor nucleic acid sequence and a second recombinase recognition sequence, and wherein the recombinase mediates recombination between a first recombinase recognition sequence and the second recombinase recognition sequence.

9. A lipid delivery particle comprising: a first nucleic acid sequence encoding a prime editor, wherein the prime editor comprises a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; a guide nucleic acid molecule or a second nucleic acid sequence encoding the guide nucleic acid molecule; a third nucleic acid sequence encoding a recombinase; and a donor nucleic acid sequence that comprises a second recombinase recognition sequence, or a template RNA encoding the donor nucleic acid sequence, and wherein the recombinase mediates recombination between a first recombinase recognition sequence and the second recombinase recognition sequence.

10. The lipid delivery particle of claim 8, wherein the lipid delivery particle has a diameter that is less than 5 pm, less than 4 pm, less than 3 pm, less than 2 pm, or less than 1 pm.

11. The lipid delivery particle of claim 9, wherein the lipid delivery particle has a diameter that is less than 5 pm, less than 4 pm, less than 3 pm, less than 2 pm, or less than 1 pm.

12. The lipid delivery particle of claim 8 or 9, wherein the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule.

13. The lipid delivery particle of claim 8 or 9, wherein the lipid delivery particle comprises a lipid containing membrane encapsulating a protein core.

14. The lipid delivery particle of any one of claims 1-11, wherein the lipid containing membrane comprises a phospholipid bilayer.

15. The lipid delivery particle of any one of claims 6-11, wherein the template RNA comprises a long terminal repeat (LTR) sequence.

16. The lipid delivery particle of claim 15, wherein the template RNA comprises at least two LTR sequences flanking a nucleic acid sequence encoding the donor nucleic acid molecule, optionally wherein the at least two LTR sequences is capable of self-circularizing.

17. The lipid delivery particle of claim 15, wherein the LTR sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 345-352.

18. The lipid delivery particle of any one of claims 1-11, wherein the lipid delivery particle further comprises an envelope protein attached to the lipid containing membrane.

19. The lipid delivery particle of claim 18, wherein the envelope protein is a viral envelope protein.

20. The lipid delivery particle of claim 19, wherein the viral envelope protein is selected from the group consisting of: a VSV-G protein, a FuG-B2 envelope protein, a FuG-E envelope protein, an HIV-1 envelope, a baboon retroviral envelope protein, and an ecotropic murine leukemia virus (MLV) envelope protein, and functional mutants thereof.

21. The lipid delivery particle of claim 19, wherein the viral envelope protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 83-104.

22. The lipid delivery particle of claim 18, wherein the envelope protein is a human endogenous retroviral envelope protein.

23. The lipid delivery particle of claim 22, wherein the human endogenous retroviral envelope protein is selected from the group consisting of hENVHl, hENVH2, hENVH3, hENVKl, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)l, and hENVK_COn, and functional mutants thereof.

24. The lipid delivery particle of claim 22, wherein the human endogenous retroviral envelope protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 49-82.

25. The lipid delivery particle of any one of claim 1-11, comprising a plasma membrane recruitment element.

26. The lipid delivery particle of claim 25, wherein the plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof, optionally wherein the plasma membrane recruitment element is part of a structural protein that forms the protein core.

27. The lipid delivery particle of claim 26, wherein the structural protein further comprises a retroviral protease (pro) protein.

28. The lipid delivery particle of claim 25, wherein the plasma membrane recruitment element is a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof.

29. The lipid delivery particle of claim 25, wherein the plasma membrane recruitment element is a pleckstrin homology (PH) domain of a protein selected from the group consisting of: phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3 -phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, and MAPK associated protein 1 (MAPKAP1), and functional mutants thereof.

30. The lipid delivery particle of claim 25, wherein the plasma membrane recruitment element is selected from the group consisting of: a pleckstrin homology (PH) domain of human phospholipase C81, a pleckstrin homology (PH) domain of human Aktl, a pleckstrin homology (PH) domain of human Arc, human endogenous retroviral gag protein, a pleckstrin homology (PH) domain of human 3- phosphoinositide-dependent protein kinase 1 (hPDPKl), a transmembrane domain of human CD9, a transmembrane domain of human CD47, a transmembrane domain of human CD63, a transmembrane domain of human CD81, a transmembrane domain ofhuman Daapl, a transmembrane domain of mouse Grpl, a transmembrane domain ofhuman Grpl, a transmembrane domain ofhuman OSBP, a transmembrane domain ofhuman Btkl, a transmembrane domain ofhuman FAPP1, a transmembrane domain ofhuman CERT, a transmembrane domain ofhuman PKD, a transmembrane domain of human PHLPP1, a transmembrane domain ofhuman SWAP70, and a transmembrane domain of human MAPKAP1, and functional mutants thereof.

31. The lipid delivery particle of claim 25, wherein the plasma membrane recruitment element comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-48.

32. The lipid delivery particle of any one of claims 1-11, wherein the lipid delivery particle further comprises a first chimeric protein that comprises a second prime editor and a second plasma membrane recruitment element.

33. The lipid delivery particle of claim 32, wherein the second prime editor has the same sequence as the prime editor.

34. The lipid delivery particle of any one of claims 1-11, wherein the lipid delivery particle further comprises a second chimeric protein that comprises a second recombinase and a third plasma membrane recruitment element.

35. The lipid delivery particle of claim 34, wherein the second recombinase has the same sequence as the recombinase.

36. The lipid delivery particle of claim 32, wherein the second plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof, and optionally wherein the first chimeric protein or the second chimeric protein forms part of the protein core.

37. The lipid delivery particle of claim 32, wherein the second plasma membrane recruitment element is a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof.

38. The lipid delivery particle of claim 34, wherein the third plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof, and optionally wherein the first chimeric protein or the second chimeric protein forms part of the protein core.

39. The lipid delivery particle of claim 34, wherein the third plasma membrane recruitment element is a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof.

40. The lipid delivery particle of claim 37 or 39, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element is a pleckstrin homology (PH) domain of a protein selected from the group consisting of: phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol-binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four- phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, and MAPK associated protein 1 (MAPKAP1), and functional mutants thereof.

41. The lipid delivery particle of claim 37 or 39, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element is selected from the group consisting of: a pleckstrin homology (PH) domain of human phospholipase C81, a pleckstrin homology (PH) domain of human Aktl, a pleckstrin homology (PH) domain of human Arc, human endogenous retroviral gag protein, a pleckstrin homology (PH) domain of human 3 -phosphoinositide-dependent protein kinase 1 (hPDPKl), a transmembrane domain of human CD9, a transmembrane domain of human CD47, a transmembrane domain ofhuman CD63, a transmembrane domain of human CD81, a transmembrane domain ofhuman Daapl, a transmembrane domain of mouse Grpl, a transmembrane domain of human Grpl, a transmembrane domain ofhuman OSBP, a transmembrane domain ofhuman Btkl, a transmembrane domain ofhuman FAPP1, a transmembrane domain of human CERT, a transmembrane domain ofhuman PKD, a transmembrane domain ofhuman PHLPP1, a transmembrane domain ofhuman SWAP70, and a transmembrane domain ofhuman MAPKAP1, and functional mutants thereof.

42. The lipid delivery particle of claim 37 or 39, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-48.

43. The lipid delivery particle of any one of claims 1-11, wherein the nucleic acid-guided polypeptide is a Cas protein.

44. The lipid delivery particle of claim 43, wherein the Cas protein is a type I, type II, type III, type IV, type V, or type VI Cas protein.

45. The lipid delivery particle of claim 43, wherein the Cas protein is selected from the group consisting of: c2cl, Casl3a, Casl3b, Casl3c, Casl3d, c2c3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, Cas 14, Cas 10, CaslOd, CasF, CasG, CasH, Cas 12a, 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, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CasX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs and modified versions thereof.

46. The lipid delivery particle of any one of claims 1-11, wherein the nucleic acid-guided polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 318-338.

47. The lipid delivery particle of any one of claims 1-11, wherein the nucleic acid polymerase is a reverse transcriptase.

48. The lipid delivery particle of claim 47, wherein the reverse transcriptase comprises an RNase H domain.

49. The lipid delivery particle of claim 47, wherein the reverse transcriptase lacks an RNase H domain.

50. The lipid delivery particle of claim 47, wherein the reverse transcriptase is selected from the group consisting of: murine leukemia virus reverse transcriptase (M-MLV RT) (optionally D200N, T306K, W3 13F, T330P, and L603W), friend murine leukemia virus reverse transcriptase (FMLV RT), human endogenous retrovirus Kcon reverse transcriptase (HERV Kcon RT), a AMV-RT, a MarathonRT, a transcription xenopolymerase (RTX), and a small reverse transcriptase (Tfl), and functional mutants thereof.

51. The lipid delivery particle of claim 47, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 339-344.

52. The lipid delivery particle of any one of claims 1-11, wherein the recombinase is selected from the group consisting of: Hin, Gin, Tn3, P-six, CinH, ParA, y8, Bxbl, <|>C31, TP901, TGI, cpBTl, R4, cpRVl, cpFCl, MR11, Al 18, U153, gp29, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2 recombinase.

53. The lipid delivery particle of any one of claims 1-11, wherein the recombinase comprises one or more recombinases independently selected from the group consisting of: Cre, Bxbl, FLP, Al 18, Abrogate, Airmid, Anglerfish, B2, B3, Benedict, BL3, Bob3, Bred, BxZ2, Cin, Conceptll, CreALSHG, Cre- R3M3, Doom, Dre, Fre, Gin, Hin, Hinder, HK022, ICleared, IntlO, Inti 1, Intl2, Intl3, Int3, Int4, Int8, Int9, Inti, K38, Kd, KSSJEB, LI, L5, LI, Lockley, Mariner (Himarl), Mariner (mosl), Min, Minos, MH (phiFCl), MR11, Mundrea, Museum, Nigri, P22, Panto, PattyP, Peaches, phi370.1, phiBTl, phiC31, phiJoe, phiK38, phiRVl, R, Rl, R2, R3, R4, R5, RDF, Rebeuca, retrotransposases encoded by R2, Sarfire, Scowl, Sere, Severus, Sheen, Sin, SkiPole, SPBc, SprA, SV1, Switzer, Tc3, TD1-40, TGI, Theia, Tol2Tcl, TP901-1, Tre, Troube, U153, Vcre, Veracruz, Vika, WB, Wp, <p370.1, cpBTl, cpCl, cpC31, cpFCl, and cpRV.

54. The lipid delivery particle of any one of claims 1-11, wherein the recombinase recognizes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538.

55. The lipid delivery particle of any one of claims 1-11, wherein the lipid delivery particle is a retroviral particle or a lentiviral particle.

56. The lipid delivery particle of any one of claims 1-11, wherein the donor nucleic acid sequence encodes a therapeutic molecule, and optionally wherein the therapeutic molecule comprises at least a functional portion of a viral envelope protein, a hormone, a cytokine, a ligand, a receptor, an antibody, an enzyme, a transcription factor, a chimeric antigen receptor, a T cell receptor, an antigen, a secreted protein, or any combination thereof.

57. The lipid delivery particle of any one of claims 1-11, wherein the guide nucleic acid molecule comprises one or more guide RNA, optionally wherein each of the one or more guide RNA comprises (A) a primer binding site, (B) a clamp segment, (C) a sequence encoding at least a portion of a first recombinase recognition sequence, (D) an aptamer, (E) spacer, or (F) scaffold, or any combinations thereof.

58. The lipid delivery particle of any one of claims 1-11, wherein the guide nucleic acid molecule comprises a first guide RNA encoding at least a first portion of a first recombinase recognition sequence and a second guide RNA encoding at least a second portion of the first recombinase recognition sequence, wherein the first guide RNA and the second guide RNA work in a pair and collectively encode the first recombinase recognition sequence, optionally wherein the first and the second portion of the first recombinase recognition sequence have at least 6bp overlap.

59. The lipid delivery particle of any one of claims 1-11, wherein the guide nucleic acid molecule comprises (A) a nicking guide RNA and (B) a guide RNA encoding a first recombinase recognition sequence.

60. The lipid delivery particle of any one of claims 5-11, wherein the first recombinase recognition sequence or the second recombinase recognition sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538, and wherein the first recombinase recognition sequence and the second recombinase recognition sequence are a pair of recombinase recognition sequences for a cognate recombinase.

61. The lipid delivery particle of any one of claims 1-11, wherein the lipid delivery particle further comprises an additional recombinase or a fourth nucleic acid sequence encoding the additional recombinase, wherein the additional recombinase recognizes an additional pair of recombinase recognition sequences, and wherein the additional recombinase and the recombinase are different.

62. The lipid delivery particle of claim 61, wherein the donor nucleic acid molecule further comprises the additional pair of recombinase recognition sequences, wherein the additional pair of recombinase recognition sequences comprises a third recombinase recognition sequence located at a 3’ end of the donor nucleic acid molecule and a fourth recombinase recognition sequence located at a 5’ end of the donor nucleic acid molecule, wherein the additional pair of recombinase recognition is capable of self-circularizing when contacted with the additional recombinase, and wherein the additional pair of recombinase recognition sequences has a faster integration rate than the first recombinase recognition sequence and the second recombinase recognition sequence, thereby the additional pair of recombinase recognition sequences recombines prior to recombination of the first recombinase recognition sequence and the second recombinase recognition sequence in the presence of the recombinase and the additional recombinase.

63. The lipid delivery particle of claim 61, wherein the lipid delivery particle comprises a nucleic acid molecule that comprises the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, the fourth nucleic acid sequence, and the donor nucleic acid sequence.

64. The lipid delivery particle of any one of claims 1-11, further comprising a MLHldn protein, and optionally wherein the MLHldn protein is a part of a third chimeric protein comprising a fourth plasma membrane recruitment element, and optionally wherein the fourth plasma membrane recruitment element is the same as the plasma membrane recruitment element, the second plasma membrane recruitment element or the third plasma membrane recruitment element.

65. A composition comprising: a first nucleic acid sequence encoding a first chimeric protein comprising a first plasma membrane recruitment element coupled to a prime editor, wherein the prime editor comprises a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; a guide nucleic acid molecule or a second nucleic acid sequence encoding the guide nucleic acid molecule; and a third nucleic acid sequence encoding a second chimeric protein comprising a second plasma membrane recruitment element coupled to a recombinase.

66. The composition of claim 65, wherein the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule.

67. The composition of claim 65, wherein the recombinase mediates recombination between the first recombinase recognition sequence and a second recombinase recognition sequence.

68. The composition of claim 65, wherein the composition further comprises either (1) a donor nucleic acid molecule that comprises the second recombinase recognition sequence; or (2) a template RNA that encodes the donor nucleic acid molecule.

69. The composition of claim 65, wherein the composition further comprises a fourth nucleic acid sequence encoding an envelope protein.

70. A composition comprising: a first nucleic acid sequence encoding a prime editor, wherein the prime editor comprises a nucleic acid-guided polypeptide coupled to a nucleic acid polymerase; a guide nucleic acid molecule or a second nucleic acid sequence encoding the guide nucleic acid molecule; a third nucleic acid sequence encoding a recombinase; a donor nucleic acid sequence that comprises a second recombinase recognition sequence, or a template RNA encoding the donor nucleic acid sequence ; and a nucleic acid sequence encoding an envelope protein; wherein the recombinase mediates recombination between a first recombinase recognition sequence and the second recombinase recognition sequence.

71. The composition of claim 70, wherein the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule.

72. The composition of claim 70, wherein the first nucleic acid sequence encodes a first chimeric protein comprising a first plasma membrane recruitment element coupled to the prime editor.

73. The composition of claim 70, wherein the third nucleic acid sequence encodes a second chimeric protein comprising a second plasma membrane recruitment element coupled to the recombinase.

74. The composition of any one of claims 68-73, wherein the template RNA comprises a long terminal repeat (LTR) sequence.

75. The composition of claim 74, wherein the template RNA comprises at least two LTR sequences flanking a nucleic acid sequence encoding the donor nucleic acid molecule, optionally wherein the at least two LTR sequences is capable of self-circularizing.

76. The composition of claim 74, wherein the LTR sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 345-352.

77. The composition of any one of claims 69-73, wherein the envelope protein is a viral envelope protein.

78. The composition of claim 77, wherein the viral envelope protein is selected from the group consisting of: a VSV-G protein, a FuG-B2 envelope protein, a FuG-E envelope protein, an HIV-1 envelope, a baboon retroviral envelope protein, and an ecotropic murine leukemia virus (MLV) envelope protein, and functional mutants thereof.

79. The composition of claim 77, wherein the viral envelope protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 83-104.

80. The composition of any one of claims 69-73, wherein the envelope protein is a human endogenous retroviral envelope protein.

81. The composition of claim 80, wherein the human endogenous retroviral envelope protein is selected from the group consisting of hENVHl, hENVH2, hENVH3, hENVKl, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)l, and hENVK_COn, and functional mutants thereof.

82. The composition of claim 80, wherein the human endogenous retroviral envelope protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 49-82.

83. The composition of any one of claims 69-73, wherein the composition further comprises a fifth nucleic acid sequence encoding a structural protein comprising a third plasma membrane recruitment element.

84. The composition of claim 83, wherein the third plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof.

85. The composition of claim 83, wherein the structural protein further comprises a retroviral protease (pro) protein.

86. The composition of claim 72 or 73, wherein the first plasma membrane recruitment element or the second plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof, a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof.

87. The composition of claim 72 or 73, wherein the first plasma membrane recruitment element or the second plasma membrane recruitment element is a pleckstrin homology (PH) domain of a protein selected from the group consisting of: phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxy sterol -binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate- adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, and MAPK associated protein 1 (MAPKAP1), and functional mutants thereof.

88. The composition of claim 72 or 73, wherein the first plasma membrane recruitment element or the second plasma membrane recruitment element is selected from the group consisting of: a pleckstrin homology (PH) domain of human phospholipase C81, a pleckstrin homology (PH) domain of human Aktl, a pleckstrin homology (PH) domain of human Arc, human endogenous retroviral gag protein, a pleckstrin homology (PH) domain of human 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), a transmembrane domain of human CD9, a transmembrane domain of human CD47, a transmembrane domain ofhuman CD63, a transmembrane domain of human CD81, a transmembrane domain ofhuman Daapl, a transmembrane domain of mouse Grpl, a transmembrane domain of human Grpl, a transmembrane domain ofhuman OSBP, a transmembrane domain ofhuman Btkl, a transmembrane domain ofhuman FAPP1, a transmembrane domain of human CERT, a transmembrane domain ofhuman PKD, a transmembrane domain ofhuman PHLPP1, a transmembrane domain ofhuman SWAP70, and a transmembrane domain ofhuman MAPKAP1, and functional mutants thereof.

89. The composition of claim 72 or 73, wherein the first plasma membrane recruitment element or the second plasma membrane recruitment element comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-48.

90. The composition of any one of claims 65-73, wherein the nucleic acid-guided polypeptide is a Cas protein.

91. The composition of claim 90, wherein the Cas protein is a type I, type II, type III, type IV, type V, or type VI Cas protein.

92. The composition of claim 90, wherein the Cas protein is selected from the group consisting of: c2cl, Casl3a, Casl3b, Casl3c, Casl3d, c2c3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), Cas 10, CaslOd, Casl4, CaslO, CaslOd, CasF, CasG, CasH, Casl2a, 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, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CasX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs and modified versions thereof.

93. The composition of any one of claims 65-73, wherein the nucleic acid-guided polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 318-338.

94. The composition of any one of claims 65-73, wherein the nucleic acid polymerase is a reverse transcriptase.

95. The composition of claim 94, wherein the reverse transcriptase comprises an RNase H domain.

96. The composition of claim 94, wherein the reverse transcriptase lacks an RNase H domain.

97. The composition of claim 94, wherein the reverse transcriptase is selected from the group consisting of: murine leukemia virus reverse transcriptase (M-MLV RT) (optionally D200N, T306K, W313F, T330P, and L603W), friend murine leukemia virus reverse transcriptase (FMLV RT), human endogenous retrovirus Kcon reverse transcriptase (HERV Kcon RT), a AMV-RT, a MarathonRT, a transcription xenopolymerase (RTX), and a small reverse transcriptase (Tfl), and functional mutants thereof.

98. The composition of claim 94, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 339-344.

99. The composition of any one of claims 65-73, wherein the recombinase is selected from the group consisting of: Hin, Gin, Tn3, 0-six, CinH, ParA, y8, Bxbl, 4>C31, TP901, TGI, cpBTl, R4, cpRVl, cpFCl, MR11, Al 18, U153, gp29, Ore, FLP, R, Lambda, HK101, HK022, and pSAM2 recombinase.

100. The composition of any one of claims 65-73, wherein the recombinase comprises one or more recombinases independently selected from the group consisting of: Cre, Bxbl, FLP, Al 18, Abrogate, Airmid, Anglerfish, B2, B3, Benedict, BL3, Bob3, Bred, BxZ2, Cin, Conceptll, CreALSHG, Cre- R3M3, Doom, Dre, Fre, Gin, Hin, Hinder, HK022, ICleared, IntlO, Inti 1, Intl2, Intl3, Int3, Int4, Int8, Int9, Inti, K38, Kd, KSSJEB, LI, L5, LI, Lockley, Mariner (Himarl), Mariner (mosl), Min, Minos, MH (phiFCl), MR11, Mundrea, Museum, Nigri, P22, Panto, PattyP, Peaches, phi370.1, phiBTl, phiC31, phiJoe, phiK38, phiRVl, R, Rl, R2, R3, R4, R5, RDF, Rebeuca, retrotransposases encoded by R2, Sarfire, Scowl, SCre, Severus, Sheen, Sin, SkiPole, SPBc, SprA, SV1, Switzer, Tc3, TD1-40, TGI, Theia, Tol2Tcl, TP901-1, Tre, Troube, U153, VCre, Veracruz, Vika, WB, W0, <p370.1, cpBTl, cpCl, cpC31, cpFCl, and cpRV.

101. The composition of any one of claims 65-73, wherein the recombinase recognizes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538.

102. The composition of any one of claims 68-73, wherein the donor nucleic acid sequence encodes a therapeutic molecule, and optionally wherein the therapeutic molecule comprises at least a functional portion of a viral envelope protein, a hormone, a cytokine, a ligand, a receptor, an antibody, an enzyme, a transcription factor, a chimeric antigen receptor, a T cell receptor, an antigen, a secreted protein, or any combination thereof.

103. The composition of any one of claims 65-73, wherein the guide nucleic acid molecule comprises one or more guide RNA, optionally wherein each of the one or more guide RNA comprises (A) a primer binding site, (B) a clamp segment, (C) a nucleic acid sequence encoding at least a portion of a first recombinase recognition sequence, (D) an aptamer, (E) spacer, or (F) scaffold, or any combinations thereof.

104. The composition of any one of claims 65-73, wherein the guide nucleic acid molecule comprises a first guide RNA encoding at least a first portion of a first recombinase recognition sequence and a second guide RNA encoding at least a second portion of the first recombinase recognition sequence, wherein the first guide RNA and the second guide RNA work in a pair and collectively encode the first recombinase recognition sequence, and optionally wherein the first and the second portion of the first recombinase recognition sequence have at least 6bp overlap.

105. The composition of any one of claims 65-73, wherein the guide nucleic acid molecule comprises a (A) nicking guide RNA and (B) a guide RNA sequence encoding a first recombinase recognition sequence.

106. The composition of any one of claims 67-73, wherein the first recombinase recognition sequence or the second recombinase recognition sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538, and wherein the first recombinase recognition sequence and the second recombinase recognition sequence are a pair of recombinase recognition sequences for a cognate recombinase.

107. The composition of any one of claims 65-73, wherein the composition further comprises a fourth nucleic acid sequence encoding an additional recombinase, wherein the additional recombinase recognizes an additional pair of recombinase recognition sequences, and wherein the additional recombinase and the recombinase are different.

108. The composition of claim 107, wherein the donor nucleic acid molecule further comprises the additional pair of recombinase recognition sequences, wherein the additional pair of recombinase recognition sequences comprises a third recombinase recognition sequence located at a 3’ end of the donor nucleic acid molecule and a fourth recombinase recognition sequence located at a 5’ end of the donor nucleic acid molecule, wherein the additional pair of recombinase recognition is capable of self-circularizing when contacted with the additional recombinase, and wherein the additional pair of recombinase recognition sequences has a faster integration rate than the first recombinase recognition sequence and the second recombinase recognition sequence, thereby the additional pair of recombinase recognition sequences recombines prior to recombination of the first recombinase recognition sequence and the second recombinase recognition sequence in the presence of the recombinase and the additional recombinase.

109. The composition of claim 107, wherein the composition comprises a nucleic acid molecule that comprises the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, the fourth nucleic acid sequence, and the donor nucleic acid sequence.

110. The composition of any one of claims 65-73, wherein the composition comprises a sixth nucleic acid sequence encoding a MLHldn protein, and optionally wherein the sixth nucleic acid sequence encodes a third chimeric protein comprising a fourth plasma membrane recruitment element and the MLHldn protein, and optionally wherein the fourth plasma membrane recruitment element is the same as the first plasma membrane recruitment element, the second plasma membrane recruitment element, or the third plasma membrane recruitment element.

111. A system, comprising:

(1) a lipid delivery particle comprising

(c) a lipid containing membrane; and

(d) a ribonucleoprotein complex comprising: (A) a prime editor comprising a nucleic acid- guided polypeptide coupled to a nucleic acid polymerase; and (B) a guide nucleic acid molecule, wherein the ribonucleoprotein complex is within an inside cavity encapsulated by the lipid containing membrane; and

(2) a recombinase or a nucleic acid sequence encoding the recombinase.

112. A system, comprising:

(1) a lipid delivery particle comprising

(c) a lipid containing membrane; and

(d) a recombinase, wherein the recombinase is within an inside cavity encapsulated by the lipid containing membrane; and

113. The system of claim 111, wherein the lipid delivery particle has a diameter that is less than 5 pm, less than 4 pm, less than 3 pm, less than 2 pm, or less than 1 pm.

114. The system of claim 112, wherein the lipid delivery particle has a diameter that is less than 5 pm, less than 4 pm, less than 3 pm, less than 2 pm, or less than 1 pm.

115. The system of any one of claims 111-114, wherein the lipid containing membrane encapsulates a protein core.

116. The system of any one of claims 111-114, wherein the prime editor and the guide nucleic acid molecule are configured to introduce a first recombinase recognition sequence into a target nucleic acid molecule; and wherein the recombinase mediates recombination between the first recombinase recognition sequence and a second recombinase recognition sequence.

117. The system of any one of claims 111-114, wherein the system further comprises either (3) a donor nucleic acid molecule comprising a donor nucleic acid sequence and the second recombinase recognition sequence, or (4) a template RNA that encodes the donor nucleic acid molecule.

118. The system of any one of claims 111-114, wherein the lipid containing membrane comprises a phospholipid bilayer.

119. The system of claim 117, wherein the template RNA comprises a long terminal repeat (LTR) sequence.

120. The system of claim 119, wherein the template RNA comprises at least two LTR sequences flanking a nucleic acid sequence encoding the donor nucleic acid molecule, optionally wherein the at least two LTR sequences is capable of self-circularizing.

121. The system of claim 119, wherein the LTR sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 345-352.

122. The system of any one of claims 111-114, wherein the lipid delivery particle further comprises an envelope protein attached to the lipid containing membrane.

123. The system of claim 122, wherein the envelope protein is a viral envelope protein.

124. The system of claim 123, wherein the viral envelope protein is selected from the group consisting of: a VSV-G protein, a FuG-B2 envelope protein, a FuG-E envelope protein, an HIV-1 envelope, a baboon retroviral envelope protein, and an ecotropic murine leukemia virus (MLV) envelope protein, and functional mutants thereof.

125. The system of claim 123, wherein the viral envelope protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 83-104.

126. The system of claim 122, wherein the envelope protein is a human endogenous retroviral envelope protein.

127. The system of claim 126, wherein the human endogenous retroviral envelope protein is selected from the group consisting of hENVHl, hENVH2, hENVH3, hENVKl, hENVK2, hENVK3, hENVK4, hENVK5, hENVK6, hENVT, hENVW, hENVFRD, hENVR, hENVR(b), hENVR(c)2, hENVR(c)l, and hENVK_COn, and functional mutants thereof.

128. The system of claim 126, wherein the human endogenous retroviral envelope protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 49-82.

129. The system of any one of claim 111-114, wherein the lipid delivery particle comprises a plasma membrane recruitment element.

130. The system of claim 129, wherein the plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof, optionally wherein the plasma membrane recruitment element is part of a structural protein that forms the protein core.

131. The system of claim 130, wherein the structural protein further comprises a retroviral protease (pro) protein.

132. The system of claim 129, wherein the plasma membrane recruitment element is a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof.

133. The system of claim 129, wherein the plasma membrane recruitment element is a pleckstrin homology (PH) domain of a protein selected from the group consisting of: phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3 -phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxysterol -binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate-adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, and MAPK associated protein 1 (MAPKAP1), and functional mutants thereof.

134. The system of claim 129, wherein the plasma membrane recruitment element is selected from the group consisting of: a pleckstrin homology (PH) domain of human phospholipase C81, a pleckstrin homology (PH) domain of human Aktl, a pleckstrin homology (PH) domain of human Arc, human endogenous retroviral gag protein, a pleckstrin homology (PH) domain of human 3 -phosphoinositidedependent protein kinase 1 (hPDPKl), a transmembrane domain of human CD9, a transmembrane domain of human CD47, a transmembrane domain of human CD63, a transmembrane domain of human CD81, a transmembrane domain of human Daapl, a transmembrane domain of mouse Grpl, a transmembrane domain of human Grpl, a transmembrane domain of human OSBP, a transmembrane domain of human Btkl, a transmembrane domain of human FAPP1, a transmembrane domain of human CERT, a transmembrane domain of human PKD, a transmembrane domain of human PHLPP1, a transmembrane domain of human SWAP70, and a transmembrane domain of human MAPKAP1, and functional mutants thereof.

135. The system of claim 129, wherein the plasma membrane recruitment element comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-48.

136. The system of any one of claims 111-114, wherein the lipid delivery particle further comprises a first chimeric protein that comprises a second prime editor and a second plasma membrane recruitment element.

137. The system of claim 136, wherein the second prime editor has the same sequence as the prime editor.

138. The system of any one of claims 111-114, wherein the lipid delivery particle further comprises a second chimeric protein that comprises a second recombinase and a third plasma membrane recruitment element.

139. The system of claim 138, wherein the second recombinase has the same sequence as the recombinase.

140. The system of claim 136, wherein the second plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof, and optionally wherein the first chimeric protein or the second chimeric protein forms part of the protein core.

141. The system of claim 136, wherein the second plasma membrane recruitment element is a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof.

142. The system of claim 138, wherein the third plasma membrane recruitment element is a retroviral gag protein or a functional mutant thereof, and optionally wherein the first chimeric protein or the second chimeric protein forms part of the protein core.

143. The system of claim 138, wherein the third plasma membrane recruitment element is a human endogenous retroviral gag protein or a functional mutant thereof, from a mammalian protein, a membrane protein or a transmembrane domain thereof, or a pleckstrin homology (PH) domain or a functional mutant thereof.

144. The system of claim 141 or 143, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element is a pleckstrin homology (PH) domain of a protein selected from the group consisting of: phospholipase C81 (PLC81), Aktl, Arc, endogenous retroviral gag protein, 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), CD9, CD47, CD63, CD81, Disc and Actin-Associated Protein 1 (Daapl), General receptor for phosphoinositides 1 (Grpl), Oxy sterol -binding protein 1 - Homo sapiens (OSBP), Bruton tyrosine kinase (Btk), Four-phosphate- adaptor protein 1 (FAPP1), ceramide transfer protein (CERT), protein kinase D (PKD), PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), Switching B Cell Complex Subunit SWAP70, and MAPK associated protein 1 (MAPKAP1), and functional mutants thereof.

145. The system of claim 141 or 143, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element is selected from the group consisting of: a pleckstrin homology (PH) domain of human phospholipase C81, a pleckstrin homology (PH) domain of human Aktl, a pleckstrin homology (PH) domain of human Arc, human endogenous retroviral gag protein, a pleckstrin homology (PH) domain of human 3-phosphoinositide-dependent protein kinase 1 (hPDPKl), a transmembrane domain of human CD9, a transmembrane domain of human CD47, a transmembrane domain ofhuman CD63, a transmembrane domain of human CD81, a transmembrane domain ofhuman Daapl, a transmembrane domain of mouse Grpl, a transmembrane domain of human Grpl, a transmembrane domain ofhuman OSBP, a transmembrane domain ofhuman Btkl, a transmembrane domain ofhuman FAPP1, a transmembrane domain of human CERT, a transmembrane domain ofhuman PKD, a transmembrane domain ofhuman PHLPP1, a transmembrane domain ofhuman SWAP70, and a transmembrane domain ofhuman MAPKAP1, and functional mutants thereof.

146. The system of claim 141 or 143, wherein the second plasma membrane recruitment element or the third plasma membrane recruitment element comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1-48.

147. The system of any one of claims 111-114, wherein the nucleic acid-guided polypeptide is a Cas protein.

148. The system of claim 147, wherein the Cas protein is a type I, type II, type III, type IV, type V, or type VI Cas protein.

149. The system of claim 147, wherein the Cas protein is selected from the group consisting of: c2cl, Casl3a, Casl3b, Casl3c, Casl3d, c2c3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), Cas 10, CaslOd, Casl4, CaslO, CaslOd, CasF, CasG, CasH, Casl2a, 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, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CasX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs and modified versions thereof.

150. The system of any one of claims 111-114, wherein the nucleic acid-guided polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 318-338.

151. The system of any one of claims 111-114, wherein the nucleic acid polymerase is a reverse transcriptase.

152. The system of claim 151, wherein the reverse transcriptase comprises an RNase H domain.

153. The system of claim 151, wherein the reverse transcriptase lacks an RNase H domain.

154. The system of claim 151, wherein the reverse transcriptase is selected from the group consisting of: murine leukemia virus reverse transcriptase (M-MLV RT) (optionally D200N, T306K, W313F, T330P, and L603W), friend murine leukemia virus reverse transcriptase (FMLV RT), human endogenous retrovirus Kcon reverse transcriptase (HERV Kcon RT), a AMV-RT, a MarathonRT, a transcription xenopolymerase (RTX), and a small reverse transcriptase (Tfl), and functional mutants thereof.

155. The system of claim 151, wherein the reverse transcriptase comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 339-344.

156. The system of any one of claims 111-114, wherein the recombinase is selected from the group consisting of: Hin, Gin, Tn3, fl-six, CinH, ParA, y8, Bxbl, (|)C31, TP901, TGI, cpBTl, R4, cpRVl, cpFCl, MR11, Al 18, U153, gp29, Cre, FLP, R, Lambda, HK101, HK022, and pSAM2 recombinase.

157. The system of any one of claims 111-114, wherein the recombinase comprises one or more recombinases independently selected from the group consisting of: Cre, Bxbl, FLP, Al 18, Abrogate, Airmid, Anglerfish, B2, B3, Benedict, BL3, Bob3, Bred, BxZ2, Cin, Conceptll, CreALSHG, Cre- R3M3, Doom, Dre, Fre, Gin, Hin, Hinder, HK022, ICleared, IntlO, Inti 1, Intl2, Intl3, Int3, Int4, Int8, Int9, Inti, K38, Kd, KSSJEB, LI, L5, LI, Lockley, Mariner (Himarl), Mariner (mosl), Min, Minos, MH (phiFCl), MR11, Mundrea, Museum, Nigri, P22, Panto, PattyP, Peaches, phi370.1, phiBTl, phiC31, phiJoe, phiK38, phiRVl, R, Rl, R2, R3, R4, R5, RDF, Rebeuca, retrotransposases encoded by R2, Sarfire, Scowl, SCre, Severus, Sheen, Sin, SkiPole, SPBc, SprA, SV1, Switzer, Tc3, TD1-40, TGI, Theia, Tol2Tcl, TP901-1, Tre, Troube, U153, VCre, Veracruz, Vika, WB, W0, cp370.1, cpBTl, cpCl, <pC31, cpFCl, and <pRV.

158. The system of any one of claims 111-114, wherein the recombinase recognizes a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538.

159. The system of any one of claims 111-114, wherein the lipid delivery particle is a retroviral particle or a lentiviral particle.

160. The system of claim 117, wherein the donor nucleic acid sequence encodes a therapeutic molecule, optionally wherein the therapeutic molecule comprises at least a functional portion of a viral envelope protein, a hormone, a cytokine, a ligand, a receptor, an antibody, an enzyme, a transcription factor, a chimeric antigen receptor, a T cell receptor, an antigen, a secreted protein, or any combination thereof.

161. The system of any one of claims 111-114, wherein the guide nucleic acid molecule comprises one or more guide RNA, optionally wherein each of the one or more guide RNA comprises (A) a primer binding site, (B) a clamp segment, (C) a sequence encoding at least a portion of a first recombinase recognition sequence, (D) an aptamer, (E) spacer, or (F) scaffold, or any combinations thereof.

162. The system of any one of claims 111-114, wherein the guide nucleic acid molecule comprises a first guide RNA encoding at least a first portion of a first recombinase recognition sequence and a second guide RNA encoding at least a second portion of the first recombinase recognition sequence, wherein the first guide RNA and the second guide RNA work in a pair and collectively encode the first recombinase recognition sequence, optionally wherein the first and the second portion of the first recombinase recognition sequence have at least 6bp overlap.

163. The system of any one of claims 111-114, wherein the guide nucleic acid molecule comprises (A) a nicking guide RNA and (B) a guide RNA encoding a first recombinase recognition sequence.

164. The system of claim 116, wherein the first recombinase recognition sequence or the second recombinase recognition sequence comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% sequence identity to any one of the sequences set forth in SEQ ID NOs: 105-317 or 515-538, and wherein the first recombinase recognition sequence and the second recombinase recognition sequence are a pair of recombinase recognition sequences for a cognate recombinase.

165. The system of any one of claims 111-114, wherein the system further comprises an additional recombinase or a nucleic acid encoding the additional recombinase, wherein the additional recombinase recognizes an additional pair of recombinase recognition sequences, and wherein the additional recombinase and the recombinase are different.

166. The system of claim 165, wherein the donor nucleic acid molecule further comprises the additional pair of recombinase recognition sequences, wherein the additional pair of recombinase recognition sequences comprises a third recombinase recognition sequence located at a 3’ end of the donor nucleic acid molecule and a fourth recombinase recognition sequence located at a 5’ end of the donor nucleic acid molecule, wherein the additional pair of recombinase recognition is capable of self-circularizing when contacted with the additional recombinase, and wherein the additional pair of recombinase recognition sequences has a faster integration rate than the first recombinase recognition sequence and the second recombinase recognition sequence, thereby the additional pair of recombinase recognition sequences recombines prior to recombination of the first recombinase recognition sequence and the second recombinase recognition sequence in the presence of the recombinase and the additional recombinase.

167. The system of any one of claims 111-114, wherein the system further comprises a MLHldn protein or a nucleic acid sequence encoding the MLHldn protein, and optionally wherein the MLHldn protein is a part of a third chimeric protein comprising a fourth plasma membrane recruitment element, and optionally wherein the fourth plasma membrane recruitment element is the same as the plasma membrane recruitment element, the second plasma membrane recruitment element or the third plasma membrane recruitment element.

168. A pharmaceutical composition comprising (a) the lipid delivery particle of any one of claims 1-11 or the system of any one of claims 111-114; and (b) a pharmaceutically acceptable excipient.

169. A kit comprising (a) the lipid delivery particle of any one of claims 1-11, the system of any one of claims 111-114, or the pharmaceutical composition of claim 168; and (b) an information material containing instructions for administering a dosage of the lipid delivery particle or the system, or a dosage form of the pharmaceutical composition to a subject.

170. A method of treating a disease or a condition in a subject in need thereof, comprising administering to the subject the lipid delivery particle of any one of claims 1-11, the system of any one of claims 111-114, orthe pharmaceutical composition of claim 168.

171. A method comprising contacting a cell with the lipid delivery particle of any one of claims 1-11.

172. The method of claim 171, the method comprising generating a template DNA in the cell using at least a portion of the template RNA as a template, wherein the template DNA encodes a therapeutic molecule, and optionally circularizing the template DNA in the cell; and expressing the therapeutic molecule from the template DNA in the cell.

173. A method comprising contacting a cell with the system of any one of claims 111-114.

174. A method comprising administering the lipid delivery particle of any one of claims 1-11 to a subject in need thereof.

175. A method of producing the lipid delivery particle of any one of claims 1-11.

176. A method of producing a lipid delivery particle, the method comprising contacting a producer cell with the composition of any one of claims 65-73.